JPH08112063A - Whey protein hydrolyzate having excellent flavor and its production - Google Patents

Abstract

PURPOSE: To obtain a whey protein hydrolyzate having excellent flavor usable as a raw material for a resource of protein to infancy, early childhood and nursing mothers or a sick person having lowered immunological function aiming at prevention of allergies of infancy and early childhood having immature digestive ability or an aged person, a sick person or an allergy patient having lowered digestive ability. CONSTITUTION: This whey protein hydrolyzate having excellent flavor is a hydrolyzate of whey protein having a purity of >=70wt.% and has a fraction having molecular weight of 5,000-10,000 dalton of <1wt.% of total hydrolyzate, a residual antigen activity of <=10<-5> determined by an ELISA suppressing test using anti-whey protein serum, a ratio of an amount of free amino acid to the amount of total amino acid in the hydrolyzate of 10-15wt.%, a ratio of an amount of free lysine to the amount of total lysine contained in the whey protein of 12-20wt.% and an ammonium content of <=0.2wt.%.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a whey protein hydrolyzate having unique physicochemical properties and a method for producing the same, which is excellent in intestinal absorbability and amino acid balance and has preventive and therapeutic effects on diet allergy. Further, it is a novel whey protein hydrolyzate which has an antioxidant effect and a good flavor and can be used in a wide variety of applications, and a method for producing the same. In the present specification, percentages are by weight, unless otherwise specified, except for transmittance and suppression.

[0002]

2. Description of the Related Art Recently, from the viewpoint of digestion and absorption, it has been revealed that oligopeptides are superior to a mixture of free amino acids in terms of absorption rate and amino acid balance after absorption (dairy science / food research, Volume 39, A-
283, 1990). On the other hand, the number of allergic patients caused by dietary protein has increased rapidly, and it has become clear that allergies caused by whey protein, particularly β-lactoglobulin, are frequently occurring in infants (dairy science and food research, No. 39). Volume, Pages A-283, 1990
), There is a need to reduce the antigenicity of whey protein in infant food or to substantially remove the whey protein antigen from infant food.

Hydrolysis of whey protein has been widely adopted as a means of reducing the antigenicity of whey protein in baby foods or substantially removing whey protein antigens from baby foods, but amino acid release. Degradation products with extremely low rates often have a bitter taste, which can be an obstacle to ingestion. In addition, since whey protein hydrolyzate may become unstable to heat, inconveniences such as the formation of a precipitate and browning may occur in the state of a solution. There was a problem when using as.

Further, when the whey protein hydrolyzate is used in foods, oxidation is caused especially in foods coexisting with fat (for example, formula powder for infants contains 27% fat per 100 g). Prevention is a serious issue. That is, in foods containing fat, the balance between saturated fatty acids and unsaturated fatty acids has been considered from a nutritional point of view, but especially unsaturated fatty acids have the drawback of being easily oxidized. D is abundant in biological membranes of nerve and retinal tissue and is considered to be involved in the expression of its function.
HA and the like, once oxidized, release an extremely strong oxidative odor and have a significant adverse effect on product quality, and therefore, there is a strong demand for preventing the progress of oxidation.

On the other hand, the ingested amino acid is decomposed by transglutaminase, glutanote dehydrogenase and the like to produce ammonia, but the produced ammonia is toxic and needs to be immediately processed by the liver, and ammonia should be added to foods to be ingested. Is required to be contained. From this point, it is extremely important that the whey protein hydrolyzate does not contain ammonia.

[0006] Further, the nitrogen equilibrium of grown animals can be obtained by ingesting an amount of nitrogen commensurate with the minimum metabolism of nitrogen, but it is ineffective to give this nitrogen as ammonia alone, and if it is not ingested as an essential amino acid. I won't. To do this, the food that you eat must contain the required amount of essential amino acids.

Nutrition of the above proteins and amino acids
From the physiological background, various methods for producing a hydrolyzate of whey protein with an enzyme have been developed, and some of them are as follows.

1) Whey protein is decomposed by two kinds of enzymes, endopeptidase and trypsin derived from Bacillus subtilis, or three kinds of enzymes, endopeptidase, trypsin and chymotrypsin derived from Bacillus subtilis, and the molecular weight is 2,000 daltons. The antigen residual rate is 10 ^-4 or less,
An oligopeptide mixture having an amino acid liberation rate of 5% or less is disclosed (JP-A-4-248959).

2) Degradation of milk protein with alkaline protease to obtain dipeptides and tripeptides of 75 mol% or more,
A degradation product having an amino acid liberation rate of less than 5%, consisting of 4 or more amino acids and having a peptide with an average chain length of 6.2 less than 20 mol% is disclosed (Japanese Patent Publication No. 5-505304).

3) Whey, casein and soybeans are decomposed with pepsin, trypsin and chymotrypsin, ultrafiltered, and 4 to
A peptide having 10 amino acids is 40 to 60%, and an oligopeptide having a molecular weight of 60,000 daltons or less is disclosed (JP-A-3-187348).

4) pH 6 while denaturing the whey protein by heat
Decomposes at -10, 60-80 ° C, deactivates the enzyme by heating, molecular weight 10,000 or less, main peak 1,000
~ 5,000, average peptide chain length 3-8, free amino acid content 20% or less, β-lactoglobulin antigenicity 1/1
Disintegration products of 10,000 or less have been disclosed (JP-A-4-
No. 112753).

5) A low allergenic peptide having a molecular weight of 10,000 daltons or less and having an oral tolerance inducing property is disclosed in which milk protein is decomposed by enzymes of trypsin, α-chymotrypsin, Aspergillus and Bacillus. Kaihei 5-5000).

6) Casein is decomposed with an acid protease, neutralized with peptidase, and has a molecular weight of 3,000 daltons or less, a free amino acid content of 30 to 55%, and an inhibition ELISA test for α _S -casein is α _S-.
A peptide in which the bitterness functional value of a 5% solution of 10,000 times or less of casein is equivalent to 0.04% of caffeine aqueous solution or less is disclosed (JP-A-6-113893).

7) Whey was hydrolyzed with neutral protease (Aspergillus sp.) At pH 5 to 11 to give pH 2
A method of heating at ~ 4 to remove the precipitate and obtaining a dipeptide and a tripeptide in a ratio of 50% is disclosed (Japanese Patent Publication No. 5-82412).

[0015]

However, in the above-mentioned conventional techniques, reduction of the antigenicity of whey protein hydrolyzate, improvement of bitterness, free amino acid content, molecular weight distribution, etc. are taken into consideration. No consideration is given to the ammonia content and antioxidant activity of the purified protein hydrolyzate. Therefore, conventionally, there has been a disadvantage that the whey protein hydrolyzate cannot be used for a wide range of foods.

The inventors of the present invention have conducted extensive studies in view of the above-mentioned prior art and obtained by hydrolyzing whey protein, which is effective in avoiding, preventing and treating diet allergy and is excellent in digestion and absorption. The present invention has been completed by finding a whey protein hydrolyzate having a low ammonia content, an antioxidant effect, and a good flavor which can be used in a wide range of applications, and a method for producing the same.

The object of the present invention is whey protein which is excellent in intestinal absorption and amino acid balance, has a preventive and therapeutic effect on diet allergy and an antioxidant effect, has a low ammonia content and can be used in a wide range of applications. It is to provide a hydrolyzate and a method for producing the same.

