JPH06343422A - Oligopeptide mixture and its production - Google Patents

Abstract

PURPOSE:To obtain an oligopeptide mixture extremely excellent as a low- allergic food and an enteral nutrient preparation. CONSTITUTION:Whey protein is hydrolyzed with proteases derived from a bacteria of genus Bacillus and an actinomyces, optionally in combination with (chymo)tripsin. The objective oligopeptide mixture is obtained by removing the enzymes and insoluble hydrolysates from the hydrolysate mixture.

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 having excellent intestinal absorbability, good amino acid balance, and good taste, which can be used for foods with low allergenicity and enteral nutrition, and a method for producing the same. It is a thing.

[0002]

2. Description of the Related Art Atopic dispositions, which are often found in infants and children, can be traced to their predisposition due to ingestion of highly antigenic eggs, milk, soybean products, fish, etc. during pregnancy, especially during the pregnancy. It is said that. Therefore, it is desirable to refrain from ingesting these heterologous proteins having strong antigenicity during pregnancy, but on the contrary, since these are also typical protein sources, it is a problem in nutrition intake.

Also, for those who have already become allergic, limiting the intake of foods that are protein sources is a big problem in selecting foods.

On the other hand, with respect to enteral nutritional agents used for nutritional supplementation before and after surgery, those using undegraded protein are indigestible, and those using an amino acid mixture are diarrhea and abdomen. The occurrence of bloating symptoms is often a problem. Further, it has been revealed that the oligopeptide is superior to the amino acid mixture in terms of absorption rate from the intestinal tract.

Under the above circumstances, many methods have been developed for producing a milk protein originally having an excellent amino acid balance, particularly an oligopeptide mixture obtained by hydrolyzing whey protein with an enzyme. Some examples of them are as follows.

JP-A-2-138991 discloses that the peptide has a molecular weight of 1000 or less, does not exhibit antigenicity, and is enzymatically decomposed until 90% or more of free amino acids are contained in the starting protein, and then gel filtration method is used. Discloses a method for producing a low molecular weight peptide, which comprises 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 1/10000 or less of β-lactoglobulin. As described above, a method for producing a whey protein hydrolyzate, which comprises hydrolyzing with a thermostable enzyme at 60 to 80 ° C, is shown.

[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 protein, which comprises hydrolyzing with a mixed enzyme of endopeptidase, trypsin and / or chymotrypsin from which has been shown.

[0009]

However, in the invention of JP-A-2-138991 described above, the aromatic amino acid content is extremely low, and the excellent amino acid balance originally possessed by whey protein is significantly impaired. No mention is made of the flavor.

Further, in the invention of the above-mentioned JP-A-4-112753, a peptide having a large molecular weight distribution of 10,000 or less and a main peak of 1000 to 5000 is contained, and the invention of the above-mentioned JP-A-4-248959 is also included. Peptides having a large molecular weight remain so as to produce a large amount of precipitate when heated at pH 4.5 to 5.5, which are not satisfactory in terms of intestinal absorption.

As described above, the oligopeptide mixture obtained by enzymatically degrading whey protein satisfies all the conditions of excellent intestinal absorbability, good amino acid balance, and good flavor, and is hypoallergenic. What was available as a nitrogen source for foods and enteral nutrients has not been heretofore known.

The present invention has been made in view of the current state of the art.
It was made for the purpose of newly developing a novel oligopeptide mixture having excellent properties as described above.

[0013]

Means for Solving the Problems As a result of investigations from various aspects to achieve the above object, the present inventors have found that whey protein is produced by two kinds of enzymes, a protease derived from Bacillus and a protease derived from actinomycete. By hydrolyzing and removing the enzyme and the insoluble hydrolyzate, it was 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.

Further, a Bacillus-derived protease,
The inventors have found that an oligopeptide mixture can be similarly obtained by hydrolyzing in combination with trypsin and / or chymotrypsin in addition to the protease derived from actinomycetes, and completed the present invention.

The present invention will be described in detail below.

In order to carry out the present invention, the 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).

In WPC, whey produced during the production of cheese or casein is treated by a method such as ultrafiltration, gel filtration or whey crystal separation, and the protein content is usually 35 to 85%.
It is increased to (solid content conversion).

WPI is distinguished from WPC and has a protein content of 95% by a method such as an ion exchange method.
It is an undenatured whey protein that has been purified to a high purity (as calculated as solid content) or higher.

