JP3222638B2 - Oligopeptide mixture and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel oligopeptide mixture which can be used as a hypoallergenic food and enteral nutrition, has excellent intestinal absorption, has a good amino acid balance and a good taste, and a process for producing the same. Things.

[0002]

2. Description of the Related Art Atopic constitutions, which are common in infants and children, can be traced to the predisposition of pregnant women during pregnancy, especially in the late pregnancy due to consumption of highly antigenic eggs, milk, soy products, fish, etc. It is said that. Therefore, it is desirable to refrain from ingesting these heterologous proteins having strong antigenicity during pregnancy. However, since these are also typical protein sources, it is a difficult problem in nutritional intake.

[0003] In addition, even for those who are already allergic, restricting the intake of foods that are protein sources is a major problem in food selection.

On the other hand, among enteral nutritional supplements used for nutritional supplementation before and after surgery, those using undegraded proteins are indigestion, while those using an amino acid mixture are diarrhea and abdomen. The occurrence of bloating symptoms is often a problem. In addition, it has been clarified that the oligopeptide is superior to the amino acid mixture in the absorption rate from the intestinal tract.

[0005] Under the circumstances described above, a number of methods have been developed for producing milk proteins having an originally excellent amino acid balance, in particular, oligopeptide mixtures obtained by hydrolyzing whey proteins with enzymes. Some examples are as follows.

Japanese Patent Application Laid-Open No. 2-138991 discloses that a peptide having a molecular weight of 1,000 or less, not exhibiting antigenicity, and enzymatic decomposition of aromatic amino acids in a raw material protein to 90% or more of free amino acids, followed by gel filtration. A method for producing a low-molecular peptide comprising removing free aromatic amino acids to reduce the free amino acid content to 20% or less.

JP-A-4-112753 discloses that the peptide has a molecular weight of 10,000 or less, an average chain length of 3 to 8, a free amino acid content of 20% or less, and an antigenicity of 1 / 10,000 or less of β-lactoglobulin. Thus, a method for producing a whey protein hydrolyzate comprising hydrolyzing at 60 to 80 ° C. with a heat-resistant enzyme is disclosed.

[0008] Japanese Patent Laid-Open No. 4-248959, the following molecular weight peptides 2000, residual antigenic activity is ^10-4 or less, as free amino acid content of 5% or less Bacillus subtilis (Bacillus subtilis)
A method for producing a mixture of oligopeptides derived from whey proteins, comprising hydrolyzing with a mixed enzyme of endopeptidase, trypsin and / or chymotrypsin from the same is shown.

[0009]

However, in the invention of Japanese Patent Application Laid-Open No. 2-138991, the aromatic amino acid content is extremely low, and the excellent amino acid balance inherent in whey protein is significantly impaired. No mention is made of the flavor.

In the invention of JP-A-4-112753, a peptide having a molecular weight distribution of 10,000 or less and a main peak of as large as 1,000 to 5,000 is contained, and the invention of JP-A-4-248959 is also disclosed. When heated at a pH of 4.5 to 5.5, a peptide having a molecular weight large enough to produce a large amount of precipitates remains, and each is not satisfactory from the viewpoint of intestinal absorption.

As described above, the oligopeptide mixture obtained by enzymatically degrading whey protein satisfies all the conditions of excellent intestinal absorption, good amino acid balance and good flavor, There have been no known sources of nitrogen that can be used in foods and enteral nutrition.

The present invention has been made in view of such a state of the art,
This is for the purpose of newly developing a novel oligopeptide mixture having the excellent properties as described above.

[0013]

Means for Solving the Problems The present inventors have conducted various studies to achieve the above object, and as a result, have found that whey protein is converted into two kinds of enzymes, a Bacillus-derived protease and an actinomycete-derived protease. By hydrolyzing and removing the insoluble hydrolyzate from the enzyme, it has been found that an oligopeptide mixture having excellent intestinal absorbability and good flavor can be obtained while maintaining the original amino acid balance of whey protein.

A protease derived from the genus Bacillus;
The present inventors have found that an oligopeptide mixture can be similarly obtained by hydrolysis in combination with trypsin and / or chymotrypsin in addition to actinomycete-derived protease, thus completing the present invention.

Hereinafter, the present invention will be described in detail.