[0018]

The first invention of the present invention for solving the above-mentioned problems is a hydrolyzate of whey protein having a purity of at least 70% (by weight), and the following a) to h) are provided. A) The fraction having a molecular weight of 5,000 to 10,000 daltons is less than 1% (by weight) of the total hydrolyzate, b) An ELISA inhibition test method using anti-whey protein serum. The residual antigen activity measured by the method is 10 ^-5 or less, c) the ratio of the amount of free amino acids to the amount of total amino acids of the hydrolyzate is 10 to 15% (by weight), and d) the whey protein. The ratio of the amount of free lysine to the amount of total lysine contained is 12 to 20% (by weight), e) the ammonia content is 0.2% (by weight) or less, and f) the 10% (by weight) solution. Is a 1 cm cell and the transmittance measured at 540 nm is 8% or more, g) a 5% (by weight) solution having a pH of 4 to 7 is heated at 120 ° C. for 10 minutes to prevent precipitation, and h) has an antioxidant effect. It is a whey protein hydrolyzate with a good flavor.

A second aspect of the present invention which solves the above-mentioned problems is to provide whey protein having a purity of at least 70% (by weight).
Dissolved in water at a concentration of 5% (weight) or less, the pH of the aqueous solution
Was adjusted to 7.5-10, and two types of proteolytic enzymes, an endopeptidase derived from Bacillus subtilis and an exopeptidase derived from lactic acid bacteria, were added to the aqueous solution to initiate hydrolysis, and The amount of free lysine was measured with time, and the ratio of the amount of free lysine to the amount of total lysine contained in the whey protein as the starting material was 12%.
To 20% (by weight), hydrolysis is stopped, and ultrafiltration is performed to completely remove a fraction having a molecular weight of 10,000 daltons or more, and a whey protein hydrolyzate having a good flavor is produced. Is the law.

Next, the present invention will be described in detail, but in order to facilitate understanding of the present invention, description will be given from the second invention of the present invention. The whey protein used as a starting material in the method of the present invention may be a commercially available product having a purity of at least 70%, and is known as a whey protein concentrate (WPC) or a whey protein isolate (WPI). Commercially available products having higher purity than the existing products are suitable. These whey proteins are dissolved in water at a concentration of 15% or less, preferably 8 to 12%, and the pH is adjusted to 7.5 to 10, preferably 8 to 9 with an alkaline aqueous solution.

Next, two kinds of proteolytic enzymes, an endopeptidase derived from Bacillus subtilis and an exopeptidase derived from lactic acid bacteria, are added to the whey protein solution. In addition, a very small amount of endopeptidase such as trypsin and papain can be added. However, the addition of exopeptidases (including pancreatin etc.) other than those derived from lactic acid bacteria deteriorates the flavor and should be avoided. Bacillus subtili
As the endopeptidase derived from s), commercially available products can be used, and 1,000 to 7,500 per 1 g of whey protein can be used.
PUN units (this unit will be described later), preferably 2,000 to 3,000 PUN units, are added.

The PUN unit is casein [Hammerstein]. Made by Merck] to Bacillus subtilis
The enzyme activity showing the color reaction of allyl amino acid corresponding to 1 μg of tyrosine with the Folin reagent at 30 ° C. for 1 minute is set to 1 PUN unit by allowing the endopeptidase derived from (Bacillus subtilis) to act.

The exopeptidase derived from lactic acid bacteria can be produced, for example, by the method described in the section of “(3) Enzyme used” in JP-B No. 54-36235, column 6, line 4). Lactic acid bacteria (including bifidobacteria) are cultivated by a known method (for example, the method described in Japanese Patent Publication No. 48-43878), the obtained culture solution is centrifuged to collect the lactic acid bacterium cells, and the cells are sterilized in sterile water. The procedure of suspending the cells and recovering the lactic acid bacteria cells by centrifugation is repeated twice, the cells are washed, and the cells are suspended in sterilized water at a concentration of 20%, and the cell crusher [for example, Dynomill ( Willy Bachnfen Engin
eering Works) product. KDL type] to crush the cells,
Lyophilization is performed to obtain lactic acid bacterium-derived exopeptidase powder. 20 to 200 activity units of this enzyme per 1 g of whey protein (this unit will be described later), preferably 60
~ 90 activity units.

The activity unit is measured by the following method. 0.2g / 100m of powder containing exopeptidase
An enzyme solution is prepared by dispersing or dissolving in 0.1 mol phosphate buffer (pH 7.0) at a ratio of 1 l. On the other hand, leucylparanitroanilide (manufactured by Kokusan Kagaku Co., Ltd. hereafter, Leu-pN
Described as A) in 0.1 molar phosphate buffer (pH 7.
0) to prepare a 2 mM substrate solution. 1 ml of the substrate solution is added to 1 ml of the enzyme solution, and the mixture is reacted at 37 ° C. for 5 minutes, and then 2 ml of a 30% acetic acid solution is added to stop the reaction. The reaction solution is filtered with a membrane filter, and the absorbance of the filtrate is measured at a wavelength of 410 nm. The activity unit of exopeptidase is 1 μmol of Leu-p per minute.
The amount of enzyme required for decomposing NA was defined as 1 activity unit, and calculated by the following formula. Active unit (per 1 g of powder) = 20 × (A / B) However, in the above formula, A and B are respectively wavelengths of 4
The absorbance of the sample at 10 nm and the absorbance of 0.25 mM paranitroaniline are shown.

The enzyme-added solution is kept at 30 to 60 ° C., preferably 45 to 55 ° C. to start the hydrolysis of whey protein. When the hydrolysis reaction proceeds and the pH decreases, it is desirable to maintain the pH at 6 or higher, preferably 6 to 7. After starting hydrolysis, measure the amount of free lysine in the decomposed solution over time using a device that can measure the amount of free lysine in the decomposed solution over time, for example, Biotech Analyzer (Asahi Kasei Co., Ltd.) However, the ratio of the amount of free lysine to the amount of total lysine contained in the whey protein as a starting material is 12 to 20%, preferably 14 to 17
%, The reaction solution is immediately heated (for example, at 85 ° C. for 15 minutes) to inactivate the enzyme and stop the hydrolysis.

The obtained reaction solution was treated with an acid such as citric acid to obtain p
H is adjusted to a range of 5.5 to 7 and a known device [for example,
Ultrafiltration module (manufactured by Asahi Kasei Kogyo Co., Ltd.)] to completely remove a fraction having a molecular weight of 10,000 daltons or more to obtain a desired whey protein hydrolyzate having a good taste. The liquid containing the whey protein hydrolyzate can be concentrated by a known method to give a concentrated solution, and the concentrated solution can be dried by a known method to give a powder.

The whey protein hydrolyzate obtained as described above has the following physicochemical properties as will be apparent from the examples described below. a) As shown in FIG. 1, the molecular weight is 5,000 to 10,000.
The fraction of 0 dalton is less than 1% (by weight) of the total hydrolyzate, does not include the fraction of molecular weight of 10,000 daltons or more, and the fraction of molecular weight of less than 1,000 daltons is 70% or more, It has peaks at a molecular weight of 500 daltons and a molecular weight of 1,000 daltons, and has a number average molecular weight of 300 to 400 daltons and a weight average molecular weight of 600 to 800 daltons. 1 shows the molecular weight distribution of the whey protein 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.

B) As shown in FIG. 2, the residual antigen activity measured by the ELISA inhibition test method using anti-whey protein serum is 10 ^-5 or less, preferably 10 ^-6 or less. Figure 2
Shows the antigen residual activity of the whey protein hydrolyzate of the present invention obtained in Example 1, and the vertical axis and the horizontal axis show the inhibition ratio and the final sample concentration, respectively. In the figure, + and □ indicate whey protein and the whey protein degradation product of the present invention, respectively.

