JP2524551B2 - Peptide composition having high glutamine content, method for producing the same, and enteral nutritional supplement - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide composition containing glutamine or glutamic acid in an amount of 40% or more of the constituent amino acids, a method for producing the peptide composition, and an enteral nutritional supplement containing the composition. INDUSTRIAL APPLICABILITY The peptide composition of the present invention is useful as a nitrogen source for enteral nutrients and as an infusion raw material.

[0002]

2. Description of the Related Art Parenteral nutrition and enteral nutrition can be mentioned as nutritional supplementation methods after surgery and nutritional therapy for various diseases. If the catabolic function due to surgery etc. has improved,
Therapies for supplementing high-calorie nutrition and promoting recovery of the living body are generally performed. As a nutritional composition for this purpose, one that does not burden digestion and absorption is used. A typical example is a high calorie infusion.
High-calorie infusion is a continuous administration of amino acid solution and high-concentration sugar solution via a central vein. As the enteral nutrient, a component nutrient designed so as not to burden digestion and absorption is used. It consists of amino acid mixture, dextrin, triglyceride, inorganic salt, and vitamin mixture as nutritional components, and is said to have almost no burden of digestion and absorption. Also, a component nutritional supplement using a peptide obtained by enzymatically hydrolyzing a protein as a nitrogen source is commercially available.

Various formulations have been studied for the amino acid composition of these high calorie infusions and component infusions. In particular, various studies have been conducted on essential amino acids, and compositions in which specific amino acids are increased or decreased according to the pathological condition have been proposed. However, such studies have not been made for non-essential amino acids. Recently, non-essential amino acids have been investigated, and addition of arginine and glutamine has been investigated. However, glutamine is very unstable, there are pharmaceutical problems, and there is no clear standard regarding the required amount, and glutamine is generally used as a substitute for glutamine as the amino acid composition of the above component nutritional supplements and high calorie infusions. It has already been used and no glutamine fortified product has yet been offered.

However, there is a remarkable recent research on amino acid metabolism, and the importance of glutamine is being clarified. Especially, it has essential effects as a nutritional supplement after surgery, such as improving nitrogen balance during various invasions and stresses, wound healing effect, anti-ulcer effect, and preventive effect on gastrointestinal mucosal atrophy during complete parenteral nutrition. It is known to have. Therefore, attempts have been made to add glutamine to the infusion solution.

As glutamine to be added to these infusion solutions, use of crystalline glutamine or acylated glutamine has been proposed. Further, Japanese Patent Laid-Open No. 2-119762 proposes the use of a tripeptide or dipeptide containing an L-glutamine residue. Amino acids are generally used as a nitrogen source for infusion, and it is considered that there is no problem in using such amino acid derivatives and dipeptides. However, when used as an enteral nutrient, it has been pointed out that when a low-molecular compound is present in a high concentration, the osmotic pressure becomes high and often causes diarrhea. It has also been pointed out that amino acids and low-molecular-weight peptides have a pungent taste and are resistant to oral administration.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have investigated glutamine preparations to be added to nutritional supplements administered after surgery or at the time of illness, and as a result, they are pharmaceutically stable and have little effect on osmotic pressure. The inventors have found a peptide composition containing glutamine in a high concentration and completed the present invention. Therefore, the present invention relates to a peptide composition containing glutamine or glutamic acid in an amount of 40% or more of the constituent amino acids, a method for producing the peptide composition, and an enteral nutritional supplement containing the composition.

[0007]

SUMMARY OF THE INVENTION Provided by the present invention.
Peptide composition containing highly glutamine,Gluten
Or zein with acidic and neutral proteases
Obtained by sequentially hydrolyzing and hydrolyzing the hydrolyzate
It has the following properties that can
It contains a small amount of free amino acids.
It is a peptide composition. Glutamine or glutamine in a peptide composition
Acid content: When amino acids are quantified after hydrolysis
More than 40% of the no acids are quantified as glutamic acid. Free amino acid content: 10% or less and free glutamine
acidIs 5% or less. Average molecular weight: about 200Dalton (by gel filtration
Ru) Molecular weight distribution:Most of it is molecular weight131-1411
DaltonDistributed in the range ofAnd the molecular weight200Dalton
It has a peak in the vicinity.  Physiological action: It has a function to suppress the degeneration of small intestinal mucosa.This
Peptide compositions with such characteristics have been obtained so far.
Not provided by the present invention.