In order to carry out the present invention, whey protein must be treated with a specific enzyme. The whey protein used in the present invention includes whey protein concentrate (Whey Protein Concentr).
ate, WPC) and whey protein isolate (Whey
Protein Isolate (WPI).

WPC is obtained by treating whey produced during the production of cheese or casein by ultrafiltration, gel filtration, whey crystal separation, etc., so that the protein content is usually 35 to 85%.
(Solid content conversion).

WPI is distinguished from WPC and has a protein content of 95% by an ion exchange method or the like.
It is a non-denatured whey protein purified to a high purity (in terms of solid content) or higher.

In addition, β-lactoglobulin and α-lactoglobulin in whey protein are determined by methods such as ion exchange and hydrophobic adsorption within a range that does not significantly change the amino acid balance.
WPC from which a part of lactalbumin has been removed can also be used.

Such whey proteins are hydrolyzed by an enzyme. The enzymes used in the present invention include proteases derived from the genus Bacillus, proteases derived from actinomycetes, and trypsin and chymotrypsin derived from animal organs. It is. These enzymes are
In each case, commercially available products can be used.

For example, Bacillus-derived proteases include bioprase (Nagase Seikagaku), Alcalase (Novo), and protease N Amano (Amano Pharmaceutical). Actinomyces-derived proteases include: Actinase (Kaken Pharma), Pronase (Sigma) and the like.

In the present invention, the above-mentioned whey protein is contained in a protein concentration of 1 to 20%, preferably 5 to 10%.
An aqueous solution is prepared and subjected to enzymatic degradation after heat sterilization or without heat sterilization.

The enzymatic decomposition is carried out at 40 to 60 ° C. while adjusting the pH of the enzyme to 7 to 8 using an alkaline solution such as sodium hydroxide or potassium hydroxide or an acid solution such as hydrochloric acid or citric acid. Hold for 3 to 48 hours, preferably 6
Perform for ~ 24 hours.

The enzymes to be used may be added all at once or separately. At this time, the order of addition is not limited.

After the enzymatic decomposition, the enzyme is inactivated by heating, and a hydrolyzate insoluble in the enzyme is removed by a method such as centrifugation, microfiltration, or ultrafiltration. Alternatively, the enzyme and the insoluble hydrolyzate may be removed by a method such as performing ultrafiltration using an ultrafiltration membrane having a cut-off molecular weight of 20,000 or less without heat deactivation after enzymatic decomposition.

The oligopeptide mixture thus obtained usually contains an ash content of 5 to 20% (in terms of solid content), but if necessary, may be subjected to a desalting treatment by a method such as electrodialysis. It is possible.

The oligopeptide mixture thus obtained can be frozen, concentrated and dried by a known method.

The desired oligopeptide mixture is obtained as described above. For example, when measured by the test method described below, the average chain length is 3 to 5, the maximum molecular weight is 1500 or less, and the antigenicity of β-lactoglobulin is one. / 10000 or less, and very little bitterness or umami.

Hereinafter, Test Examples and Examples will be described. Numerical values used therein are obtained by the following test methods.

Test method:

(Average chain length) The average chain length of the peptides in the oligopeptide mixture was determined by the following equation. (OTA-FA) / (OFA-FA) where OTA: all amino groups in the oligopeptide mixture OFA: free amino groups in the oligopeptide mixture FA: amino groups equivalent to free amino acids

All amino groups (OT) of the oligopeptide mixture
A) hydrolyzes the sample under strong acidity or strong basicity,
It was calculated from the amino acid composition analyzed by the automatic amino acid analyzer.

At this time, tryptophan was hydrolyzed with 8N sodium hydroxide at 110 ° C. for 22 hours, and cysteine and methionine were treated with 6N after performing formic acid treatment.
For other amino acids, values measured using a sample hydrolyzed with hydrochloric acid at 110 ° C. for 22 hours and values of other amino acids measured using a sample hydrolyzed with 6N hydrochloric acid at 110 ° C. for 22 hours were used.

The free amino groups (O
FA) is TNBS (trinitrobenzenesulfonic acid)
It was measured by the method.

The amino group (FA) corresponding to a free amino acid is
After diluting the sample with 0.1N hydrochloric acid, it was calculated from the free amino acid composition analyzed by an automatic amino acid analyzer.