C) The ratio of the amount of free amino acid to the amount of total amino acid of the hydrolyzate is 10 to 15% (by weight), preferably 11 to 13% (by weight). d) The ratio of the amount of free lysine to the amount of total lysine contained in the whey protein is 12 to 20% (by weight), preferably 1
It is 4 to 17% (weight). e) Ammonia content is 0.2% (weight) or less, preferably 0.1% (weight) or less. f) The transmittance of a 10% solution measured in a 1 cm cell at 540 nm is 98% or more. g) Heating a 5% (by weight) solution of pH 4-7 at 120 ° C. for 10 minutes without precipitation.

H) As shown in FIG. 3, it has an antioxidant activity equal to or higher than that of the known antioxidant α-tocopherol. FIG. 3 shows the antioxidant activity of the whey protein hydrolyzate of the present invention obtained in Example 1, and the vertical axis and the horizontal axis respectively show the antioxidant capacity residual rate and time. ◇ 、
+ And □ indicate the whey protein hydrolyzate of the present invention, α-tocopherol, and a control (no sample or standard preparation added), respectively.

Next, the present invention will be described in detail by showing test examples.
In the test example of the present invention, the following test method was adopted. (1) Method for measuring molecular weight High-performance liquid chromatography (edited by Nobuo Ui et al., “High-performance liquid chromatography for proteins and peptides”, Kagaku Special Issue No. 102, page 241, Kagaku Dojin, 1984.
Year) was measured as follows. Polyhydroxyethyl aspartamide column [Poly L (PolyL
Made by C). Diameter 4.6 mm and length 200 mm],
Elution was performed with 20 mM sodium chloride and 50 mM formic acid at an elution rate of 0.4 ml / min. UV detector is used for detection,
The data analysis used the GPC analysis system (made by Shimadzu Corporation).

(2) Method of measuring residual activity of antigens ELISA test method (Journal of Japanese Society of Pediatric Allergy, No. 1
Vol., P. 36, 1978). 96-well plate (manufactured by Nunc) is coated with whey protein and washed, and a mixture of rabbit anti-whey protein serum and hydrolyzate sample is supplied to the well of the plate to react, and after washing, alkaline phosphatase-labeled goat Anti-rabbit IgG antibody (manufactured by Zymed Laboratories) was reacted, and after washing, p-nitrophenyl sodium phosphate was added, and after 30 minutes, 5N sodium hydroxide was added to stop the reaction, and the reaction product was micronized. It measured with the plate reader (made 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) x 100 However, the absorbance of the test antigen solution and the absorbance of the control are the same as the test sample solution of anti-whey protein serum. Alternatively, it is a value measured after the reaction of the hole in which the mixed liquid of the diluting liquid is put.

(3) Method for measuring amino acid composition For amino acids other than tryptophan, cysteine and methionine, samples were hydrolyzed with 6N hydrochloric acid at 110 ° C. for 24 hours, and for tryptophan, barium hydroxide at 110 ° C. for 22 hours. After decomposition, cysteine and methionine were treated with 6N hydrochloric acid after the treatment with excess acid.
It was hydrolyzed at 0 ° C. for 18 hours and decomposed with an amino acid analyzer (manufactured by Hitachi Ltd., model 835) to measure the mass of the amino acid.

(4) Method for measuring free amino acid composition The sample was deproteinized with sulfosalicylic acid and analyzed by an amino acid analyzer (Hitachi, Ltd. model 835), based on the mass of each amino acid obtained by the above amino acid composition analysis. The percentage of free amino acid mass was calculated.

(5) Method for measuring free lysine content Enzyme electrode for lysine measurement, 20 mM L-lysine standard solution,
Using a 0.1 M L-lysine phosphate measurement buffer and a washing surfactant (both manufactured by Asahi Kasei Kogyo Co., Ltd.), the free lysine concentration was measured in a batch or online with a Biotech analyzer (Asahi Kasei Kogyo Co., Ltd.), The ratio of the amount of free lysine to the total lysine was calculated from the lysine content of the decomposition solution to the lysine content of whey protein.

(6) Method for measuring ammonia content The sample was deproteinized with sulfosalicylic acid and analyzed with an amino acid analyzer (Hitachi Ltd. model 835) to measure the mass of ammonia.

(7) Method for measuring antioxidant activity Linoleic acid and β-carotene were emulsified with Tween 20,
Α-Tocopherol was added to this as a sample or a standard, and its change with time was measured by a colorimetric method [Phytochemistry, vol. 10, p. 1445,
1971]. Linoleic acid, β-carotene, Tween
The final concentrations of 20, sample and α-tocopherol were 0.96 mg / ml, 4.8 μg / ml and 9.6 m, respectively.
g / ml, 0.19 mg / ml and 0.19 mg / ml
Met.

Test Example 1 This test was carried out to examine a suitable concentration of the whey protein solution to be subjected to hydrolysis, using the ratio of the high molecular weight fraction closely related to the antigenicity as an index. 1) Preparation of sample The whey protein solution was hydrolyzed in the same manner as in Example 1 except that the whey protein concentration was changed as shown in Table 1 to prepare 7 types of samples.

2) Test method The ratio of the fraction having a molecular weight of 5,000 to 10,000 daltons was determined by the above-mentioned molecular weight measuring method.

3) Test Results The test results are shown in Table 1. As is clear from Table 1, the concentration of whey protein in which the fraction having a molecular weight of 5,000 to 10,000 daltons is less than 1% is 15%.
Below, it was found that it is desirably 12% or less. Considering the reaction efficiency, 8-12% is most desirable.
It should be noted that the whey protein was tested by varying the enzyme amount within the range of the type of whey protein, the type of endopeptidase derived from Bacillus subtilis and the type of exopeptidase derived from lactic acid bacteria, and the range determined in Test Example 3 described below. , And almost the same result was obtained.

[0041]

[Table 1]

Test Example 2 This test was carried out to examine the initial pH range of enzyme treatment suitable for hydrolysis using the antigenicity as an index. 1) Preparation of sample, except that the initial pH of hydrolysis was changed as follows:
The whey protein solution was hydrolyzed in the same manner as in Example 1 to prepare 5 types of samples. Sample 1: After initial pH was adjusted to pH 6.5, hydrolysis was performed. Sample 2: After initial pH was adjusted to pH 7.5, hydrolysis was performed. Sample 3: After initial pH was adjusted to pH 8.0, hydrolysis was performed. Sample 4: After initial pH was adjusted to pH 9.0, hydrolysis was performed. Sample 5: After the initial pH was adjusted to pH 10.0, hydrolysis was performed.

2) Test method The residual antigen activity was measured by the method for measuring residual antigen activity.

3) Test Results The test results are shown in Table 2. As is clear from Table 2, in order to obtain a whey protein hydrolyzate having a low antigenicity, the initial pH for hydrolysis is 7.5 to 10.
It was found to be 0, preferably 8-9. It should be noted that the whey protein was tested by varying the enzyme amount within the range of the type of whey protein, the type of endopeptidase derived from Bacillus subtilis and the type of exopeptidase derived from lactic acid bacteria, and the range determined in Test Example 3 described below. , And almost the same result was obtained.

[0045]

[Table 2]

Test Example 3 This test was carried out in order to examine the proper amount of the enzyme to be used, with the ratio of the high molecular weight fraction closely related to the antigenicity, the ammonia content and the antioxidant activity as indicators. 1) Preparation of sample, except that the amount of enzyme used was changed as shown in Table 3.
The whey protein solution was hydrolyzed in the same manner as in Example 1 to prepare 12 types of samples. Samples 1 and 7 had a free lysine content of 1 even after hydrolysis for 30 hours.
Since it did not reach 4%, the hydrolysis was terminated at that point.

