JP2000287698A - Method for producing oligopeptide of high glutamine content - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish a practical process for largely producing an oligopeptide of high glutamine content, which is expected the clinical application as a nutrient agent for intestinal administration, from porous dry glutene. SOLUTION: Porous gluten resulting from freeze-drying of gluten balls is suspended in a protease solution to effect the reaction in a shortened time and collect or fractionate the objective peptide of high glutamine content from the reaction mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an glutamine-rich oligopeptide useful as a material for intestinal infusion,
The present invention relates to a method for producing a large amount from porous dried gluten.

[0002]

BACKGROUND ART Glutamine, which has the highest concentration in human free amino acids in blood, is regarded as a non-essential amino acid in conventional nutrition, and its functions include transport of ammonia,
Only storage and detoxification were pointed out. However, in recent years, it has been clarified that glutamine plays a very important role in living organisms, and it has been pointed out that glutamine uptake and its role as a major energy substrate of the intestinal tract, which is a consuming organ, are particularly important functions. For these reasons, attempts to clinically apply an infusion containing glutamine have been considered, but it is difficult to add glutamine to an amino acid infusion. This is because glutamine is hardly soluble, and free glutamine is unstable in an aqueous solution and cyclizes into pyroglutamic acid.

[0003]

From the background described above, since glutamine other than the amino terminus does not cyclize,
There is a growing social demand for the development of peptide glutamine that can be effectively used. In the case of the peptide type, glutamine other than the amino terminus is not cyclized and is effectively used. The present inventors have already reported a method of hydrolyzing gluten with morphine and actinase to prepare an oligopeptide having a high glutamine content (high glutamine peptide) (J. Food Biochem. 16: 235-248 (1993)). ). However, although the method according to the above-mentioned report can obtain a product with satisfactory quality, the substrate concentration must be as low as 1-5% in order to make the reaction system with the enzyme a homogeneous liquid. Therefore, a large amount of energy resources are used in the drying process after the treatment. In addition, the reaction time is 10 hours to 48 hours, which is disadvantageous in that it requires a long time. An object of the present invention is to develop a method for mass-producing high-glutamine peptides having the same or higher quality as that of a product according to a previously reported method by using a simple process using gluten as a raw material.

[0004]

Accordingly, the present inventors have conducted intensive studies, prepared porous dry gluten from wheat flour, treated with proteolytic enzyme, and fractionated high glutamine peptide from the enzyme reaction solution. We have developed a production method consisting of the process of obtaining oligopeptides with high glutamine content. Hereinafter, embodiments of the present invention will be specifically described.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, gluten is used as a raw material.
Instead of powdered gluten such as "gluten", dried gluten of special specifications having particularly excellent porosity is essential. This is because ordinary gluten has poor dispersibility in the enzyme reaction, requires a large amount of water (including buffer solution) for dispersion, requires a long time for the reaction, and lowers the peptide yield. Because there is.

As an example of a porous gluten suitable for the present invention, it can be prepared by freeze-drying or vacuum-drying gluten (wet gluten) obtained from wheat flour and pulverizing it to about 48 mesh or less. it can. It may be made porous by firing. The porous gluten is prepared by adding 400 to 1000 parts by weight, more preferably 400 to 500 parts by weight, of a protease in an amount of 0.1 to 5 parts by weight with respect to 100 parts by weight of the gluten in water (buffer). (Including liquids and the like) in an enzyme solution. The amount of water in the reaction system
It is sufficient to minimize the amount of water evenly distributed on the substrate. Even if the amount is increased more than necessary, a particularly good effect is not obtained, and conversely, the processing cost in the purification process of the next step is increased.

As the proteolytic enzyme used in the present invention, a commercially available enzyme preparation of a food additive class can be used.
Enzymes of microorganism or plant origin having H, such as actinase (AS, manufactured by Kaken Pharmaceutical), proteinase N
"Amano" (manufactured by Amano Pharmaceutical Co., Ltd.), Bioprase SP-4FG
(Manufactured by Nagase Seikagaku Corporation), AO protease (manufactured by Kikkoman), papain, bromelain (manufactured by Amano Pharmaceutical) and the like are preferred. If necessary, two or more of these enzymes can be used in combination.

The reaction is carried out while stirring the reaction solution if necessary.
The reaction is carried out at an optimum temperature and an optimum pH of the enzyme to be used in a relatively short time of 0.5 to 5 hours, more preferably about 1 to 2 hours. This is because the desired high glutamine peptide is produced in such a short time in the method of the present invention. Even if the reaction time is unnecessarily extended, exopeptidase in the enzyme preparation acts to increase free amino acids. This is because inconvenience may occur.

The enzyme reaction solution obtained by the present invention contains about 90% by dry weight of high glutamine peptide (molecular weight distribution is approximately 500-1000). For this reason, the reaction solution can be subjected to treatments such as sterilization, concentration, and drying as it is to obtain a high glutamine peptide product. However, if necessary, a molecular sieve material such as ultrafiltration or Sephadex (Pharmacia Biotech) can be used A low molecular fraction such as free amino acid and a high molecular weight fraction having a molecular weight of 1000 or more can be removed by a known method such as fractionation by using a conventional method, and the fraction having a molecular weight of 500 to 1,000 can be concentrated. By such a treatment, the glutamine content in the high glutamine peptide product can be higher than that of the gluten substrate.

