JP3383461B2 - Casein hydrolyzate 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 casein hydrolyzate having low antigenicity, good flavor, and excellent digestion and absorption properties, and a method for producing the same.
INDUSTRIAL APPLICABILITY The casein hydrolyzate of the present invention has a preventive effect against dietary allergic substances, has a good flavor and is excellent in digestion and absorption properties, and therefore can be used for a wide range of applications. As used herein, percentages are by weight unless otherwise noted.

[0002]

2. Description of the Related Art Recently, from the viewpoint of intestinal absorption, it has been revealed that low molecular weight peptides such as dipeptides and tripeptides are extremely effective as a protein nutrient source.
It has also been revealed that free amino acids increase the osmotic pressure in the intestinal tract, cause a burden on the transport system, and reduce nutritional efficiency (Food and nutrition, Vol. 31, 247).
P., 1978). Therefore, recently, in view of digestion and absorption and nutritional physiology, a peptide composition having a low free amino acid content and a defined molecular weight distribution has been demanded.

On the other hand, proteins such as milk proteins are heterologous proteins for human beings, and when they are absorbed in the body while still having antigenicity under incomplete digestion, they show allergic symptoms and, in some cases, life. Even the danger of. Therefore, food allergic patients are significantly restricted in the foods they consume, and in particular, insufficient intake of protein, which is an essential nutritional source, poses a problem. Therefore, one solution is
BACKGROUND ART Proteins in foods are hydrolyzed by enzymes until they show substantially no antigenicity. In addition, recently, there is an idea that foods that have been hydrolyzed with enzymes to reduce their antigenicity can be used to prevent allergens by ingesting them at a stage where they do not exhibit allergic symptoms. The demand for low-molecular-weight peptide compositions with reduced gas is increasing further.

However, when a milk protein such as casein or whey protein is hydrolyzed with an enzyme for the purpose of obtaining a peptide composition having the above-mentioned advantages, a bitter peptide is generated, which is a major limitation when used in foods. For example, a method for producing an allergen-free liquid dairy product and the like is disclosed as a product obtained by decomposing milk protein with an enzyme (Japanese Patent Publication No. Sho 4).
5-319112), the milk protein hydrolyzate obtained by this method has a strong bitterness.

Therefore, conventionally, various methods have been developed for the purpose of minimizing the production of bitter peptides in order to utilize the enzymatic decomposition product of milk protein as a food material, but some of them have been developed. An example is as follows. (1) It has been disclosed that the production of bitter peptides is reduced as much as possible by using an exopeptidase that decomposes bitter peptides [Journal of Agricultural and Food Chemistry]. (Jour
nalof Agricultural Food Chememistry), Volume 31,
Page 50, 1983 and Journal of the Hours
De Science (Journal of Food Science), 54th
Vol. 1225, 1989].

The following is an example of a conventional method relating to a casein hydrolyzate which is relatively prone to bitterness among milk proteins. (2) A method for producing a protein hydrolyzate having no bitterness and no antigenicity using casein as a raw material is disclosed (Japanese Patent Publication No. 54-362).
No. 35). (3) A low allergenic casein peptide composition having little bitterness and unpleasant taste, a method for producing the same, and the like are disclosed (JP-A-6-113893).

[0007] Furthermore, methods for improving the overall flavor of the enzyme-decomposed solution which is not limited to bitterness have also been developed, and the examples thereof are as follows. (4) It is disclosed that the flavor peculiar to casein is improved by mildly degrading casein with an enzyme derived from lactic acid bacteria (Japanese Patent Application No. 6-122958). (5) It has been disclosed that the taste of the enzymatic degradation solution is improved by treating the enzymatic degradation product with a low blocking rate reverse osmosis membrane (Journal of Japan Food Industry Society, vol. 39, page 763, 1992). ).

However, in the above (1), as a result of the action of exopeptidase, various free amino acids are produced in a large amount, and the taste of the enzyme-decomposed product deteriorates due to its taste.

The bitterness of the protein hydrolyzate largely depends on the distribution of hydrophobic amino acids in the peptide, and casein having a high degree of hydrophobicity is likely to produce a bitter peptide, and whey
It was difficult to improve the flavor compared to the protein hydrolyzate. Therefore, conventionally, in order to reduce the bitterness and the antigenicity as well, the flavor is sacrificed to some extent as described in (2) above, and a high decomposition rate (as a free amino acid content of 40% is obtained).
%) Has often been done. However, the increase in free amino acids makes the taste and odor unpleasant due to its taste, reduces taste, and affects digestive absorption in the intestinal tract, similar to the problem (1). There was a possibility that it was not preferable.

Further, in the above (3), a "bitterness value" is introduced, and a combination of protease and peptidase is screened using this as an index to obtain a natural allergenic α.
It is disclosed that a hypoallergenic casein peptide free of bitterness or unpleasant taste reduced to 10,000 times or less of _s -casein was obtained, and the free amino acid content thereof was 30%.
The value was as high as 55%, and had the same problems as in (1) and (2) above.

As is apparent from these prior arts, when the bitterness of the casein hydrolyzate is intended to be reduced, conventionally, the free amino acid is inevitably increased, which results in deterioration of flavor and deterioration of palatability. Not only that, it also had an effect on the digestive absorbability in the intestinal tract.

On the other hand, for the purpose of improving the overall flavor, the casein hydrolyzate in which the release of amino acids is suppressed by mildly degrading casein with an enzyme derived from lactic acid bacteria as described in (4) above Although satisfactory, it had a serious drawback of remaining antigenicity due to its low degradation rate.