2) Test method The ratio of the fraction having a molecular weight of 5,000 to 10,000 daltons is the method for measuring the molecular weight, the ammonia content is the method for measuring the ammonia content, and the antioxidant activity is the method for measuring the antioxidant action. Sought by. The antioxidant activity of the sample is
The relative strength of α-tocopherol against the antioxidant activity was expressed as an index.

3) Test Results The test results are shown in Table 3. As is clear from Table 3, the fraction having a molecular weight of 5,000 to 10,000 daltons is 1% or less, the ammonia content is 0.2% or less,
In addition, the amount of the enzyme having an antioxidant activity equivalent to or higher than that of α-tocopherol is from 1,000 to 1,000 of the endopeptidase derived from Bacillus subtilis per 1 g of whey protein.
7,500 PUN units, preferably 2,000-3,0
00 PUN units, 2 exopeptidases derived from lactic acid bacteria
It has been found to range from 0 to 200 activity units, preferably 60 to 90 activity units. In addition, the type of whey protein and the types of endopeptidase derived from Bacillus subtilis and exopeptidase derived from lactic acid bacteria were changed and tested, but almost the same results were obtained.

[0049]

[Table 3]

Test Example 4 In this test, the appropriate lysine release rate of the hydrolyzate was determined by using the ratio of the high molecular weight fraction closely related to flavor and antigenicity, and the content of free amino acids that affect flavor. Went to find out. 1) Preparation of sample As shown in Table 4, the hydrolysis reaction was carried out in the same manner as in Example 1 except that the enzyme was appropriately inactivated and stopped at a ratio of the desired amount of free lysine. Different types of samples were prepared.

2) Test method The ratio of the fraction having a molecular weight of 5,000 to 10,000 daltons and the content of free amino acids were determined by the above-mentioned method. The flavor was tested by the following method. a) Flavor test A sensory test was conducted by a panel of 10 people each for men and women, and it was evaluated in four grades from good flavor (0 point) to poor flavor (3 points).
From the average value of the evaluation points, less than 0.5 points is good, 0.5 points or more and less than 1.5 points is a little good, 1.5 points or more and less than 2.5 points is a little bad and 2.5 points or more It was judged to be defective when less than 3.0 points.

3) Test Results The results of this test are shown in Table 4. As is clear from Table 4, the whey protein hydrolyzate having a good flavor was found to have a lysine release rate of 12 to 20%, preferably 14 to 17%. The type of whey protein, Bacillus
The type of endopeptidase derived from S. subtilis and the exopeptidase derived from lactic acid bacteria, and the enzyme amount were varied within the range determined in Test Example 3 described above, and the same results were obtained.

[0053]

[Table 4]

Test Example 5 In this test, the ratio of the high molecular weight fraction closely related to the antigenicity, the antigenicity, and the transmittance and heat stability, which are the criteria of transparency which is a property suitable as a food material, are used as indexes. , To determine a suitable filtration method for the hydrolyzate. 1) Preparation of sample As shown in Table 5, the whey protein solution was hydrolyzed in the same manner as in Example 1 except that the filtration membrane (molecular weight cut-off) was changed to prepare three types of samples. As the filtration membrane, an ultrafiltration membrane having a cut-off molecular weight of 3,000 daltons and 10,000 daltons manufactured by Asahi Kasei Kogyo Co., Ltd., and a pore size of 0.
A 25 μm precision filtration membrane was used.

2) Test method The ratio of the fraction having a molecular weight of 5,000 to 10,000 daltons, the residual antigen activity, the transmittance and the thermal stability were all determined by the above-mentioned methods.

3) Test Results The results of this test are shown in Table 5. As is clear from Table 5, the ultrafiltration treatment method having a fraction with a molecular weight of 5,000 to 10,000 daltons of less than 1%, a transmittance of 98% or more, and heat resistance is citric acid at a pH of 5. 5-
It was found that it is necessary to adjust the number to 7 and use an ultrafiltration membrane having a molecular weight cutoff of 10,000 or less, preferably 3,000 or less. The type of whey protein, Bacillus
The type of endopeptidase derived from S. subtilis and the exopeptidase derived from lactic acid bacteria, and the enzyme amount were varied within the range determined in Test Example 3 described above, and the same results were obtained.

[0057]

[Table 5]

Test Example 6 This test was carried out in order to investigate the type of enzyme suitable for hydrolysis using flavor and antioxidant activity as indicators. 1) Preparation of samples Six types of samples were prepared by the same method as in Example 1 except that the type and addition amount of the enzyme used were changed as shown in Table 6. As the enzyme, an endopeptidase derived from Bacillus subtilis, bioplase 6.0S (manufactured by Nagase Seikagaku Co., Ltd.) and exopeptidase derived from lactic acid bacteria were prepared by the same method as in Reference Example 1 described below. Lactobacillus helveticus crushed cells or Bifidobacterium previs crushed cells, other endopeptidases, trypsin (manufactured by Novo Nordisk), and other exopeptidases, Denatim AP (Nagase Sangyo) (Manufactured by the company) was used. Also,
Since the free lysine amount of Sample Nos. 1 and 2 did not reach 14% even after 30 hours of hydrolysis, the hydrolysis was terminated at that time.

2) Test method Both the flavor and the antioxidant activity were determined by the above-mentioned methods.

3) Test Results The results of this test are shown in Table 6. As is clear from Table 6, whey protein hydrolyzate having a good flavor and an antioxidant activity equivalent to or higher than that of α-tocopherol is two types of proteins, an endopeptidase derived from Bacillus subtilis and an exopeptidase derived from lactic acid bacteria. It was found to be hydrolyzed with a degrading enzyme. The type of whey protein and the type and amount of the enzyme were changed (for the endopeptidase derived from Bacillus subtilis and the exopeptidase derived from lactic acid bacterium, the amount of enzyme was changed within the range determined in Test Example 3 described above. Was changed) and tested, but almost the same results were obtained.

[0061]

[Table 6]

Reference Example 1 100 parts of tap water and 5 parts of lime were added to 20 parts of corn steep liquor (weight, the same applies hereinafter) to neutralize the acid contained in the corn steep liquor, and Celite 50 as a filter aid. And then filtered to obtain a filtrate A. Separately from this, 50 parts of Celite was added to a mixed solution of 20 parts of fish river, 35 parts of molasses and 200 parts of tap water and filtered to obtain a filtrate B.

Equivalent mixture 500 of the above-mentioned filtrate A and filtrate B
5 parts glucose, 2.5 parts potassium monophosphate, 2.5 parts dipotassium phosphate and 5 parts sodium acetate were added to the parts, and the pH was adjusted to 6.4 with 30% sodium hydroxide,
Water was added to adjust to 1000 parts.

Lactobacillus helveticus was cultivated in 10 liters of the sterilized medium having the above composition, and the obtained culture solution was centrifuged to collect the lactic acid bacteria cells, and the cells were suspended in sterile water and centrifuged. The procedure of recovering lactic acid bacterium cells is repeated twice to wash the microbial cells, and then the microbial cells are suspended in sterilized water at a concentration of 20%, and then ultrasonically disrupted (Branson Corp. SONIFIER
The cells were crushed according to model 250) and freeze-dried to obtain about 25 g of lactic acid bacterium-derived exopeptidase powder.