When the high glutamine peptide of the present invention is used for amino acid infusion or the like, a sterilization or sterilization step is essential. For this purpose, sterilization by treatment with a synthetic polymer membrane such as polyolefin is the most suitable. . This is because, when a peptide containing glutamine at the amino terminus is heated, the amino-terminal glutamine may be cyclized to produce an indigestible pyroglutamine peptide. The high glutamine peptide obtained in the present invention has high solubility in water and has a higher glutamine content than the raw material gluten, so that clinical application as an enteral nutritional agent is greatly expected. Hereinafter, the present invention will be described in detail with reference to examples. However, the above description and the examples described below are preferable examples of the practice of the present invention, and mean that the effective range of the present invention is limited or limited. It does not do.

[0011]

EXAMPLES (Preparation of porous gluten) Strong flour (15
kg) was kneaded with water, and starch and the like were washed away with water to prepare gluten balls. When this was freeze-dried by a conventional method, many voids were found in the gluten ball. After crushing the gluten balls, the gluten balls were sieved through a 48 mesh sieve to prepare porous gluten, which was used as a substrate for the actinase reaction.

(Actinase reaction) Enzyme actinase (AS, 250 tyrosine units / mg, Kaken Pharmaceutical) 100
The substrate (10 kg) was suspended in 50 L of water in which g was dissolved. During the reaction, the pH was adjusted to 7 with 1M NaOH, and the reaction was carried out at 40 ° C. with stirring.

(Change over time in dissolution of gluten) The change over time of the soluble protein in the reaction solution was examined. A 9-fold amount of 4 M urea-containing Tris-HCl buffer (pH 8.6) was added to a part of the enzyme reaction solution, and the total protein was quantified using a commercially available protein quantification kit (protein assay kit, Bio-Rad). Separately, a part of the enzyme reaction solution was
Separation was performed at 000 xg for 15 minutes, and the amount of solubilized protein in the extract in the supernatant was similarly determined. As a result, the ratio of the soluble protein to the total protein almost reached a plateau one hour after the start of the reaction, and it was revealed that about 90% of the substrate was solubilized. Table 1 shows changes over time in the solubility of gluten.

[0014]

[Table 1]

(Preparation by gel filtration)
The reaction mixture was centrifuged at 5,000 × g for 15 minutes, and the supernatant (150 mL) was separated with a Sephadex G-15 column (Pharmacia Biotech, 20 ×).
(60 cm). 10% ethanol was used for elution, the flow rate was 100 mL / min, and the detection was 260 n.
m. The excluded volume was 5L. The chromatogram of the protein separation is shown in FIG. In FIG. 1, the amount of eluate is shown on the horizontal axis, and the absorbance at 260 nm is shown on the vertical axis.

(Membrane filtration) When a peptide containing glutamine at the amino terminal is heated, pyroglutamic acid may be generated at the amino terminal. Therefore, the high glutamine peptide prepared here was sterilized by filtration. That is, ethanol was removed from the fraction obtained by gel filtration under reduced pressure, and the bacteria were passed through a polyolefin-based synthetic polymer membrane having a pore size of 0.25 μm (Asahi Kasei, PMP-113) to remove bacteria. Ethanol was removed under reduced pressure to obtain 8.2 kg of high glutamine peptide product.

(Amino acid analysis of high glutamine peptide)
After freeze-drying the sterilized product, the amino acid composition was analyzed by the following method. A sample (1 mg) was added to 6N HCl (1 m
L), degassed, hydrolyzed at 110 ° C. for 24 hours, and the constituent amino acids were quantified by an automatic amino acid analyzer (Hitachi 835). After oxidizing the sample by the formic acid oxidation method, it was hydrolyzed with 6N HCl at 110 ° C. for 24 hours,
Two cysteines (1/2 Cys) were analyzed. 3 samples
Hydrolysis was performed with N mercaptoethanesulfonic acid, and tryptophan (Trp) was analyzed. Amino acid composition was expressed in weight%. Separately, a sample (5 mg) is dissolved in 20 mM Tris buffer (0.5 mL), and the sample is hydrolyzed by a series of enzymatic reactions using actinase E, aminopeptidase, carboxypeptidase A, and carboxypeptidase W. Glutamine (Gln) was quantified using the amino acid analyzer described above in the program. Table 2 shows the results of the amino acid analysis.

[0018]

[Table 2]

The Glx (Gln + Glu) content in the product was 50.6%, and the Glx content of the raw gluten (41.
8%). Most of Glx was glutamine (Gln). That is, although the raw material gluten used here has the highest glutamine content obtained as a raw material, the glutamine content is further increased by 1.2 times as compared with that, and it is recognized that a high glutamine peptide was obtained. . From the above, it has been clarified that high-glutamine peptides can be easily produced in large quantities and easily by enzymatic reaction for one hour and gel filtration chromatography using lyophilized gluten balls as a starting material. In the future, high glutamine peptides are expected to be clinically applied as enteral nutritional supplements.

[0020]

Industrial Applicability According to the present invention, a practical process for producing a large amount of oligopeptide having a high glutamine content from porous dried gluten has been established.

[Brief description of the drawings]

FIG. 1 shows gluten treated with actinase.
It is a figure which shows the pattern of a gel filtration chromatography.

Claims (4)

[Claims] 1. A method for producing an oligopeptide, comprising: (1) preparing porous dry gluten from wheat flour; (2) decomposing the porous dry gluten with a protease; A method for producing an oligopeptide, comprising a step of collecting an oligopeptide from a degradation product. 2. The method for producing an oligopeptide according to claim 1, wherein a porous dried gluten is prepared from the raw wheat flour by freeze drying, vacuum drying or baking. 3. The method according to claim 1, wherein 100 parts by weight of porous dry gluten is decomposed by a protease in a solution of 0.1 to 5 parts by weight of protease in 400 to 1000 parts by weight of water. The method for producing an oligopeptide according to claim 1, wherein 4. The method for producing an oligopeptide according to claim 1, wherein the porous dried gluten is decomposed with a protease for 1 to 5 hours.