The method for producing the peptide composition of the present invention is as follows.
It is a cage . The peptide composition of the present invention can be obtained by subjecting glutamine as a constituent amino acid of a protein to enzymatically degrading a highly contained protein under specific conditions and further performing molecular weight fractionation. Glutamine is included in a large amount in usual in food, but is preferably not more than 20% protein constituent amino acid, in particular, zein to <br/> relatively many are gluten, corn protein, wheat protein are contained, such proteins and the raw material
To be done . It is particularly preferable to use gluten from the viewpoint of availability and supply of raw materials.

[0009] The entire amino acid sequence of gluten has been clarified from its cDNA sequence.
Research (Nucleic Acid Res), 13 , 8729, (1985)] shows that 96% of the total amount of glutamine and glutamic acid is glutamine, which is particularly suitable as a raw material. In addition, when an amino acid is analyzed by acid hydrolysis, glutamine is quantified as glutamic acid. Therefore, using glutamic acid as an index has an advantage that the amount of glutamine can be accurately grasped.
The protein used as a raw material may be a crude product or a refined product and may be used.

The protein is dissolved or suspended in water so as to have a protein concentration of 0.5 to 10% and then treated with an enzyme. As the enzyme, any acidic protease can be used, but microbial protease or animal protease is generally preferred. A fungal protease can be illustrated as a microbial protease. Examples of fungal proteases include lapidase (manufactured by Takeda Chemical) and Morcin (manufactured by Sigma), and examples of animal proteases include pepsin. Other enzymes can be used as long as they are acidic proteases. An enzyme is added to the above-mentioned aqueous solution to perform an enzymatic decomposition treatment. The treatment is carried out under the optimum conditions of the enzyme to be used, and it has been confirmed that morphine is particularly suitable for the production of low molecular weight peptides in the enzyme treatment using gluten as a substrate.

When gluten is used as a substrate, a solution containing the peptide composition can be obtained by treatment in an enzyme / substrate ratio of 1: 100 in a 1% acetic acid solvent at 37 ° C. for 24 hours. After completion of the reaction, the enzymatic reaction was stopped by heat treatment or the like, is subjected to the enzymatic treatment et al., To adjust the molecular weight of the peptide composition to a certain size.

In the present invention, after the gluten enzymatic degradation by Molsin as a raw material, performing an enzyme treatment by further neutral protease. The pH of the solution treated with the acidic protease is made neutral with an alkali such as sodium hydrogen carbonate solution, and the neutral protease is added to perform the enzyme treatment. Actinase protease, especially actinase (Kaken Pharmaceutical)
Are particularly preferable. When gluten is used as a substrate, a solution containing the peptide composition can be obtained by treating the enzyme / substrate ratio at 1: 100 in a pH 7 solution at 37 ° C. for 24 hours. After completion of the reaction, the enzyme was inactivated, molecular weight fractionation by gel filtration and adsorption chromatography, to remove free amino acids, it can be obtained peptide fraction. For the fractionation of the peptide fraction , any carrier can be used as long as it can separate peptides having a peptide chain length of 2 to 5.

The peptide composition can be made into a powder as it is as a solution or by a treatment such as freeze-drying or spray-drying. The peptide composition thus obtained can be used alone or as an amino acid-containing enteral nutritional supplement. In particular, by adding the composition of the present invention to the composition of a conventional enteral nutritional supplement in an amount of 1 to 5%, it is possible to suppress the degeneration of small intestinal mucosal cells. JP-B-51-26490 discloses a nutritional composition containing an amino acid mixture as a nitrogen source. It is possible to use the compositions of the invention as a source of glutamine in such compositions. In the formulation, all of the amount supplied as glutamine can be replaced with the peptide composition of the present invention, and in the enteral nutritional supplement containing protein as the nitrogen source, the present invention can be used in an amount corresponding to the glutamine-enhancing amount. Addition of the peptide composition of 1) can suppress the degeneration of the small intestinal mucosa.

The present invention will be described in more detail below with reference to examples and experimental examples.

Example 1 Production of Peptide Composition Containing High Level of Glutamine Preparation of Raw Material Protein Wheat gluten (manufactured by Nacalai Tesque) was defatted with ether, and after removing the ether, suspended in 1% acetic acid (200 ml). It was Homogenize vigorously with a mixer and
Centrifuged at 500 xg for 10 minutes. The protein concentration in the obtained supernatant was measured with a protein assay kit (manufactured by Bio-Rad), and 1% acetic acid was added so that the protein concentration would be 1% to prepare a substrate solution.