Reference Example 2 25.0 kg of a whey protein hydrolyzate (protein equivalent 79.4%) obtained by the same method as in Example 2 was dissolved in 140 kg of water, and a predetermined amount of the solution was dissolved in 5 kg of water. Add minerals,
Vegetable fat containing 70 g of DHA heated to 60 ° C 2.0
kg, malt dextrin 65.1kg, sugar 6.6k
g and a predetermined amount of vitamins were mixed, and this mixed solution was sufficiently homogenized by a high-pressure homogenizer, sterilized at 120 ° C. for 2 seconds, and spray-dried to obtain about 99 kg of a powdery antiallergic composition.

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】

Example 1 1 kg of a whey protein powder having a purity of 75% (manufactured by California Protein Co.) was dissolved in 9 kg of deionized water to prepare 75
Sterilize by holding at ℃ for 15 seconds, adjust pH to 9.0,
Proteases N Amano (Amano Pharmaceutical Co., Ltd.) 1.8 million PUN
Units (2400 PUN units per 1 g of whey protein) and Lactobacillus prepared by the same method as in Reference Example 1
Helveticus cell disruption product (60,000 activity units (90 activity units per 1 g of whey protein)) was added, and the mixture was kept at 50 ° C to hydrolyze and then biotech analyzer (manufactured by Asahi Kasei Kogyo Co., Ltd.) was used. The amount of free lysine was measured, and when the amount of free lysine reached 14%, the enzyme was inactivated by heating at 80 ° C. for 6 minutes, cooled, and then the pH was adjusted to 6.0 with citric acid, Ultrafiltration was performed with an ultrafiltration membrane having a molecular weight cut-off of 10,000 (manufactured by Nitto Denko Corporation) to obtain about 16 kg of a solution containing 5.9% whey protein hydrolyzate.

Some of the results of the test of the obtained whey protein hydrolyzate by the above test method are shown in FIGS. 1, 2 and 3. As a result, in the whey protein hydrolyzate, the fraction having a molecular weight of 5,000 to 10,000 daltons has a total hydrolyzate content of 0.3%, an antigen residual activity of 10 ^-6 or less, and a lysine release rate. 14%, free amino acid content 11%, ammonia content 0.07%, 10% solution permeability 98%, 5
% Unadjusted pH of the solution and pH 4 at 120 ° C., 10
It was stable even after heating for a minute and had an antioxidant activity equivalent to that of α-tocopherol. The amino acid composition tested by the above test method (per 1 g of whey protein hydrolyzate) was as follows.

L-alanine 52.8 (mg) L-arginine 23.4 L-aspartic acid (including L-asparagine) 102.6 L-cysteine 17.1 L-glutamic acid (including L-glutamine) 185. 1 L-glycine 18.8 L-histidine 17.7 L-isoleucine 59.9 L-leucine 100.1 L-lysine 94.6 L-methionine 15.8 L-phenylalanine 29.5 L-proline 61.4 L -Serine 49.2 L-threonine 70.3 L-tryptophan 16.7 L-tyrosine 26.1 L-valine 54.7

Example 2 1 kg of 85% pure whey protein powder (manufactured by Denmark Protein Co.) was dissolved in 19 kg of deionized water to adjust the pH to 10, and commercially available trypsin (manufactured by Novo Nordisk Co.) was used. 110,000 PUN units (130 per gram of whey protein
PUN unit), Protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.)
1.8 million PUN units (2100P per gram of whey protein)
UN units) and 51,000 active units of Lactobacillus bulgaricus crushed cells prepared by the same method as in Reference Example 1 (60 active units per 1 g of whey protein) were added, and 4
It is hydrolyzed at 0 ° C., and the amount of free lysine is measured with time using a Biotech analyzer (manufactured by Asahi Kasei Corporation).
When the amount of free lysine reached 17%, the enzyme was inactivated by heating at 130 ° C. for 2 seconds, cooled, and then added with citric acid.
H was adjusted to 6.5, ultrafiltered with an ultrafiltration membrane having a molecular weight cut off of 3,000 (manufactured by Asahi Chemical Industry Co., Ltd.), concentrated, spray-dried, and powdered whey protein hydrolyzate about 800 g Got

The obtained whey protein hydrolyzate was tested by the above-mentioned test method, and as a result, the molecular weight was 5,000 to 10,
The fraction of 000 daltons has a total hydrolyzate content of 0.2%, an antigen residual activity of 10 ^-6 or less, a lysine release rate of 17%, a free amino acid content of 13%, and an ammonia content of 0.04.
%, The transmittance of the 10% solution was 99%, the pH of the 5% solution was not adjusted, and it was stable even when heated at 120 ° C. for 10 minutes at pH 4, and had an antioxidant activity equivalent to that of α-tocopherol.

Example 3 Whey protein powder having a purity of 90% (manufactured by Biopol) 1k
g was dissolved in 19 kg of deionized water, sterilized by holding at 75 ° C. for 15 seconds, the pH was adjusted to 8.0, and commercially available papain (manufactured by Amano Pharmaceutical Co., Ltd.) was added to 100,000 PUN units (whey protein 1
110 PUN units per g), Neutrase (Novo Nordisk) 2.2 million PUN units (whey protein 1 g)
(2400 PUN units per 1) and Bifidobacterium prebis crushed product 90,000 active units (100 active units per 1 g of whey protein) prepared by the same method as in Reference Example 1 above, and kept at 50 ° C. After hydrolysis, the amount of free lysine was measured with time using a Biotech analyzer (manufactured by Asahi Kasei Co., Ltd.), and when the amount of free lysine reached 20%, it was heated at 85 ° C for 15 minutes to lose the enzyme. Let it live
After cooling, the pH is adjusted to 7.0 with citric acid, ultrafiltered with an ultrafiltration membrane with a molecular weight cut-off of 10,000 (manufactured by Nitto Denko), concentrated, spray-dried and powdered milk. About 800 g of clear protein hydrolyzate was obtained.

The whey protein hydrolyzate thus obtained was tested by the above-mentioned test method, and as a result, a molecular weight of 5,000 to 10,0 was obtained.
The fraction of 00 daltons is 0.3% of the total hydrolyzate, the residual antigen activity is 10 ^-6 or less, the lysine release rate is 20%, the free amino acid content is 15%, the ammonia content is 0.09%,
The transmittance of the 10% solution was 98%, it was stable even when the pH of the 5% solution was unadjusted and heated at 120 ° C for 10 minutes at pH 4, and it had an antioxidant activity equivalent to that of α-tocopherol.

Example 4 1 kg of whey protein powder having a purity of 70% (manufactured by Mirai Co.)
Dissolved in 5.7 kg of deionized water, adjusted the pH to 9.0, and used bioprase 6.0S (Nagase Seikagaku Corporation) 16
0,000 PUN units (2000 PUN per 1 g of whey protein)
Unit) and 63,000 active units of Streptococcus lactis crushed product prepared in the same manner as in Reference Example 1 (90 active units per 1 g of whey protein), and
After hydrolyzing at ℃, the amount of free lysine was measured with time using a Biotech analyzer (manufactured by Asahi Kasei Co., Ltd.), and when the amount of free lysine reached 19%, the mixture was heated at 130 ° C. for 2 seconds and the enzyme was added. Deactivate, cool, and then pH with citric acid
Was adjusted to 7.0 and subjected to ultrafiltration with an ultrafiltration membrane having a molecular weight cut off of 10,000 (manufactured by Asahi Kasei) to obtain about 11 kg of a solution containing 8.4% whey protein hydrolyzate.