(Maximum molecular weight) The molecular weight of the maximum peptide in the oligopeptide mixture was determined by Asahi Pak GS-320.
(Diameter 7.6 mm, length 500 mm) was measured by high performance liquid chromatography. The maximum molecular weight was determined from the elution time of the first peak on the chromatogram (but the first shoulder if there is a shoulder) and a calibration curve prepared separately.

(Antigenicity) The antigenicity of the oligopeptide mixture was measured by an ELISA inhibition test using rabbit anti-β-lactoglobulin serum and alkaline phosphatase-labeled goat anti-rabbit IgG antibody. For the β-lactoglobulin and the oligopeptide mixture, the maximum concentration without any inhibition was compared, and the ratio was shown.

(Bitterness and Umami) The bitterness and umami of the oligopeptide mixture were evaluated organoleptically by a skilled panel of 16 people in their 20s and 30s using a solution in which the protein concentration was adjusted to 2%. The evaluation results are shown below.

Bitterness-: Number of people who have bitterness is 0 to 4/16 people ±: 5 to 11/16 people +: 12 to 16/16 people

Umami-: Number of people who have umami is 0 to 4 people / 16 people ±: 5 to 11 people / 16 people +: 12 to 16 people people

[0041]

[Test Example] 55 g of WPI (protein content 92%) was dissolved in 900 g of water and sterilized by heating at 80 ° C. for 10 minutes. 5
After cooling to 0 ° C., the enzyme was added at one time, and the mixture was hydrolyzed at 50 ° C. for 24 hours while adjusting the pH to 7. Then 9
After heating at 0 ° C. for 10 minutes to inactivate the enzyme, the mixture was centrifuged to remove the insoluble hydrolyzate from the enzyme. For samples prepared using various enzymes, average chain length, maximum molecular weight,
Antigenicity, bitterness and umami were evaluated. Table 1 shows the test results.
The results are shown in Table 1 shown in Table 2.

[0042]

[Table 1]

[0043]

[Table 2]

As is clear from the above results, the antigenicity was 1/1000 that of β-lactoglobulin in any of the test groups.
0 or less, and the average peptide chain length was in the range of 3-5.

However, in Test Groups 1 to 3 in which exopeptidase was not used, the maximum molecular weight was 1800 or more, and slightly more people felt bitterness.

Further, in Test Groups 4 to 6 in which enzymes derived from the genus Aspergillus (eg, protease A amano, denazyme AP) were used as exopeptidases, the maximum molecular weight was 1500 or less;
Few people felt bitter, but many free amino acids and many felt umami.

On the other hand, in Test Groups 7 to 9, which are combinations of the enzymes described in the present invention, the maximum molecular weight was 1500 or less, and almost no one felt bitterness or umami.

As described above, the average chain length is 3 to 5, the maximum molecular weight is 1500 or less, and the antigenicity is 1 / th of that of β-lactoglobulin.
In order to prepare an oligopeptide mixture that satisfies all the conditions of 10,000 or less and extremely low bitterness and umami, two kinds of enzymes, a Bacillus-derived protease and an actinomycete-derived protease, or a Bacillus-derived protease, It was necessary to hydrolyze with three or four enzymes consisting of actinomycete-derived protease, trypsin and / or chymotrypsin.

[0049]

Example 1 770 g of WPC (78% protein content)
Was dissolved in 9200 g of water, and sterilized at 75 ° C. for 10 minutes.
Cooled to ° C. Alcalase (Novo) 2500 units /
g protein and actinase (Kaken Pharma Inc.) 5
000 units / g protein was added and hydrolyzed at 40 ° C. for 12 hours while adjusting the pH to 7 with a 10% sodium hydroxide solution. After heating at 90 ° C. for 10 minutes to inactivate the enzyme, the supernatant was collected by centrifugation and dried to obtain 720 g of an oligopeptide mixture powder.

The average chain length of the peptide in this powder was 4.2,
The maximum molecular weight was 1200, the antigenicity was 1 / 10,000 or less of that of β-lactoglobulin, and there was almost no bitterness or umami.