Further, within a range that does not significantly change the amino acid balance, β-lactoglobulin or α-lactoglobulin in whey protein can be prepared by a method such as an ion exchange method or a hydrophobic adsorption method.
It is also possible to use WPC from which a part of lactalbumin has been removed.

Such whey protein is hydrolyzed by an enzyme. The enzyme used in the present invention is a protease derived from the genus Bacillus, a protease derived from actinomycete and trypsin and chymotrypsin derived from animal organs. Is. These enzymes are
In each case, a commercial product can be used.

Examples of Bacillus-derived proteases include bioprase (Nagase Seikagaku Corp.), Alcalase (Novo), Protease N Amano (Amano Pharmaceutical Co., Ltd.), and actinomycete-derived proteases include: Examples include actinase (Kaken Pharma Co., Ltd.) and pronase (Sigma Co.).

In the present invention, the above whey protein is contained in a protein concentration of 1 to 20%, preferably 5 to 10%.
An aqueous solution is prepared so that the above condition is obtained, and enzymatic decomposition is performed after heat sterilization or without heat sterilization.

The enzymatic decomposition is carried out at 40 to 60 ° C. using an alkaline solution such as sodium hydroxide or potassium hydroxide or an acid solution such as hydrochloric acid or citric acid while adjusting the pH to 7-8 which is the optimum pH of the enzyme. Hold for 3 to 48 hours, preferably 6
~ 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 the enzyme and insoluble hydrolyzate are 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 molecular weight cutoff of 20000 or less without deactivating by heating after enzymatic decomposition.

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

The oligopeptide mixture thus obtained can also 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 is 1 of β-lactoglobulin. / 10,000 or less, and very little bitterness and umami.

The test examples and examples will be described below. The numerical values used therein are obtained by the following test method.

Test method:

(Average Chain Length) The average chain length of the peptides in the oligopeptide mixture was determined by the following formula. (OTA-FA) / (OFA-FA) where OTA: all amino groups in the oligopeptide mixture OFA: free amino group in the oligopeptide mixture FA: amino group corresponding to free amino acid

All amino groups of the oligopeptide mixture (OT
A) is, after hydrolysis of the sample under strong acidity or basicity,
It was calculated from the amino acid composition analyzed by an amino acid automatic 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 treatment with formic acid.
The values measured by the sample hydrolyzed with hydrochloric acid at 110 ° C. for 22 hours and the values of other amino acids measured by the sample hydrolyzed with 6N hydrochloric acid at 110 ° C. for 22 hours were used.

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

The amino group (FA) corresponding to the free amino acid is
The sample was diluted with 0.1 N hydrochloric acid and then calculated from the free amino acid composition analyzed by an amino acid automatic analyzer.

(Maximum Molecular Weight) The molecular weight of the maximum peptide in the oligopeptide mixture was determined by Asahi Pack 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 (however, if there is a shoulder, the first shoulder) and the calibration curve prepared separately.

(Antigenicity) The antigenicity of the oligopeptide mixture was measured by an ELISA inhibition test method using rabbit anti-β-lactoglobulin serum and alkaline phosphatase-labeled goat anti-rabbit IgG antibody. For β-lactoglobulin and the oligopeptide mixture, the maximum concentrations at which there was no inhibition were compared, and the ratios are shown.

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

Bitterness: 0 to 4 people / 16 people with bitterness ±: 5 to 11 people / 16 people +: 12 to 16 people / 16 people

Umami −: The number of people who have umami is 0 to 4 people / 16 people ±: The same number 5 to 11 people / 16: The same number 12 to 16 people / 16 people

[0041]

[Test Example] 55 g of WPI (protein content 92%) was dissolved in 900 g of water, and heat sterilized at 80 ° C. for 10 minutes. 5
After cooling to 0 ° C, the enzyme was added all at once, and the mixture was kept at 50 ° C for 24 hours while being adjusted to pH 7 for hydrolysis. Then 9
After heating at 0 ° C. for 10 minutes to inactivate the enzyme, centrifugation was performed to remove the enzyme and insoluble hydrolyzate. Average chain length, maximum molecular weight of samples prepared using various enzymes,
Antigenicity, bitterness and umami were evaluated. The test results are shown in Table 1,
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 is 1/1000 that of β-lactoglobulin in any of the test groups.
It was 0 or less, and the average chain length of the peptide was within the range of 3 to 5.

However, in test groups 1 to 3 in which no exopeptidase was used, the maximum molecular weight was 1800 or more, and there were a few people who felt bitterness.