The obtained whey protein hydrolyzate was tested by the above-mentioned test method, and as a result, a molecular weight of 5,000 to 10,
The fraction of 000 Dalton is 0.9% of total hydrolyzate, the residual antigen activity is 10 ^-6 or less, the lysine release rate is 19%, the free amino acid content is 14.5%, and the ammonia content is 0.1.
The transmittance of the 0% and 10% solutions was 98%, it was stable even when the pH of the 5% solution was unadjusted and heated at 120 ° C. for 10 minutes at pH 4, and had an antioxidant activity equivalent to that of α-tocopherol. .

Example 5 1 kg of whey protein powder having a purity of 80% (manufactured by New Zealand Daily Board) was dissolved in 12.3 kg of deionized water and kept at 75 ° C. for 15 seconds for sterilization to adjust the pH to 8.
Adjusted to 5, commercial papain (Amano Pharmaceutical Co., Ltd.) 80,000P
UN units (100 PUN units per 1 g of whey protein),
Biopulase (manufactured by Nagase Seikagaku Co., Ltd.) 2.2 million PUN units (2700 PUN units per 1 g of whey protein) and Streptococcus cremoris cell disruption product 56,000 active units (whey) prepared in the same manner as in Reference Example 1 above. (70 activity units per 1 g of protein) was added, the pH was kept at 6.5, hydrolysis was carried out at 55 ° C., and the amount of free lysine was measured with time using a Biotech Analyzer (Asahi Kasei Co., Ltd.). When the amount of free lysine reaches 17%, 90 ° C
The mixture is heated for 5 minutes to inactivate the enzyme, cooled, then adjusted to pH 5.5 with citric acid, and ultrafiltered with an ultrafiltration membrane (Nitto Denko) with a molecular weight cutoff of 10,000. After concentrating and spray-drying, about 800 g of powdery whey protein hydrolyzate was obtained.

The obtained whey protein hydrolyzate was tested by the above-mentioned test method, and as a result, the molecular weight was 5,000 to 10,0.
The fraction of 00 daltons is 0.3% of the total hydrolyzate, the residual antigen activity is 10 ^-6 or less, the lysine release rate is 17%, the free amino acid content is 13%, the ammonia content is 0.11%,
The transmittance of the 10% solution was 99%, the pH of the 5% solution was unadjusted, and it was stable even when heated at 120 ° C for 10 minutes at pH 4, and had an antioxidant activity equivalent to that of α-tocopherol.

Example 6 1 kg of whey protein powder having a purity of 70% (California Protein Co., Ltd.) was dissolved in 7 kg of deionized water to adjust the pH.
Was adjusted to 8.0 and bromelain (manufactured by Amano Pharmaceutical Co., Ltd.) 35
10,000 PUN units (500 PUN units per 1 g of whey protein), Neutrase (Novo Nordisk) 230
10,000 PUN units (3300 PUN units per 1 g of whey protein) and 56,000 active units of Lactobacillus bulgaricus cell disrupted product (80 active units per 1 g of whey protein) prepared by the same method as in Reference Example 1 above. Add, hydrolyze at 47 ° C, measure the amount of free lysine over time using a Biotech analyzer (Asahi Kasei), and when the amount of free lysine reaches 17%, at 85 ° C for 15 minutes Heat to inactivate the enzyme, cool, and then adjust the pH to 5.5 with citric acid, ultrafilter with an ultrafiltration membrane (Nitto Denko) with a cut-off molecular weight of 10,000, and concentrate. Spray dried
About 800 g of powdery whey protein hydrolyzate was obtained.

The obtained whey protein hydrolyzate was tested by the above-mentioned test method, and as a result, a molecular weight of 5,000 to 10,
The 000 Dalton fraction contained 0.4% of total hydrolyzate, residual antigen activity of 10 ^-6 or less, lysine release rate of 17%, free amino acid content of 13%, and ammonia content of 0.10.
%, The transmittance of the 10% solution was 98%, the pH of the 5% solution was not adjusted, and the pH was stable at 120 ° C. for 10 minutes at pH 4.

Example 7 1 kg of whey protein powder having a purity of 80% (manufactured by Protein Co., Denmark) was dissolved in 9 kg of deionized water to adjust the pH to 7.5, and a commercially available Neutrase (Novo Nordisk Co.) was used. Manufactured) by 1.6 million PUN units (2000 PUN units per 1 g of whey protein) and 28,000 active units of Bifidobacterium previs crushed cells prepared by the same method as in Reference Example 1 (per 1 g of whey protein) 35 activity units) was added, and the mixture was kept at pH 7.5 and hydrolyzed at 45 ° C., and the amount of free lysine was measured with time using a Biotech analyzer (Asahi Kasei Co., Ltd.). %, The enzyme was inactivated by heating at 90 ° C. for 20 minutes, cooled, and then the pH was adjusted to 7.0 with citric acid, and an ultrafiltration membrane with a molecular weight cutoff of 3,000 (Asahi Kasei Company ), Ultrafiltrate, concentrate and spray dry to obtain about 800 g of powdery whey protein hydrolyzate.

The obtained whey protein hydrolyzate was tested by the above-mentioned test method, and as a result, the molecular weight was 5,000 to 10,0.
The fraction of 00 dalton is 0.4% of total hydrolyzate, the residual antigen activity is 10 ^-6 or less, the liberation rate of lysine is 12%, the free amino acid content is 10%, the ammonia content is 0.09%,
The transmittance of the 10% solution was 100%, the pH of the 5% solution was unadjusted, and the pH was stable at 120 ° C. for 10 minutes at pH 4.

[0081]

As described in detail above, the present invention is a whey protein hydrolyzate having a good flavor and a method for producing the same, and the effects of the present invention are as follows. 1) Since the whey protein hydrolyzate of the present invention is excellent in intestinal absorbability and has a good amino acid balance, an infant who is immature in digestion and absorption ability or an elderly person whose digestion and absorption ability is reduced,
It can be used as a protein source material for the sick. 2) Since the whey protein hydrolyzate of the present invention has no residual antigen activity, it can be used as a material for protein supply to infants, pregnant women, and sick people with reduced immune function for the purpose of allergy patients and allergy prevention. 3) The whey protein hydrolyzate of the present invention has an antioxidant effect, has high heat stability and transparency, and has a good flavor, and thus is used as a material for a protein source of a milk fortified composition or an oral enteral nutritional supplement. it can. 4) By the method of the present invention, a whey protein hydrolyzate having a wide range of uses can be produced.

[Brief description of drawings]

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

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

FIG. 3 shows the antioxidant activity of the whey protein hydrolyzate of the present invention.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] March 9, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

B) As shown in FIG. 2, the residual antigen activity measured by the ELISA inhibition test method using anti-whey protein serum is 10 ^-5 or less, preferably 10 ^-6 or less. FIG. 2 shows the antigen residual activity of the whey protein 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 the whey protein degradation product and whey protein of the present invention , respectively.

[Procedure amendment]

[Submission date] December 13, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0005

[Correction method] Change

[Correction content]

On the other hand, the ingested amino acid is decomposed by transglutaminase, glutamate dehidrogenase and the like to produce ammonia, which is toxic and needs to be immediately treated in the liver.
It is necessary that the food you eat does not contain ammonia. From this point, it is extremely important that the whey protein hydrolyzate does not contain ammonia.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction content]

5) A low allergenic peptide having a molecular weight of 10,000 daltons or less and having an oral tolerance- inducing ability, which is obtained by decomposing a milk protein with enzymes of trypsin, α-chymotrypsin, Aspergillus and Bacillus. (Japanese Patent Laid-Open No. 5-5000).

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

[0013] 6) casein digested with acid protease, decomposes at peptidase at neutral, the molecular weight of 3,000 daltons or less, free amino acid content 30~55%, α _S - ELISA inhibition test against casein alpha _S - casein A peptide having a bitterness functional value of a 10,000% or less and a 5% solution corresponding to a caffeine 0.04% aqueous solution or less is disclosed (JP-A-6-113893).