[0051]

Example 2 WPI (protein content 92%) 1000
g was dissolved in 8800 g of water. To this, 2200 units / g protein of bioprase (Nagase Seikagaku), 1300 units / g of trypsin (Novo), 90 units / g of chymotrypsin (Novo) and 1100 units / g of actinase (Kaken Pharma) were added. 6. Add and adjust the pH to 7 with 10% sodium hydroxide solution.
The mixture was hydrolyzed at 50 ° C. for 20 hours while adjusting to 5. This was subjected to ultrafiltration with an ultrafiltration membrane having a molecular weight cut-off of 20,000 to remove the enzyme and insoluble hydrolyzate. Further, the mixture was desalted using an electrodialyzer until the electric conductivity became 1/10, and then dried to obtain 940 g of an oligopeptide mixture powder.

The 2% solution of this powder did not show any sediment even when heated at pH 4-5, and had little bitterness or umami. The average chain length of the peptide is 3.
8. The maximum molecular weight was 1000, and the antigenicity was 1/10000 or less of β-lactoglobulin.

[0053]

Example 3 Cheese whey having a pH of 4.6 was passed through a cation exchange resin Indion S3 (Life Technology Co., Ltd.) to adsorb most of whey proteins except a part of α-lactalbumin. This was eluted with a sodium hydroxide solution at pH 9, concentrated with an ultrafiltration membrane having a molecular weight cut off of 20,000, and spray-dried to give a protein content of 88%.
Was obtained. Β-lactoglobulin was 80% and α-lactalbumin was 6% in the protein of this WPC.

570 g of this WPC was dissolved in 9400 g of water, and 2,000 units / g of bioprase (Nagase Seikagaku) and 2500 units / g of trypsin (Novo) were used.
g protein was added and hydrolyzed at 50 ° C. for 8 hours while adjusting the pH to 8 with a 10% sodium hydroxide solution.
Next, actinase (Kaken Pharma) 1250 units /
g protein and adjust the pH to 7 at 50 ° C
For 16 hours. After heating at 120 ° C. for 15 seconds to inactivate the enzyme, the supernatant was collected by centrifugation, and the residue was filtered through a microfiltration membrane having a pore size of 0.45 μm and dried to obtain 530 g of an oligopeptide mixture powder. Was.

The 2% solution of this powder did not show any sediment even when heated at pH 4-5, and had little bitterness or umami. The average chain length of the peptide is 3.
6. The maximum molecular weight was 1000, and the antigenicity was 1/10000 or less of β-lactoglobulin.

[0056]

Industrial Applicability According to the production method of the present invention, an oligopeptide mixture having excellent intestinal absorbability, good amino acid balance, no antigenicity and good flavor can be obtained from whey protein in good yield.

The oligopeptide mixture according to the present invention can be used for the following applications.

(1) Nutritional supplements and enteral nutritional supplements for infants, aged persons, and patients before and after surgery with low digestive absorption ability. (2) Nutritional supplements and hypoallergenic foods for allergic diseases from infants to children. (3) A nutritional supplement for pregnant women whose diet is restricted in order to suppress the transfer of allergic constitution to the fetus during the pregnancy, especially in the later period.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12P 21/06 A61K 37/18 (72) Inventor Tetsuo Kaneko 1-2-1-3, Sakaemachi, Higashimurayama-shi, Tokyo Meiji Dairies Corporation Central Research Center In-house (72) Inventor Tetsuhiko Maruyama 1-21-3 Sakaecho, Higashimurayama-shi, Tokyo Meiji Dairies Co., Ltd. Central Research Laboratory (56) References JP-A-4-248959 (JP, A) JP-A 5-5000 (JP) , A) (58) Field surveyed (Int. Cl. ⁷ , DB name) A23L 1/305 A23J 3/08 A23J 3/34 A61K 38/00 BIOSIS (DIALOG)

Claims (4)

(57) [Claims] 1. An oligopeptide mixture comprising hydrolyzing whey protein with two kinds of enzymes, a Bacillus-derived protease and an actinomycete-derived protease, and then removing an enzyme and an insoluble hydrolyzate. Production method. 2. The method according to claim 1, wherein the hydrolysis is carried out using trypsin and / or chymotrypsin. 3. An oligopeptide mixture produced by the method according to claim 1 or 2. 4. The mixture of oligopeptides having an average chain length of 3 to
5. The oligopeptide mixture according to claim 3, wherein the maximum molecular weight is 1500 or less, the antigenicity is 1/10000 or less of β-lactoglobulin, and the bitterness and umami are extremely small.