Selection of Enzyme Gluten becomes susceptible to enzymatic reactions because it is suspended under acidic or alkaline conditions. Since the pyrrole reaction (N-terminal cyclization reaction) occurs under alkaline conditions, we decided to use acidic protease as the enzyme that hydrolyzes proteins. Morcine (made by Sigma) as an acidic protease,
Pepsin (manufactured by Sigma) and Lapitase (manufactured by Takeda Chemical)
The molecular weight distribution of the hydrolyzate produced by each reaction for 24 hours at 37 ° C is shown by Biogel P-2 (manufactured by Bio-Rad).
It was measured by gel filtration. Sample 50 mg in 0.1M phosphate buffer
Dissolve in (pH7.0) and dissolve in Biogel P-2 column (1.6
X 87.5 cm). The same solution was used for elution, and the flow rate was 19
Set to ml / hour. The exclusion volume of the column was 48 ml. As the molecular weight markers, bacitracin (MW 1411), isoleucylglycine (MW 188), and isoleucine (MW 131) were used. As a result, it was found that the molsine hydrolyzate had the lowest molecular weight.

Enzyme treatment: The enzyme morphine and the substrate gluten were treated at an enzyme / substrate ratio of 1: 100,
Treatment was carried out at 37 ° C for 24 hours in a 1% acetic acid solvent. Since the molecular weight of most of the hydrolyzate treated with morphine was 1000 or more, after the lysis with morphine, the pH was adjusted to 7 and actinase E (Kaken Pharmaceutical Co., Ltd.) was allowed to act on it. The reaction conditions were the same except for pH. The resulting hydrolyzate is used as Biogel P-
2 Measured by gel filtration and shown in FIG. The numerical value in the upper part of FIG. 1 indicates the elution position of the molecular weight standard. A peak was shown at a molecular weight of around 188.

Actinase Treatment Conditions The amino acids released by the actinase treatment were measured over time and are shown in Table 1 below. Amino acids with a high free amount,
In order were Gln, Val, Phe, and Ile. However, when the ratio of the amino acids released from the original gluten was calculated for each amino acid, Glx 9.6%, Val 83.4%, Phe 67.4%
%, Ile 74.4%, and 67% or more of Val, Phe, and Ile were released, while it was estimated that most of Gln remained in the peptide form. Since the amino acids other than Gln were selectively released by the actinase in this way, the glutamine content in the peptide composition is considerably higher than that of the raw material gluten.

[0018]

[Table 1]

Removal of Free Amino Acids The method of Yamasita et al. For removing free amino acids from peptides (Reference: Yamasita et.al, J. Food. Sci., Vol 41, 102).
9, 1976) and Sephadex G-15 (Pharmacia) column (2.5 × 60 cm) was applied. Elution is 10%
Ethanol (pH 7) was used and the flow rate was set to 95 ml / hour. The exclusion volume of the column was 96 ml. The chromatogram is shown in FIG. This allowed the separation into 4 fractions. The results of analyzing the amino acid composition of each fraction as Fr. 1, 2, 3, 4 are shown in Table 2 below.

[0020]

[Table 2]

The Glx content was high in Fr.1 and Fr.3,
As a result of free amino acid analysis, it was found that most of Glx of Fr.3 was free amino acids. On the other hand, the ratio of free amino acids in Glx of Fr.1 was 3.5%, and most of them were in peptide form. The Glx content of Fr.1 was higher than that of the raw material gluten.

N-terminal Glutamine Analysis The N-terminal amino acid analysis of the peptides of the Fr.1 fraction was performed. Peptides were prepared by the method of Hartley et al. [Biochemical.
hys. Acta.), 21 , 58, (1956)] and then hydrolyzed at 110 ° C for 24 hours for amino acid analysis.
The Glx amount at this time, that is, Glx that is not dansylated is D
And Glx amount when dansylating the same amount of sample
Q. The amount of Glx existing at the N-terminal was calculated as Q-D. Glx existing at the N-terminus of Fr.1 is 17.6 of all Glx.
%Met. The proportion of Glx existing in positions other than the N-terminal, which is stable in aqueous solution, was 78.9% of all Glx (100−
3.5 -17.6). The ratio of Glx to Fr.1 is 50.33%
However, when converted to the ratio of Glx that is stable in aqueous solution,
It becomes 39.71%. Most gluten-based peptides are composed of glutamine.