The treatment (5) is effective for removing free amino acids, but is not effective for removing bitter peptides, and it is difficult to improve the overall flavor only by this treatment. there were. It is possible to improve the overall flavor by performing the treatment (5) on the proteolytic products (1) to (3), but in this case the process becomes complicated. In addition, there is a problem that the yield of the target protein degradation product per raw material protein is reduced due to the removal of a large amount of free amino acids. As mentioned above, milk protein,
In particular, it has been extremely difficult to reduce the antigenicity of the enzymatic hydrolyzate of casein and to improve the flavor and the digestive absorbability together, despite various studies.

[0014]

DISCLOSURE OF THE INVENTION The above-mentioned prior art only discloses a protein hydrolyzate that leaves a problem in bitterness and flavor without antigenicity, or a casein hydrolyzate that retains a good flavor and antigenicity. However, conventionally, no casein hydrolyzate having low antigenicity and improved taste and digestive absorbability has been known.

Further, conventionally, the ratio of the fraction having a molecular weight of 1,000 daltons or less is 80% or more, and the amino acid liberation rate is 1.
A low molecular weight peptide consisting of 2-3 amino acid chains with a molecular weight of 500 or less is less than 0% as a main component (50% or more)
No casein hydrolyzate having low antigenicity, good flavor, and excellent digestion and absorption properties is known.

Further, in the past, in a simple process, the yield was good, and the ratio of the fraction having a molecular weight of 1000 daltons or less with a sufficient decomposition rate was 80% or more, whereby low antigenicity and reduction of bitterness were obtained. It has a good taste by suppressing the release of amino acids, and has excellent digestion and absorbability by using a low molecular weight peptide consisting of 2-3 amino acid chains with a molecular weight of 500 or less and having an amino acid release rate of less than 10% as a main component. There is no known method for producing a casein hydrolyzate having the excellent properties of Therefore, using casein having an excellent nutritional value as a raw material, in various foods, with low antigenicity that can be widely applied, a casein hydrolyzate having improved taste and digestive absorbability is also desired. It was

In view of the above-mentioned prior art, the inventors of the present invention have conducted intensive studies for the purpose of developing a new product that can solve the above-mentioned various problems of conventional products, and as a result, identified casein with an enzyme. By decomposing it into a casein hydrolyzate having the physicochemical and biological properties of, it was not possible with the conventional casein hydrolyzate,
It was found that a casein hydrolyzate having low antigenicity, a good flavor, and excellent digestive and absorption properties can be obtained, and a method for stably producing the casein hydrolyzate. The present invention has been completed.

An object of the present invention is to provide casein which has low molecular weight, low antigenicity, low amino acid release rate, good flavor, low molecular weight, and excellent digestion and absorption properties. It is to provide a hydrolyzate.

Another object of the present invention is to have a low molecular weight,
It is to provide a method for producing a casein hydrolyzate having low antigenicity, a low amino acid liberation rate, a good flavor, a low molecular weight, and excellent digestive and absorption properties. .

[0020]

The first invention of the present invention for solving the above-mentioned problems is the following physicochemical and biological properties of a) to f); a) Casein decomposition rate is 20 to 30%. B) isoleucine, tyrosine, phenylalanine, proline, leucine, free isoleucine in the total mass of specific amino acids of valine contained in the casein hydrolyzate,
Free tyrosine, free phenylalanine, free proline,
The total mass ratio of free specific amino acids of free leucine and free valine is 4 to 10%, c) has the following molecular weight distribution, more than 5,000 0 to 0.5% more than 1000 and less than 5,000 10 to 20% 500 Super 1000 or less 15-30% 500 or less 50% or more d) The ratio of the fraction having a molecular weight of 1000 daltons or less is 80
% Or more, e) The ratio of the amount of free amino acids to the amount of total amino acids of the casein hydrolyzate is less than 10%, and f) ELISA (Enzyme link).
The immunogenicity measured by the immuno-sorbent assay) is 10 times that of casein.
^{It is -4} or less, It is a casein hydrolyzate characterized by the above-mentioned.

The second invention of the present invention for solving the above-mentioned problems is to provide an aqueous casein solution with an alkaline endoprotease derived from a microorganism, a neutral endoprotease derived from a bacterium of the genus Bacillus and an endoprotea derived from an animal. And three types of proteases are added, and the rate of decomposition of casein by the enzymes and the L-amino acid sensor are measured 1.
The total measured value of the number of moles of 6 kinds of amino acids was monitored over time, and the decomposition rate of casein and the measured value by the L-amino acid sensor were 20 to 30% and 5 to 12 mM (m in terms of solid content of 10%, respectively). mol / l), the enzymatic reaction is stopped at the point of time when both are satisfied, and the insoluble matter is removed. A method for producing a casein hydrolyzate, comprising the steps of: It is also a desirable embodiment to carry out by ultrafiltration or diatomaceous earth and / or ultrafiltration.

Next, the present invention will be described in detail. In order to facilitate understanding of the present invention, first, the second invention of the present invention will be described. Casein used as a starting material in the method of the present invention is a commercial product or milk, lactate casein separated by a known method from skim milk, acid casein such as casein hydrochloride, sodium caseinate, potassium caseinate, calcium caseinate. Etc., or a mixture of these in any proportion. This raw casein is dispersed in water or warm water and dissolved. Although the concentration of the lysate is not particularly limited, it is usually 5 to 15 as the protein concentration.
From the viewpoint of efficiency and operability, it is desirable to set the concentration range to about% (weight, the same applies hereafter unless otherwise specified).

Next, the following proteolytic enzyme is added to the casein solution for hydrolysis. The proteolytic enzyme used in the method for producing a casein hydrolyzate of the present invention is a microorganism-derived alkaline endoprotease, Bacillus (Baci).
llus), three types of proteases, a neutral endoprotease derived from a bacterium and an endoprotease derived from an animal, are essential enzyme combinations.