In test groups 4 to 6 using an enzyme derived from the genus Aspergillus as an exopeptidase (for example, Protease A Amano, Denazyme AP), the maximum molecular weight is 1500 or less,
Few people had a bitter taste, but many had a lot of free amino acids and had a good taste.

On the other hand, in the 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 few people 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 / l of β-lactoglobulin.
In order to prepare an oligopeptide mixture that satisfies all the conditions of 10,000 or less and extremely little bitterness and umami, two kinds of enzymes, a protease derived from Bacillus and a protease derived from actinomycete, or a protease derived from Bacillus, It was necessary to hydrolyze with 3 or 4 enzymes consisting of actinomycete-derived proteases, trypsin and / or chymotrypsin.

[0049]

Example 1 WPC (protein content 78%) 770 g
Is dissolved in 9200 g of water and sterilized at 75 ° C for 10 minutes, then 40
Cooled to ° C. Alcalase (Novo) 2500 units /
g protein and actinase (Kaken Pharma Co., Ltd.) 5
000 units / g protein was added, and hydrolysis was carried out 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, centrifugation was performed to collect a supernatant, which was dried to obtain 720 g of an oligopeptide mixture powder.

The average peptide chain length of the peptide was 4.2,
The maximum molecular weight was 1200 and the antigenicity was 1/10000 or less 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. Biopulase (Nagase Seikagaku Corp.) 2200 units / g protein, trypsin (Novo Corp.) 1300 units / g protein, chymotrypsin (Novo Corp.) 90 units / g protein and actinase (Kaken Pharma Corp.) 1100 units / g protein Add and adjust pH to 7 with 10% sodium hydroxide solution.
While adjusting to 5, hydrolysis was carried out at 50 ° C. for 20 hours. This was subjected to ultrafiltration with an ultrafiltration membrane having a molecular weight cut off of 20,000 to remove the enzyme and the insoluble hydrolyzate. Further, it was desalted using an electrodialyzer until the electric conductivity became 1/10 and then dried to obtain 940 g of an oligopeptide mixture powder.

A 2% solution of this powder showed no sediment at all even when heated at pH 4 to 5, and had little bitterness or umami. The average chain length of the peptides 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 whey proteins except a part of α-lactalbumin. This was eluted with sodium hydroxide solution at pH 9, concentrated with an ultrafiltration membrane having a molecular weight cut off of 20,000, and then spray-dried to give a protein content of 88%.
Got the WPC. Β-lactoglobulin in the protein of this WPC was 80%, and α-lactalbumin was 6%.

570 g of this WPC was dissolved in 9400 g of water, and biopulase (Nagase Seikagaku Co., Ltd.) 2000 units / g protein, trypsin (Novo Corp.) 2500 units /
g-protein was added and hydrolysis was carried out at 50 ° C. for 8 hours while adjusting the pH to 8 with a 10% sodium hydroxide solution.
Next, actinase (Kaken Pharma Co.) 1250 units /
Add g protein and adjust pH to 7 at 50 ℃
For 16 hours. After heating at 120 ° C. for 15 seconds to inactivate the enzyme, centrifugation was performed to collect the supernatant, which was further filtered through a microfiltration membrane having a pore size of 0.45μ and dried to obtain 530 g of an oligopeptide mixture powder. It was

A 2% solution of this powder showed no precipitate even when heated at pH 4 to 5, and had almost no bitterness or umami. The average chain length of the peptides 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 purposes.

(1) A nutritional supplement and enteral nutritional supplement for infants, elderly people and sick people before and after surgery having low digestive and absorption ability. (2) A nutritional supplement and a hypoallergenic food for infants and children with allergic diseases. (3) A nutritional supplement for pregnant women who are diet-restricted in order to suppress the transfer of allergic constitution to the fetus during the pregnancy period, especially in the latter period.

Front Page Continuation (72) Inventor Tetsuo Kaneko 1-21-3 Sakaemachi, Higashimurayama, Tokyo Meiji Dairy Co., Ltd. Central Research Institute (72) Tetsuhiko Maruyama 1-21-3 Sakaemachi, Higashimurayama, Tokyo Meiji Dairy Co., Ltd. In the laboratory

Claims (4)

[Claims] 1. A mixture of oligopeptides characterized in that after whey protein is hydrolyzed by two kinds of enzymes, a protease derived from Bacillus and a protease derived from actinomycete, the enzyme and insoluble hydrolyzate are removed. Production method. 2. The production method according to claim 1, further comprising hydrolyzing with trypsin and / or chymotrypsin. 3. An oligopeptide mixture produced by the method according to claim 1 or 2. 4. A 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 taste are extremely small.