Molecular weight distribution of peptide composition Fr.1 (50 mg) was dissolved in 0.1 M phosphate buffer (pH 7.0) and loaded on a Biogel P-2 column (1.6 × 87.5 cm). The same solution was used for elution, and the flow rate was set to 19 ml / hour. The exclusion volume of the column was 48 ml. Molecular weight markers include
Bacitracin (MW 1411), isoleucylglycine (MW 18
8) and isoleucine (MW 131) were used. The chromatogram is shown in Figure 3. The core of the peptide composition was the tetra and penta peptides. In addition, 88% was peptide or amino acid calculated from the absolute value of amino acid amount in 1 mg.
The yield was 35% of the raw material protein based on nitrogen.

[0024]

[Experimental Example] Effect of glutamine peptide administration on small intestine function In this Experimental Example, an example in which the effect of the peptide composition obtained in Example 1 on the small intestine function was tested is shown. Materials and methods Feed composition: β-corn starch (Oriental Yeast Industry), cellulose powder (Oriental Yeast Industry),
Soybean oil (made by Nacalai Tesque), Choline chloride (Kanto Chemical)
A sample was prepared by mixing casein (Kanto Kagaku), minerals and vitamins. Both the mineral mixture and the vitamin mixture were blended based on the Harper composition and purchased from Oriental Yeast Co., Ltd. Divide the rats into 5 groups,
Diets of different composition were meal-fed for 7 days.
The feed composition of each group is shown in Table 3.

[0025]

[Table 3]

Experimental animals: Wistar male rats with an initial weight of 150 g were purchased from Charles River and used in the experiment after preliminary breeding. The rats were fasted for 4 days to give low nutrition model rats, fed with the above-mentioned test diet for 7 days, and dissected 18 to 24 hours after the final administration. On the other hand, methotrexate (MTX, Wako Pure Chemical Industries, Ltd.) was used for the preparation of small intestinal disorder model rats. MTX was intraperitoneally administered at 20 mg / kg according to the method of Fox et al. [Surgical Forum 38 , 43, (1987)] on the 4th day from the start of administration of the test feed. After that, the test feed was continuously administered for 3 days, and the animals were dissected 18 to 24 hours after the last administration. All rats dissected at 12: 0 to avoid the effects of diurnal variation
I went between 0 and 15:00. Water was available ad libitum during all rearing tests. Three to four rats were used for each experiment.
There was almost no difference in the initial body weight of the rats between the experimental groups.

Small intestine: Rats were anesthetized with sodium pentobarbital (Dainippon Pharmaceutical Co., Ltd.), the abdomen was opened, and the aorta was cut to exsanguine the blood, and then the small intestine was extracted. After flushing the contents with ice-cold saline, the total small intestine weight was measured. Top 10
Except for cm, the next upper 10 cm was inverted using a glass rod, and the mucous membrane was peeled off with a slide glass. A 19-fold amount of ice-cold physiological saline was added to the exfoliated mucous membrane, homogenized with a homogenizer (Kinematica) for 30 seconds, and then centrifuged at 3000 xg for 10 minutes, and the supernatant was subjected to the assay described in the following section.

Quantification of protein in mucous membrane: It was measured using a protein assay kit (manufactured by Bio-Rad). Mucosal sucrase activity: 0.1 M maleic acid buffer (pH 6.0)
Prepare a 0.056 M substrate sucrose aqueous solution using 0.1 ml, add 0.1 ml of the test solution and 1 drop of toluene to 0.1 ml, and
C It was reacted for 30 minutes. After the reaction, 1 ml of ice-cooled distilled water
Was added and heated in a boiling water bath for 2 minutes. After cooling to room temperature, the amount of glucose produced was measured using a commercially available kit (glucose C
-II Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.). Enzyme activity is μmol sucrose hydrolyzed / 60min / mg protein
Expressed in units.