Examples of the microbial-derived alkaline endoprotease used in the present invention include bioplase (manufactured by Nagase Seikagaku Corporation), Prorezer (manufactured by Amano Pharmaceutical Co., Ltd.), and Protea.
Ze S (manufactured by Amano Pharmaceutical Co., Ltd.), Sabinase (manufactured by Novo Nordisk Co., Ltd.), GODO B. A. P (manufactured by Godo Shusei Co., Ltd.) and the like can be exemplified. Bacillus used in the present invention (Ba
Examples of the neutral endoprotease derived from bacteria belonging to the genus Cillus) include Protease N (manufactured by Amano Pharmaceutical Co., Ltd.), GODO B.
N. Examples thereof include P (manufactured by Godo Shusei Co., Ltd.), nutrase (manufactured by Novo Nordisk), and alcalase (manufactured by Novo Nordisk). Examples of the endoprotease of animal origin used in the present invention include trypsin (manufactured by Novo Nordisk) and chymotrypsin (manufactured by Novo Nordisk). The three proteases may be added to the casein hydrolyzate at the same time or sequentially if the desired molecular weight distribution, antigenicity, amino acid composition and amino acid release rate can be achieved. In the present invention, the amino acid release rate refers to the ratio (percentage) of the total mass of free amino acids to the total mass of all amino acids in a casein hydrolyzate, a so-called mixture of peptides and free amino acids (dry product).

The amount of each protease to be used for casein varies depending on the substrate concentration, the enzyme titer, the reaction temperature and the reaction time, but in general, three kinds of protease are each used in an amount of 1000 per 1 g of protein in casein. Hydrolysis is carried out by adding at a ratio of 10000 active units. In particular, 600 to 2000 activity units of microbial-derived alkaline endoprotease per 1 g of protein in casein,
It is preferable to perform hydrolysis by adding in the range of neutral endoprotease 1500-3000 activity units derived from Bacillus genus bacteria and endoprotease 3500-8000 activity units derived from animals. Furthermore, the activity ratio of each enzyme was calculated by using the following formula: Microorganism-derived alkaline endoprotease: Bacillus genus neutral endoprotease: Animal-derived endoprotease =
It is most desirable to carry out hydrolysis by adding in a ratio of 2: 3: 6.

The pH of the casein hydrolysis reaction of the present invention is
It is desirable to adjust the pH within the range including the optimum pH of the enzyme used. Specifically, before adding the enzyme to the casein solution, the pH is adjusted within a range including the optimum pH of the enzyme to be used, and the pH is maintained within the range including the optimum pH of the enzyme to be used. It is desirable to carry out a decomposition reaction. Generally, the pH of the hydrolysis reaction decreases with the progress of the hydrolysis reaction after the addition of the enzyme. Therefore, it is desirable to adjust the pH before the addition of the enzyme to the alkaline side of pH 8 or more by adding an alkaline agent. In this case, sodium hydroxide, potassium hydroxide, potassium carbonate, calcium hydroxide, tribasic sodium phosphate, tribasic potassium phosphate, dibasic potassium phosphate and the like can be used alone or in combination as the alkaline agent.

The temperature of the casein hydrolysis reaction of the present invention is not particularly limited and is selected from the range that can be put to practical use, including the optimum temperature range in which the enzyme action is expressed. Usually 30 ~
It is selected from the range of 70 ° C. By maintaining the temperature in the range of 50 to 60 ° C., it is possible to prevent spoilage during the casein hydrolysis reaction. The time for the casein hydrolysis reaction of the present invention depends on the type and combination of enzymes used, reaction temperature, initial pH.
The progress is different depending on the reaction conditions such as, and if the reaction duration of the enzymatic reaction is constant, a decomposition product having different physicochemical and biological properties may be produced for each production batch, so it cannot be unconditionally determined. Therefore, it is necessary to monitor the enzymatic reaction and determine the reaction duration. Specifically, the degradation rate of casein in the enzymatic degradation solution and the total measured value of the number of moles of 16 kinds of amino acids measured by an L-amino acid sensor are monitored over time, and the degradation rate of casein and the L-amino acid are monitored. The measured value by the sensor is 20 to 3 respectively
The casein hydrolyzate having the physicochemical and biological properties of the present invention can be obtained by stopping the enzymatic reaction at the time when both the range of 5 to 12 mM in terms of 0% and solid content of 10% is satisfied. Here, the enzymatic reaction is stopped by heating (for example, at 85 ° C. for 15 minutes) to inactivate the enzyme.

Then, insoluble matter generated in the solution after the casein hydrolysis reaction is removed during the hydrolysis reaction and / or enzyme inactivation by heating is removed to obtain the desired low antigenicity and good flavor. In addition, a casein hydrolyzate having excellent digestion and absorption properties is obtained. The solution containing the casein hydrolyzate can be used as it is, or, if necessary, can be used as a concentrated solution obtained by concentrating the solution by a known method. It can also be dried by the method of 1) and used as a powder. Here, the insoluble matter can be removed by microfiltration and / or ultrafiltration, or diatomaceous earth and / or ultrafiltration.