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

(2) Method for measuring residual antigen activity The measurement was carried out as follows by the ELISA inhibition test method (Journal of Japanese Society of Pediatric Allergy, Vol. 1, p. 36, 1978). 96-well plate (manufactured by Nunc) is coated with whey protein and washed, and a mixture of rabbit anti-whey protein serum and hydrolyzate sample is supplied to the well of the plate to react, and after washing, alkaline phosphatase-labeled goat Anti-rabbit IgG antibody (manufactured by Zymed Laboratories) was reacted, and after washing, p-nitrophenyl sodium phosphate was added, and after 30 minutes, 5N sodium hydroxide was added to stop the reaction, and the reaction product was micronized. It measured with the plate reader (made 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) x 100 However, the absorbance of the test antigen solution and the absorbance of the control are the same as the test sample solution of anti-whey protein serum. Alternatively, it is a value measured after the reaction of the hole in which the mixed liquid of the diluting liquid is put.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0033

[Correction method] Change

[Correction content]

(3) Method for measuring amino acid composition For amino acids other than tryptophan, cysteine and methionine, samples were hydrolyzed with 6N hydrochloric acid at 110 ° C. for 24 hours, and for tryptophan, barium hydroxide at 110 ° C. for 22 hours. After decomposition, cysteine and methionine were treated with 6N hydrochloric acid after the treatment with excess acid.
0 ° C., 18 hours to hydrolyze, and analyzed by each amino acid analyzer (Hitachi, Ltd. .835 inch), the mass was measured amino acid.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction content]

(5) Method for measuring free lysine content Enzyme electrode for lysine measurement, 20 mM L-lysine standard solution,
Using a 0.1 M L-lysine phosphate measurement buffer and a washing surfactant (both manufactured by Asahi Kasei Kogyo Co., Ltd.), the free lysine concentration was measured in a batch or online with a Biotech analyzer (Asahi Kasei Kogyo Co., Ltd.), The ratio of the amount of free lysine to the total lysine was calculated from the free lysine content of the decomposition solution to the lysine content of whey protein.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0049

[Correction method] Change

[Correction content]

[0049]

[Table 3]

[Procedure Amendment 8]

[Document name to be amended] Statement

[Name of item to be corrected] 0058

[Correction method] Change

[Correction content]

Test Example 6 This test was carried out in order to investigate the type of enzyme suitable for hydrolysis using flavor and antioxidant activity as indicators. 1) Preparation of samples Six types of samples were prepared by the same method as in Example 1 except that the type and addition amount of the enzyme used were changed as shown in Table 6. As the enzyme, biopulase 6.0S (manufactured by Nagase Seikagaku Corporation) as endopeptidase derived from Bacillus subtilis, and exopeptidase derived from lactic acid bacterium were used in the same manner as in Reference Example 1 described later.
Lactobacillus helveticus disrupted cells were prepared or Bifidobacterium breve disrupted cells, as other endopeptidase, trypsin (manufactured by Novo Nordisk), as及Hi other exopeptidase, Denachimu AP (Nagase Co. Manufactured) was used. Also,
Since the free lysine amount of Sample Nos. 1 and 2 did not reach 14% even after 30 hours of hydrolysis, the hydrolysis was terminated at that time.

[Procedure Amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0061

[Correction method] Change

[Correction content]

[0061]

[Table 6]

[Procedure Amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0064

[Correction method] Change

[Correction content]

Milk is added to 10 liters of the sterilized medium having the above composition.
Culturing the acid bacterium Lactobacillus helveticus, centrifuging the obtained culture solution to collect lactic acid bacteria cells, suspending the cells in sterile water, and centrifuging to recover the lactic acid bacteria cells twice. Then, the bacterial cells are washed, and then suspended in sterile water at a concentration of 20%, and then ultrasonically disrupted (manufactured by Branson Co. S.
The microbial cells were crushed by ONIFIER model 250) and freeze-dried to obtain about 25 g of lactic acid bacterium-derived exopeptidase powder.

[Procedure Amendment 11]

[Document name to be amended] Statement

[Correction target item name] 0066

[Correction method] Change

[Correction content]

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 1 kg of a whey protein powder having a purity of 75% (manufactured by California Protein Co.) was dissolved in 9 kg of deionized water to prepare 75
Sterilize by holding at ℃ for 15 seconds, adjust pH to 9.0,
Protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 1.8 million PUN
Units (2400 PUN units per gram of whey protein) and Lactobacillus prepared in the same manner as in Reference Example 1 above.
Helveticus cell disruption product (60,000 activity units (90 activity units per 1 g of whey protein)) was added, and the mixture was kept at 50 ° C to hydrolyze and then biotech analyzer (manufactured by Asahi Kasei Kogyo Co., Ltd.) was used. The amount of free lysine was measured, and when the amount of free lysine reached 14%, the enzyme was inactivated by heating at 80 ° C. for 6 minutes, cooled, and then the pH was adjusted to 6.0 with citric acid, Ultrafiltration was performed with an ultrafiltration membrane having a molecular weight cut-off of 10,000 (manufactured by Nitto Denko Corporation) to obtain about 16 kg of a solution containing 5.9% whey protein hydrolyzate.

[Procedure Amendment 12]

[Document name to be amended] Statement

[Correction target item name] 0069

[Correction method] Change

[Correction content]

Example 2 1 kg of 85% pure whey protein powder (manufactured by Denmark Protein Co.) was dissolved in 19 kg of deionized water to adjust the pH to 10, and commercially available trypsin (manufactured by Novo Nordisk Co.) was used. 110,000 PUN units (130 per gram of whey protein
PUN unit), Protease N Amano (Amano Pharmaceutical Co., Ltd.)
1.8 million PUN units (2100P per gram of whey protein)
La prepared by UN units) and Reference Example 1 In the same manner as
C. Bacillus purgaricus crushed cells 51,000 activity units (60 activity units per 1 g of whey protein) were added, and 4
It is hydrolyzed at 0 ° C., and the amount of free lysine is measured with time using a Biotech analyzer (manufactured by Asahi Kasei Corporation).
When the amount of free lysine reached 17%, the enzyme was inactivated by heating at 130 ° C. for 2 seconds, cooled, and then added with citric acid.
H was adjusted to 6.5, ultrafiltered with an ultrafiltration membrane having a molecular weight cut off of 3,000 (manufactured by Asahi Chemical Industry Co., Ltd.), concentrated, spray-dried, and powdered whey protein hydrolyzate about 800 g Got

[Procedure Amendment 13]

[Document name to be amended] Statement

[Correction target item name] 0071

[Correction method] Change

[Correction content]

Example 3 Whey protein powder having a purity of 90% (manufactured by Biopol) 1k
g was dissolved in 19 kg of deionized water, sterilized by holding at 75 ° C. for 15 seconds, the pH was adjusted to 8.0, and commercially available papain (manufactured by Amano Pharmaceutical Co., Ltd.) was added to 100,000 PUN units (whey protein 1
110 PUN units per g), Neutrase (Novo Nordisk) 2.2 million PUN units (whey protein 1 g)
Was added per 2400PUN units) and the Reference Example 1 bi prepared in the same manner as Bifidobacterium breve disrupted cells 90,000 activity units (whey protein 1g per 100 activity units), and held at 50 ° C. After hydrolysis, the amount of free lysine was measured with time using a Biotech analyzer (manufactured by Asahi Kasei Co., Ltd.), and when the amount of free lysine reached 20%, it was heated at 85 ° C for 15 minutes to lose the enzyme. Let it live
After cooling, the pH is adjusted to 7.0 with citric acid, ultrafiltered with an ultrafiltration membrane with a molecular weight cut-off of 10,000 (manufactured by Nitto Denko), concentrated, spray-dried and powdered milk. About 800 g of clear protein hydrolyzate was obtained.