Mucosal aminopeptidase activity: 100 mM potassium phosphate buffer (pH 8.0) containing 1 mM dithiothreitol
In the presence of 0.5 mM Leu-NA for 5 minutes,
The reaction was stopped with 4 ml of ethanol containing 0.23 N HCl. Further, 0.4 ml of ethanol containing 0.06% p-dimethylcinnamaldehyde was added, and the mixture was allowed to stand for 30 minutes. Then, the amount of the produced Schiff base dye was measured at 540 nm to quantify the released β-naphthylamine. Enzyme activity is ΔA _540nm / min / mg pr
Expressed in otein units. Measured value processing: For measured values t
A test was performed.

Results The effects on the small intestine function of undernourished rats are shown in Table 4.

[Table 4]

In undernourished rats, a significant increase in body weight and small intestinal weight was observed only by addition of gluten or glutamine peptide. Furthermore, the amount of protein in the jejunal mucosa was significantly higher in the glutamine peptide-added group than in the control group, but the amino acid mixture of the same composition showed no significant difference from the control group. This is superior to the amino acid mixture in that the peptide of the present invention activates the small intestine function.

The effects on the small intestine function of MTX rats are shown in Table 5.

[Table 5]

It is known that the small intestinal villi damage reaches its peak 3 days after MTX administration, and 100% death occurs within 156 hours when 20 mg / kg is administered. In addition, Fox et al. Also reported that administration of free glutamine significantly suppressed the damage caused by MTX [Surgical Forum (Surgical Forum
Forum) 38 , 43, (1987)]. Intestinal weight was 57% (P <0.05) and jejunal protein content was 71% compared to normal rats after MTX administration.
(P <0.05), and sucrase activity and leucine aminopeptidase activity were significantly decreased by 0.01%. Approximately 25% of MTX rats had diarrhea, and dissection findings showed that, unlike unfed rats, a large amount of dietary digestive substances remained in the stomach and intestinal tract. From the above,
It was determined that the small bowel function was impaired by MTX.

When the glutamine peptide was administered to MTX rats, the sucrase activity was significantly increased by 1% and the leucine aminopeptidase activity was significantly increased by 5%. A similar effect was observed in the free glutamine-added group, but not in the amino acid mixture. This indicates that the peptide absorption system is more resistant to damage than the amino acid absorption system, at least in the MTX damage model. It was confirmed that the effect of glutamine on the small intestine was greater in the peptide form than in the free form.

[0035]

INDUSTRIAL APPLICABILITY By carrying out the present invention, a peptide composition containing glutamine in a high concentration and an enteral nutritional supplement containing the same are provided. The peptide composition provided by the present invention is stable in water because glutamine at the N-terminal of the peptide is not bound, and suppresses degeneration of the small intestinal mucosa that occurs due to undernutrition and the like. In addition, the glutamine absorbability of the composition incorporated as an enteral nutrient is improved.

[Brief description of the figure]

FIG. 1 Biogel P-2 of a solution obtained by further treating an enzymatic degradation product of gluten with an acidic protease with an actinase.
Figure 3 shows gel filtration chromatography. The absorbance at 260 nm was measured and used as an index of peptide concentration.

FIG. 2 shows chromatography of a solution obtained by removing free amino acids from the above solution with Sephadex G-15. The absorbance at 260 nm was measured and used as an index of peptide concentration.

FIG. 3 shows Biogel P-2 gel filtration chromatography of the peptide composition of the present invention (Fr.1 in FIG. 2). The absorbance at 260 nm was measured and used as an index of peptide concentration.

Claims (3)

(57) [Claims] 1. A gluten or zein sequentially hydrolyzed under acidic protease and neutral profile Teaze, the hydrolyzate has the following properties that can be obtained by performing a molecular weight fractions composed of pentapeptide dipeptide And a peptide composition containing a small amount of free amino acid in addition to the above. Average molecular weight about 200 Daltons (by gel filtration method) Molecular weight distribution, most of which is molecular weight 131-1411
It is distributed in the Dalton range and has a peak near a molecular weight of 200 Dalton (by gel filtration method). 40% or more of the amino acids after hydrolysis are quantified as glutamic acid. The amount of free amino acid is 10% or less, and the amount of free glutamic acid is 5% or less. It has a function to suppress degeneration of small intestinal mucosa. 2. Gluten or zein as an acidic protea
And hydrolyzate
The molecular weight fractionation is performed and the peptide fraction is collected.
The method for producing the peptide composition according to claim 1, wherein 3. A peptide composition according to claim 1 is blended as a glutamine supply source, which has a function of suppressing mucosal degeneration of the small intestine.
That enteral nutrients.