Furthermore, as a preferred embodiment of the method for producing a casein hydrolyzate of the present invention, after the termination of the enzyme reaction,
Treatment with activated carbon, followed by separation, followed by removal of insoluble matter, or termination of the enzymatic reaction and removal of insoluble matter, followed by treatment with activated carbon, followed by separation thereof. Can do things

The casein hydrolyzate obtained as described above has the following physicochemical and biological properties a) to f) as will be apparent from the examples described later. a) The decomposition rate of casein is 20 to 30%. b) Free isoleucine in the total mass of the specific amino acids of isoleucine, tyrosine, phenylalanine, proline, leucine and valine contained in the casein hydrolyzate,
Free tyrosine, free phenylalanine, free proline,
The ratio of the total mass of free specific amino acids of free leucine and free valine is 4 to 10%. c) As shown in FIG. 1, it has the following molecular weight distribution (unit is Dalton). Greater than 5000 0 to 0.5% Greater than 1000 and less than 5000 10 to 20% Greater than 500 and less than 1000 15 to 30% Less than 500 50% or more d) As shown in FIG. 1, the ratio of the fraction having a molecular weight of 1000 daltons or less is 80%. That is all. FIG. 1 shows the molecular weight distribution of the casein hydrolyzate of the present invention obtained in Example 1, and the vertical axis and the horizontal axis show the distribution ratio and the molecular weight, respectively. e) The ratio of the amount of free amino acids to the amount of total amino acids of the casein hydrolyzate is less than 10%. f) As shown in FIG. 2, an ELISA (Enzyme linked i) using a casein hydrolyzate was used.
The antigenicity measured by the mmuno-sorbent assay) inhibition test method is 10-4 or less that of casein. FIG. 2 shows the antigen residual activity of the casein hydrolyzate of the present invention obtained in Example 1, and the vertical axis and the horizontal axis show the inhibition rate and the final sample concentration, respectively. ○ in the figure
And ● represent casein and casein hydrolyzate, respectively.

As shown in the above a) to f), the casein hydrolyzate of the present invention reduces peptides having a molecular weight of more than 1000 daltons with a sufficient decomposition rate, and the ratio of the fraction having a molecular weight of 1,000 daltons or less is 80%. By the above, it is low in antigenicity, bitterness is reduced, and release of amino acids is suppressed, resulting in good flavor and free amino acids of 1
By using as a main component a low molecular weight peptide consisting of 2-3 amino acid chains having a molecular weight of 500 or less and less than 0%,
It is a casein hydrolyzate that combines the excellent properties of excellent digestion and absorption from the intestinal tract. INDUSTRIAL APPLICABILITY The casein hydrolyzate of the present invention has a prophylactic effect against dietary allergic substances, has a good flavor, and is excellent in digestive absorbability.

Next, the present invention will be described in detail by showing test examples. In the present invention, the following test method was adopted. (1) Method for measuring amino acid composition For amino acids other than tryptophan, cysteine and methionine, the sample was hydrolyzed with 6N hydrochloric acid at 110 ° C. for 24 hours, and for tryptophan, alkali decomposed with barium hydroxide at 110 ° C. for 22 hours, For cysteine and methionine, treat with 1N of 6N hydrochloric acid after the treatment with excess acid.
It was hydrolyzed at 10 ° C. for 18 hours and analyzed by an amino acid analyzer (Hitachi: Model 835) to measure the mass of each amino acid.

(2) Method for measuring free amino acid composition The sample was deproteinized with sulfosalicylic acid and analyzed by an amino acid analyzer (Hitachi: Model 835) to measure the mass of each free amino acid.

(3) Amino acid release rate (ratio of the amount of free amino acid to the amount of total amino acid of casein hydrolyzate)
The total mass of all amino acids, which is the sum of the masses of the amino acids contained in the casein hydrolyzate obtained by the method for measuring the amino acid composition, was set to 100, and was obtained by the method for measuring the free amino acid composition. The percentage of the total mass of all free amino acids, which is the sum of the masses of the free amino acids contained in the casein hydrolyzate, was calculated.

(4) Specific amino acid release rate (free isoleucine, free tyrosine, free phenylalanine, free proline, free in the total mass of specific amino acids of isoleucine, tyrosine, phenylalanine, proline, leucine and valine contained in casein hydrolyzate Calculation method of the total mass ratio of free specific amino acids of leucine and free valine In the present invention, the specific amino acid means isoleucine, tyrosine, phenylalanine, proline, leucine and valine. The casein obtained by the method for measuring the free amino acid composition relative to the total mass of all the specific amino acids, which is the sum of the masses of the specific amino acids contained in the casein hydrolyzate obtained by the method for measuring the amino acid composition, was obtained. The percentage of the total mass of all free specific amino acids, which is the sum of the masses of the individual free specific amino acids contained in the hydrolyzate, was calculated.

(5) Decomposition rate calculation method Kjeldahl method (edited by Japan Food Industry Association, "Food Analysis Method",
The total nitrogen content of the sample was measured by the 102nd page, Korin Co., Ltd., 1984, and the formol titration method (Mitsuda et al.,
The amount of formol nitrogen in the sample was measured according to "Food Engineering Experiment Manual", Vol. 1, No. 547, Yokendo Co., Ltd., 1970), and the decomposition rate was calculated from these measured values by the following formula. . Decomposition rate (%) = (formal nitrogen content / total nitrogen content) x 100

(6) Method for measuring molecular weight distribution Measured by high performance liquid chromatography (edited by Nobuo Ui, "High Performance Liquid Chromatography of Proteins and Peptides", Kagaku Special Issue No. 102, No. 241 page, Co., Ltd. Chemistry coterie, 1894). Polyhydroxyethyl aspartamide column [Poly Hydroox
yetyl AspartamideColumn:
Manufactured by Poly LC. Diameter 4.6
mm and length 200 mm] and eluted with 20 mM sodium chloride, 50 mM formic acid at an elution rate of 0.4 ml / min. A UV detector (manufactured by Shimadzu Corporation) was used for detection, and a GPC analysis system (manufactured by Shimadzu Corporation) was used for data analysis.