[Procedure Amendment 14]

[Document name to be amended] Statement

[Correction target item name] 0073

[Correction method] Change

[Correction content]

Example 4 1 kg of whey protein powder having a purity of 70% (manufactured by Mirai Co.)
Dissolved in 5.7 kg of deionized water, adjusted the pH to 9.0, and used bioprase 6.0S (Nagase Seikagaku Corporation) 16
0,000 PUN units (2000 PUN per 1 g of whey protein)
Units) and 63,000 active units of Streptococcus lactis cell disrupted product (90 active units per 1 g of whey protein) prepared in the same manner as in Reference Example 1 above.
After hydrolyzing at ℃, the amount of free lysine was measured with time using a Biotech analyzer (manufactured by Asahi Kasei Co., Ltd.), and when the amount of free lysine reached 19%, the mixture was heated at 130 ° C. for 2 seconds and the enzyme was added. Deactivate, cool, and then pH with citric acid
Was adjusted to 7.0 and subjected to ultrafiltration with an ultrafiltration membrane having a molecular weight cut off of 10,000 (manufactured by Asahi Kasei) to obtain about 11 kg of a solution containing 8.4% whey protein hydrolyzate.

[Procedure Amendment 15]

[Document name to be amended] Statement

[Correction target item name] 0075

[Correction method] Change

[Correction content]

Example 5 1 kg of whey protein powder having a purity of 80% (manufactured by New Zealand Daily Board) was dissolved in 12.3 kg of deionized water and kept at 75 ° C. for 15 seconds for sterilization to adjust the pH to 8.
Adjusted to 5, commercial papain (Amano Pharmaceutical Co., Ltd.) 80,000P
UN units (100 PUN units per 1 g of whey protein),
Biopulase (manufactured by Nagase Seikagaku Corporation) 2.2 million PUN units (2700 PUN units per 1 g of whey protein) and Streptococcus cremoris cell disruption product 56,000 active units (whey) prepared in the same manner as in Reference Example 1 above. (70 activity units per 1 g of protein) was added, the pH was kept at 6.5, hydrolysis was carried out at 55 ° C., and the amount of free lysine was measured with time using a Biotech Analyzer (Asahi Kasei Co., Ltd.). When the amount of free lysine reaches 17%, 90 ° C
The mixture is heated for 5 minutes to inactivate the enzyme, cooled, then adjusted to pH 5.5 with citric acid, and ultrafiltered with an ultrafiltration membrane (Nitto Denko) with a molecular weight cutoff of 10,000. After concentrating and spray-drying, about 800 g of powdery whey protein hydrolyzate was obtained.

[Procedure Amendment 16]

[Document name to be amended] Statement

[Correction target item name] 0077

[Correction method] Change

[Correction content]

Example 6 1 kg of whey protein powder having a purity of 70% (California Protein Co., Ltd.) was dissolved in 7 kg of deionized water to adjust the pH.
Was adjusted to 8.0 and bromelain (manufactured by Amano Pharmaceutical Co., Ltd.) 35
10,000 PUN units (500 PUN units per 1 g of whey protein), Neutrase (Novo Nordisk) 230
10,000 PUN units (3300 PUN units per 1 g of whey protein) and lactova prepared in the same manner as in Reference Example 1
Shirasu bulgaricus cell disruption product (56,000 activity units (80 activity units per 1 g of whey protein)) was added, hydrolyzed at 47 ° C, and time-lapsed using a Biotech analyzer (Asahi Kasei Co., Ltd.). The amount of free lysine was measured, and when the amount of free lysine reached 17%, it was heated at 85 ° C for 15 minutes to inactivate the enzyme, cooled, and then adjusted to pH 5.5 with citric acid, Ultrafiltration membrane with a molecular weight cut off of 10,000 (manufactured by Nitto Denko), ultrafiltration, concentration, spray drying,
About 800 g of powdery whey protein hydrolyzate was obtained.

[Procedure Amendment 17]

[Document name to be amended] Statement

[Correction target item name] 0079

[Correction method] Change

[Correction content]

Example 7 1 kg of whey protein powder having a purity of 80% (manufactured by Protein Co., Denmark) was dissolved in 9 kg of deionized water to adjust the pH to 7.5, and a commercially available Neutrase (Novo Nordisk Co.) was used. Manufactured) by 1.6 million PUN units (2000 PUN units per 1 g of whey protein) and 28,000 active units of crushed Bifidobacterium breve cells prepared by the same method as in Reference Example 1 (per 1 g of whey protein) 35 activity units) was added, and the mixture was kept at pH 7.5 and hydrolyzed at 45 ° C., and the amount of free lysine was measured with time using a Biotech analyzer (Asahi Kasei Co., Ltd.). %, The enzyme was inactivated by heating at 90 ° C. for 20 minutes, cooled, and then the pH was adjusted to 7.0 with citric acid, and an ultrafiltration membrane with a molecular weight cutoff of 3,000 (Asahi Kasei Company ), Ultrafiltrate, concentrate and spray dry to obtain about 800 g of powdery whey protein hydrolyzate.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Naoko Isomura 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Co., Ltd. (72) Inventor Yoko Azome 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Dairy Industry Co., Ltd., Institute of Nutrition Science (72) Inventor Hiroshi Ochi 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Dairy Co., Ltd., Institute of Nutrition Science (72) Mihoko Kawamoto 5-1-83, Higashihara, Zama City, Kanagawa Prefecture Morinaga Dairy Co., Ltd. Nutritional Research Institute

Claims (2)

[Claims] 1. A hydrolyzate of whey protein having a purity of at least 70% (by weight), which has the following physicochemical properties of a) to h): a) a molecular weight of 5,000 to 10,000 Daltons. The content is less than 1% (by weight) of the total hydrolyzate, b) the residual antigen activity measured by the ELISA inhibition test method using anti-whey protein serum is 10 ⁻⁵ or less, and c) the water. The ratio of the amount of free amino acid to the total amount of amino acid in the decomposed product is 10 to 15% (by weight), and d) the ratio of the amount of free lysine to the amount of total lysine contained in whey protein is 12 to 20%. (Weight), e) Ammonia content is 0.2% (weight) or less, f) 10% (weight) solution is 1 cm cell, and the transmittance measured at 540 nm is 98% or more. , G) 5% (by weight) solution of pH 4-7 It does not cause heat to precipitate at 120 ° C. 10 minutes, and h) to have antioxidant activity, characterized by having a good flavor whey protein hydrolyzate. 2. Whey protein having a purity of at least 70% (weight) is dissolved in water at a concentration of 15% (weight) or less, the pH of the aqueous solution is adjusted to 7.5 to 10, and the aqueous solution is Bacillus.・ Two types of proteolytic enzymes, endopeptidase derived from Bacillus subtilis and exopeptidase derived from lactic acid bacteria, are added to initiate hydrolysis, and the amount of free lysine in the degradation solution is measured over time to obtain the starting material. Hydrolysis is stopped when the ratio of the amount of free lysine to the amount of total lysine contained in a certain whey protein is in the range of 12 to 20% (by weight), and ultrafiltration is performed to obtain a fraction having a molecular weight of 10,000 daltons or more. A method for producing a whey protein hydrolyzate having a good flavor, which is characterized by being completely removed.