(7) Method for measuring antigenicity ELISA (Enzyme linked)
The immuno-sorbent assay) inhibition test method [Japanese Journal of Pediatric Allergic Society, Vol. 1, No. 2, No. 36, 1987] was carried out as follows. A 96-well plate (manufactured by Nunc) was coated with casein, washed, and sensitized with casein to prepare a mixed solution of a rabbit anti-casein serum and a casein hydrolyzate sample, which was supplied to the plate hole. After reacting and washing, alkaline phosphatase-labeled goat anti-rabbit IgG antibody [Zymed.
Laboratory- (Zymed Laboratorie
[s) manufactured by the company], and after washing, an enzyme substrate, p-nitrophenyl sodium phosphate is added, and 30 minutes later, 5N sodium hydroxide is added to stop the reaction, and the reaction product is microprecoated. It was measured with a tri-der (manufactured by Wako Pure Chemical Industries, Ltd.). In addition, the suppression rate calculated by the following formula was used to express the degree of reaction suppression by the addition of the test antigen solution for suppression. Inhibition rate (%) = (1-absorbance of test antigen solution / absorbance of control) × 100 However, the absorbance of test antigen solution and the absorbance of control are the same amount of test sample solution or dilution to anti-casein serum, respectively. It is the value measured after the reaction of the hole containing the liquid mixture.

(8) Measurement value measured by L-amino acid sensor (1 measured by L-amino acid sensor
Measurement method of the total number of moles of 6 kinds of amino acids) As a L-amino acid sensor, a biotech analyzer (manufactured by Asahi Kasei Corporation) was used, and 16 kinds of amino acids (L
-Leucine, L-methionine, L-phenylalanine,
L-arginine, L-lysine, L-isoleucine, L-
The total number of moles of tryptophan, L-tyrosine, L-alanine, L-histidine, L-cysteine, L-cystine, L-asparagine, L-valine, DL-serine, DL-homoserine was measured, and 1 l of the sample was measured. The total number of moles of 16 kinds of amino acids contained in was calculated. If the concentration of the sample is not 10% solid content, 10% solid content
It was converted to the value in the case of.

Test Example 1 This test was conducted on the casein hydrolyzate of the present invention and the casein hydrolyzate prepared by the prior art, the decomposition rate, the ratio of the fractions having a molecular weight of 1000 daltons or less, the antigenicity, and the specific amino acid release. The ratio and flavor were compared and examined. 1) Preparation of samples Four types of samples (Sample Nos. 1 to 4) including the present invention were prepared based on the following description of the prior art. Sample 1: The casein hydrolyzate of the present invention obtained in Example 1 of the present application. Sample 2: In accordance with Example 1 described in Japanese Patent Publication No. 54-36235, casein was crushed with Lactobacillus helveticus (Hansen commercial strain) powder, pancreatin (Amano Pharmaceutical Co., Ltd.) and Amano A (Amano Pharmaceutical Co., Ltd.). ) The casein hydrolyzate obtained by disassembling in. Sample 3: A casein hydrolyzate obtained by decomposing casein with FC-H (Lactobacillus helveticus cell concentrated frozen solution) according to Example 1 described in the specification of Japanese Patent Application No. 6-122958. Sample 4: According to Example 1 described in JP-B-45-31912, casein was used as a raw material, and actinase AS (actinomycete Streptomyces griseus origin: Kaken Pharmaceutical Co., Ltd.) was used as a actinomycete proteolytic enzyme. A casein hydrolyzate obtained by using and decomposing.

2) Test method The decomposition rate of each sample, the ratio of the fractions having a molecular weight of 1000 daltons or less, the antigenicity, and the amino acid release rate were determined by the above-mentioned methods. The antigenicity of each casein hydrolyzate is expressed as a value (antigen residual activity) when the antigenicity of casein is 1. The flavor test was conducted by the following method. a) Flavor test A sensory test is conducted by a panel of 40 persons, 20 males and 20 females aged 20 to 40 years old, for each item of sourness, saltiness, bitterness, good aftertaste, and flavor (overall flavor). ,
For sourness, saltiness, and bitterness, there are 5 levels from strong (5 points) to weak (1 point), and for the aftertaste and flavor refreshing, there are 5 levels, from bad (5 points) to good (1 point). evaluated. For sourness, saltiness, and bitterness, the average value of the evaluation points is posted as it is, and for the aftertaste and flavor, from the average value of the evaluation points, 1.0 point or more and less than 1.4 points are “good”, 1.4 points. 2. From above to less than 2.2 points is "slightly good"; from 2.2 points to less than 3.8 points is "normal";
8 points or more and less than 4.6 points were judged as "slightly bad", and 4.6 points or more and 5.0 points or less were judged as "bad", and the judgment results were posted.

3) Test Results The results of this test are shown in Table 1. As is clear from Table 1, the decomposition rate is 20 to 30% and the molecular weight is 1000.
The ratio of fractions below Dalton is 80% or more, residual antigen activity is 10 ^-4 or less of casein, specific amino acid release rate is 4 to 10
%, The amino acid liberation rate was less than 10%, and the casein hydrolyzate having a good flavor was found to be the casein hydrolyzate of the present invention only.

[0043]

[Table 1]

Test Example 2 In this test, the ratio of the fraction having a molecular weight of 1000 daltons or less, the antigenicity, the amino acid release rate and the flavor were used as indicators to determine the proper decomposition rate of the casein hydrolyzate, the specific amino acid release rate and the corresponding rate. The L-amino acid sensor was performed to check the value. 1) Preparation of sample As shown in Table 2, hydrolysis reaction was performed with a decomposition rate of 18% and 20%.
%, 25%, 28%, 30%, 33%, 6 kinds of samples (Sample Nos. 5 to 10) were prepared by the same method as in Example 1 except that the enzyme was inactivated and stopped as appropriate. did.

2) Test Method The ratio, the antigenicity, and the amino acid release rate of the fractions having a molecular weight of 1000 daltons or less in each sample were determined by the method described above and the method described in Test Example 1. The flavor test was conducted by the following method. a) Flavor test A sensory test was conducted by a panel of 40 persons, 20 males and 20 females aged 20 to 40 years, and evaluated in 5 grades from very good flavor (+2 points) to very bad flavor (-2 points). However, from the average value of the evaluation points, less than +2.0 points to +1.8 points or more is “very good”, less than +1.8 points to +1.3 points or more is “good”, less than +1.3 points to +0. A score of 5 or more is “slightly good”, a score of less than +0.5 to −0.5 or more is “not good”, a score of less than −0.5 to −1.3 or more is “slightly bad”, −1 "Bad" from less than 3 points to -1.8 points or more,
And less than -1.8 points to -2.0 points or more were judged as "very bad", and the judgment result was posted.

3) Test results The results of this test are shown in Table 2. As is clear from Table 2, a casein hydrolyzate having a ratio of a fraction having a molecular weight of 1000 daltons or less of 80% or more, an antigen residual activity of 10 ^-4 or less of casein, an amino acid release rate of less than 10%, and a good flavor. Was found to have a decomposition rate of 20 to 30% and a specific amino acid release rate of 4 to 10%, and the L-amino acid sensor value at that time was 5 to 12 mM.
In addition, the type of raw material casein, the alkaline endoprotease derived from a microorganism, the neutral endoprotease derived from a bacterium of the genus Bacillus, and the type of an endoprotease derived from an animal were changed and tested. The result was obtained.

[0047]

[Table 2]

Test Example 3 In this test, the kind of enzyme suitable for the hydrolysis of casein is examined by using the decomposition rate, the ratio of fractions having a molecular weight of 1000 daltons or less, the antigenicity, the specific amino acid release rate and the flavor as indexes. I went for. 1) Preparation of samples Nine types of samples (sample numbers 11 to 19) were prepared by the same method as in Example 1 except that the type of enzyme used was changed as shown in Table 3. In addition, as the enzyme, bioprase sp-20 (manufactured by Nagase Seikagaku Corporation) and Prorezer (manufactured by Amano Pharmaceutical Co., Ltd.) as alkaline endoprotease derived from microorganisms, neutrality derived from bacteria belonging to the genus Bacillus As endoproteases, Protease N (manufactured by Amano Pharmaceutical Co., Ltd.) and Alcalase (manufactured by Novo Nordisk Corp.),
Trypsin (manufactured by Novo Nordisk) and chymotrypsin (manufactured by Novo Nordisk) were used as endoproteases of animal origin, and papain W-40 (manufactured by Amano Pharmaceutical Co., Ltd.) was used as a protease derived from plants.

2) Test method The decomposition rate of each sample, the ratio of the fractions having a molecular weight of 1000 daltons or less, the antigenicity, and the specific amino acid release rate were determined by the method described above and the method described in Test Example 1. . The flavor was tested by the method described in Test Example 2.

3) Test Results The results of this test are shown in Table 3. As is clear from Table 3, the decomposition rate is 20 to 30% and the molecular weight is 1000.
The proportion of fractions below Dalton is 80% or more, the residual antigen activity is 10 ^-4 or less of casein, and the specific amino acid release rate is 4 to 4.
Casein hydrolyzate having 10% and good taste was obtained from three kinds of microorganisms: alkaline endoprotease derived from microorganisms, neutral endoprotease derived from bacteria of the genus Bacillus, and endoprotease derived from animals. It was found to be hydrolyzed with a combination of proteolytic enzymes. The amino acid release rate was 10 for all samples.
% Or less. Further, the type of casein, the type and combination of enzymes were changed and tested, and almost the same results were obtained.

[0051]

[Table 3]

[0052]

EXAMPLES Next, the present invention will be described in more detail by showing examples, but the present invention is not limited to the following examples. Example 1 9 kg of water was added to 1 kg of commercially available casein (manufactured by New Zealand Daily Board), well dispersed, and a 10% aqueous sodium hydroxide solution was added to adjust the pH of the solution to 7.0.
The casein was completely dissolved to prepare an aqueous casein solution having a concentration of about 10%. The casein solution was sterilized by heating at 85 ° C. for 10 minutes, the temperature was adjusted to 50 ° C., sodium hydroxide was added to adjust the pH to 9.5, and then bioprase sp-20 (manufactured by Nagase Seikagaku Corporation). ) 1,800,000 activity units (1,200 activity units per 1 g of protein), Protease N (manufactured by Amano Pharmaceutical Co., Ltd.) 1,680,000 activity units (2,000 activity units per 1 g of protein), and PTN 6.0S (manufactured by Novo Nordisk) 5,880,000 activity units (Per 1g protein
7,000 active units) to start the hydrolysis reaction, and then the degradation rate of casein and L-amino sensor- [Biotech Analyzer- (Asahi Kasei Kogyo)]
The measured values of the total number of moles of each kind of amino acid were measured, and the decomposition rate of casein was 24.1% and L-
When the value measured by the amino sensor reached 6.0 mM, the enzyme was inactivated by heating at 80 ° C for 6 minutes to stop the enzyme reaction, and the mixture was cooled to 10 ° C. A standard supercell (manufactured by Tokyo Diatomaceous Co., Ltd.) as a filter aid for this hydrolyzed liquid.
Was added and suction filtered, and then the obtained filtrate was concentrated and spray-dried by a conventional method to obtain 0.96 kg of a spray-dried product.

A part of the results of the test of the obtained casein hydrolyzate by the above test method are shown in FIGS. 1 and 2. As a result, in the casein hydrolyzate, the fraction having a molecular weight of 500 daltons or less was 61.1%, the fraction having a molecular weight of more than 500 and 1000 daltons or less was 19.9%, and the fraction having a molecular weight of more than 1000 and 5000 daltons or less was 18.7%. The fraction exceeding 5000 daltons was 0.3%, the residual antigen activity was 10 ⁻⁵ , the amino acid liberation rate was 4.1%, and the specific amino acid liberation rate was 4.4%. The amino acid composition tested by the above test method (per 1 g of casein hydrolyzate) was as follows.

[0054]                           Total amino acid composition Free amino acid composition                           [Mg / g] [mg / g] L-aspartic acid 69.72 0.94 L-threonine 40.38 2.25 L-serine 50.88 3.43 L-glutamic acid 230.69 1.81 L-glycine 18.31 1.28 L-alanine 30.48 2.11 L-valine 55.77 1.49 L-cystine 2.76 N.V. D. L-methionine 24.62 3.64 L-isoleucine 45.00 1.56 L-leucine 71.94 7.79 L-tyrosine 46.71 3.39 L-phenylalanine 37.40 1.76 L-tryptophan 5.73 0.94 L-lysine 75.13 5.37 L-histidine 27.59 1.63 L-arginine 31.00 N.V. D. L-proline 110.08 N.V. D. L-Asparagine contained in L-aspartic acid 0.83 L-glutamine 0.81 contained in L-glutamic acid

Example 2 Commercially available casein (made by Merck) (1.5 kg) and water (8.5 k)
g was added and well dispersed, a 10% sodium hydroxide aqueous solution was added to adjust the pH of the solution to 7.0, and casein was completely dissolved to prepare a casein aqueous solution having a concentration of about 15%. The casein aqueous solution was sterilized by heating at 85 ° C. for 10 minutes, the temperature was adjusted to 50 ° C., sodium hydroxide was added to adjust the pH to 9.0, and then bioprase sp-20.
(Nagase Seikagaku Corporation) 1,512,000 activity units (protein 1
1,200 activity units per gram), Neutrase (Novo Nordisk) 6,300,000 activity units (5 per gram protein)
000 activity units) and chymotrypsin (Novo Nordisk) 10,080,000 activity units (8,000 per 1 g of protein)
(Activity unit) to start a hydrolysis reaction, and the number of moles of 16 kinds of amino acids measured with a decomposition rate of casein and an L-amino sensor- [Biotech Analyzer- (Asahi Kasei Co., Ltd.)] over time. When the decomposition rate of casein reached 25.5% and the value measured by the L-amino sensor reached 7.2 mM, the total measured value of 8
The enzyme was inactivated by heating at 5 ° C for 5 minutes, the enzyme reaction was stopped, and the mixture was cooled to 10 ° C. 60g of activated carbon in this hydrolysis liquid
Was added, and the mixture was allowed to stand for 20 hours after stirring, standard supercell (manufactured by Tokyo Diatomaceous Co., Ltd.) was added as a filter aid, suction filtration was carried out, and then the obtained filtrate was concentrated and spray dried by a conventional method. Then, 1.32 kg of a spray-dried product was obtained.

The obtained casein hydrolyzate was tested by the above-mentioned test method, and as a result, the fraction having a molecular weight of 500 daltons or less was 60.9%, the fraction having a molecular weight of more than 500 and 1,000 daltons was 22.9%, and the fraction having a molecular weight of more than 1,000 and 5,000 dalton was more than 1000. The following fractions are 16.2%, the fractions exceeding 5000 daltons are 0%,
The residual antigen activity was 10 ⁻⁵ , the amino acid liberation rate was 5.9%, and the specific amino acid liberation rate was 6.4%.

Example 3 1.4 kg of commercially available casein sodium (New Zealand Daily Board) was dissolved in 8.6 kg of water to prepare an aqueous casein solution having a concentration of about 14%. The casein solution was sterilized by heating at 85 ° C for 10 minutes, the temperature was adjusted to 50 ° C, and potassium carbonate and sodium hydroxide were added to adjust the pH.
Was adjusted to 9.2, and then Prorezer (manufactured by Amano Pharmaceutical Co., Ltd.) 2,
352,000 activity units (2,000 activity units per gram of protein),
Alcalase 2.4L (Novo Nordisk) 5,8
80,000 activity units (5,000 activity units per 1 g of protein) and chymotrypsin (Novo Nordisk) 8,232,0
The hydrolysis reaction was started by adding 00 activity units (7,000 activity units per 1 g of protein), and the degradation rate of casein and L-amino sensor- [Biotech Analyzer- (Asahi Kasei Kogyo Co., Ltd.)] The total measured number of moles of 16 kinds of amino acids to be measured was measured, and when the decomposition rate of casein reached 25.0% and the measured value by L-amino sensor reached 6.7 mM, the temperature was 85 ° C. The mixture was heated for 15 minutes to deactivate the enzyme, stop the enzymatic reaction, and cool to 10 ° C. This hydrolyzed solution is ultrafiltered with an ultrafiltration membrane (made by Asahi Kasei Co., Ltd.) having a molecular weight cut off of 6000, concentrated, spray-dried,
1.1 kg of a spray-dried product was obtained.

The obtained casein hydrolyzate was tested by the above-mentioned test method. As a result, the fraction having a molecular weight of 500 daltons or less was 61.1%, the fraction having a molecular weight of more than 500 and 1,000 daltons was 19.9%, and the fraction of more than 1000 and 5,000 daltons. The following fractions were 18.9% and the fractions exceeding 5000 daltons were 0.1%.
%, Residual antigen activity 10 ^-4.5 , amino acid release rate 5.1
%, And the specific amino acid release rate was 5.4%.

Example 4 Commercial casein sodium (Unile France)
1.2 kg was dissolved in 8.8 kg of water to prepare an aqueous casein solution having a concentration of about 12%. The casein solution was sterilized by heating at 85 ° C. for 10 minutes, the temperature was adjusted to 50 ° C., potassium hydroxide was added to adjust the pH to 9.8, and then Protea-
Ze S (manufactured by Amano Pharmaceutical Co., Ltd.) 4,032,000 activity units (1 g of protein
4,000 activity units per unit, Alcalase 2.4L (Novo.
5,040,000 activity units (manufactured by Nordisk) (5,000 activity units per 1 g of protein) and PTN 6.0S (manufactured by Novo Nordisk) 7,257,600 activity units (per 1 g of protein)
7,200 activity units) to start the hydrolysis reaction, and the molar ratio of 16 kinds of amino acids measured with a decomposition rate of casein and L-amino sensor- [Biotech Analyzer- (Asahi Kasei Corp.)] over time. The total number of measured values was measured, and the decomposition rate of casein was 25.0% and L-
Amino sensor-130 when the value reached 5.8 mM
The enzyme was inactivated by heating at 0 ° C for 2 seconds to stop the enzyme reaction and cooled to 10 ° C. This hydrolyzed liquid was fractionated to have a molecular weight of 3
Ultrafiltration membrane (manufactured by Asahi Kasei Corp.) of 000, concentrated and spray-dried to obtain 0.95 kg of a spray-dried product.

The obtained casein hydrolyzate was tested by the above-mentioned test method. As a result, the fraction having a molecular weight of 500 daltons or less was 55.2%, the fraction having a molecular weight of more than 500 and 1,000 daltons was 25.6%, and the fraction of more than 1000 and 5,000 daltons was more than 1000. 19.2% of the following fractions, 0% of the fractions exceeding 5000 daltons,
The residual antigen activity was 10 ⁻⁵ , the amino acid release rate was 4.5%, and the specific amino acid release rate was 4.7%.

[0061]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a novel casein hydrolyzate having low antigenicity, good flavor, and excellent digestion and absorption properties, and a method for producing the same. Therefore, the effect achieved by the present invention is
It is as follows. 1) Since the casein hydrolyzate of the present invention has low antigenicity, it can be used as a material for a protein source for infants, pregnant women, and sick people with reduced immune function for the purpose of preventing allergic patients and allergic diseases. 2) Since the casein hydrolyzate of the present invention has a good flavor, it can be used as a material for a protein source for foods such as general foods and nutritional foods and for medical use. 3) The casein hydrolyzate of the present invention has excellent digestion and absorption properties, and thus can be used as a material for a protein source for infants with immature digestion and absorption capabilities or elderly people and sick people with reduced digestion and absorption capabilities. . 4) By the method of the present invention, casein hydrolyzate having a wide range of uses can be produced.

[Brief description of drawings]

FIG. 1 shows the molecular weight distribution of the casein hydrolyzate of the present invention.

FIG. 2 shows the residual antigen activity of the casein hydrolyzate of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hitoshi Saito 5-83, Higashihara, Zama City, Kanagawa Prefecture Mori Ei Dairy Co., Ltd. Research Institute of Nutrition Science (72) Inventor, Yasushi Kawaguchi 5-1-1, Higashihara, Zama City, Kanagawa Prefecture 83 Mori Eidai Co., Ltd., Nutritional Science Research Institute (72) Inventor Hiroshi Ochi 5-1-1, Higashihara, Zama City, Kanagawa Prefecture Mori Eidai KK, Nutritional Science Research Institute (56) Reference JP-A-6-78685 ( JP, A) Special table HEI 7-500733 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) A23J 3/10-3/34 A23L 1/305 A61K 38/01

Claims (4)

(57) [Claims] 1. Physicochemical and biological properties of the following a) to f); a) the decomposition rate of casein is 20 to 30%, b) isoleucine, tyrosine and phenylalanine contained in the hydrolyzate of casein. , Free isoleucine in the total mass of specific amino acids of proline, leucine and valine,
Free tyrosine, free phenylalanine, free proline,
The total mass ratio of free specific amino acids of free leucine and free valine is 4 to 10%, c) has the following molecular weight distribution, more than 5,000 0 to 0.5% more than 1000 and less than 5,000 10 to 20% 500 Super 1000 or less 15-30% 500 or less 50% or more d) The ratio of the fraction having a molecular weight of 1000 daltons or less is 80
% Or more, e) The ratio of the amount of free amino acids to the amount of total amino acids of the casein hydrolyzate is less than 10%, and f) ELISA (Enzyme link).
The immunogenicity measured by the immuno-sorbent assay) is 10 times that of casein.
^-4 or less, The casein hydrolyzate characterized by having. 2. A casein aqueous solution is supplemented with three kinds of proteases, that is, an alkaline endoprotease derived from a microorganism, a neutral endoprotease derived from a bacterium of the genus Bacillus, and an endoprotease derived from an animal. The decomposition rate of casein by the enzyme and the total measured value of the number of moles of 16 kinds of amino acids measured by the L-amino acid sensor are monitored over time, and the decomposition rate of casein and the measured value by the L-amino acid sensor. Of the casein hydrolyzate, wherein the enzymatic reaction is stopped and the insoluble matter is removed at the time when both satisfy the ranges of 20 to 30% and 5 to 12 mM in terms of solid content of 10% respectively. 3. The method for producing a casein hydrolyzate according to claim 2, wherein the insoluble matter is removed by microfiltration and / or ultrafiltration. 4. The method for producing a casein hydrolyzate according to claim 2, wherein the insoluble matter is removed by diatomaceous earth and / or ultrafiltration.