JPH08112064A - New production process of peptide mixture - Google Patents

Abstract

PURPOSE: To stably obtain a peptide mixture having almost constant content of each free amino acid and total amount of the free amino acids. CONSTITUTION: A peptide mixture having a constant quality is obtained by adding one or more kinds of protease to an aqueous solution of raw protein composed of one or more kinds of protein or an aqueous solution of preparatorily lightly hydrolyzed raw protein, starting hydrolysis of the raw protein or previously lightly hydrolyzed raw protein, quickly determining the amount of a specific amino acid isolated into the hydrolyzing solution by hydrolysis with passage of time, calculating the ratio of an amount of free specific amino acid to the total amount of specific amino acid contained in the raw protein or previously lightly hydrolyzed raw protein and stopping the hydrolysis as soon as the calculated value reaches the prescribed specific range.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a peptide mixture containing a predetermined amount of free amino acids in a stable state and with a constant quality. Specifically, the present invention relates to an aqueous solution of a raw material protein consisting of one or more kinds of proteins or an aqueous solution of a raw material protein that has been slightly hydrolyzed in advance (hereinafter, these aqueous solutions may be collectively referred to as a raw material aqueous solution). , Add proteolytic enzyme,
Start the hydrolysis of the raw material protein or the raw material protein that has been slightly hydrolyzed beforehand, and measure the amount of the specific amino acid released in the decomposition solution by hydrolysis over time to obtain the raw material protein or the raw material protein that has been previously lightly hydrolyzed. The ratio of the amount of free specific amino acid to the total amount of specific amino acid contained is calculated, and the hydrolysis is immediately stopped when the calculated value reaches a preset specific range. A new manufacturing method. As used herein, percentages are by weight unless otherwise noted.

[0002]

2. Description of the Related Art A peptide mixture obtained by degrading animal protein (animal milk, egg, meat, fish, etc.) or vegetable protein (soybean, wheat etc.) with a proteolytic enzyme has a thickening property and a foaming property. , Antioxidative property, digestive and absorbable property, mineral solubilizing property, low antigenic property, epidermal growth factor, cell growth factor, calcium absorption promoting function, and bioactive function such as opioid-like activity are known ( Food and Development, Volume 26, No. 11,
28-36, 1991), livestock meat, fish paste, bread, confectionery, mineral-fortified foods, infant foods, sports drinks, general nutrition foods, enteral nutritional supplements, protein allergy foods, special nutrition foods, pharmaceuticals, etc. It is one of the essential raw materials for the production of.

[0003] Conventionally, the method for producing a peptide mixture used for producing these foods or pharmaceuticals differs depending on the application, but a) In order to limit the production of free amino acids as much as possible, the starting protein is endopeptidase. A method of producing a desired peptide mixture by decomposing with only b) A method of producing a peptide mixture containing a predetermined amount of free amino acid by decomposing a starting protein with a combination of endopeptidase and exopeptidase, and c ) A method of further purifying and fractionating the desired peptide mixture from these peptide mixtures by separation operations such as ultrafiltration (UF), reverse osmosis (RO), gel filtration, ion exchange resin method, etc. can do.

Some of those prior arts are as follows. 1) A mixture of oligopeptides, which is characterized by enzymatically decomposing a protein in an aqueous system coexisting with an endo-type protease and an exo-type protease for 0.5 to 10 hours to obtain an oligopeptide having an average chain length of 3 to 10 with extremely little bitterness. A manufacturing method is disclosed (Japanese Patent Laid-Open No. 62-143697).

2) A protein raw material of arbitrary origin is added to water.
Disperse to ~ 20% (w / v) and add acid to adjust pH
Is adjusted to 1 to 4 and two or more kinds of acidic proteases are added simultaneously or sequentially to obtain 8 to 72 at a temperature of 25 to 60 ° C.
A method for producing a low-molecular peptide composition by carrying out an enzymatic decomposition reaction while suppressing the production of free amino acids has been disclosed (Japanese Patent Publication No. 57-45560).

3) In the method of partially decomposing a casein solution with an immobilized enzyme packed in a column, a method of producing a partially decomposed product of casein by controlling the flow rate of the casein solution has been disclosed (Japanese Patent Publication No. Hei 3). -31421 publication).

4) When a protein or a substance containing a protein and a dissolution accelerator are mixed and dissolved in dissolved water and the like, and one or more proteolytic enzymes are added and digested to produce a proteolytic product, at least the above A protein which prevents the formation of insoluble matter, characterized in that the viscosity of a solution after addition of a protein degradation product is measured with time, and the digestion reaction is stopped before the viscosity is greatly increased and decreased. A method for producing a decomposed product is disclosed (Japanese Patent Publication No. 3-58252).

5) A protease hydrolyzate of a glycopolypeptide derived from animal milk κ-casein, which has a decomposition rate of 5 to 25% in a method for producing a peptide mixture having a Fisher value in the range of 30 to 60. There is disclosed a method of inactivating the enzyme by heat treatment at the time when the temperature reaches (JP-A-2-300137).

6) A method for producing a protein degradation product by decomposing a milk protein with an immobilized protease, a method of measuring the peptide concentration with a peptide sensor and a method of measuring the average chain length of the peptide have been disclosed. Industrial Bioreactor System Technology Research Association, "Practical Bioreactor", pp. 166-184, Food Industry Bioreactor System Technology Research Association, 1990).

7) In a method for producing a gluten degradation product by degrading wheat gluten with an immobilized protease, the hydrophobicity of the gluten degradation product is measured by reverse phase high performance liquid chromatography (hereinafter, high performance liquid chromatography is referred to as HPLC). It is disclosed that a gluten hydrolyzate excellent in foam stability can be produced by measuring with time (food industry bioreactor system technology research association, "Practical bioreactor", pp. 106-126, food industry bio. Published by Reactor System Technology Research Association, 1990).

8) It is known to measure the amount of free amino acid as a target product in the production of fermented foods such as miso and soy sauce, and process control in amino acid fermentation of lysine and glutamic acid (Food Industry, Volume 34). , No. 16, No. 1
~ Page 11, 1991).

[0012]

As shown in the above-mentioned prior art, when a peptide mixture is conventionally produced by degrading a starting protein with a proteolytic enzyme, the end point of the degradation reaction is
It was determined by measuring the viscosity, decomposition rate, hydrophobicity, etc. of the decomposed product, using reaction time, flow rate of the protein solution, etc. as an index. , It is extremely difficult to accurately grasp the amount of free amino acids, and in the conventional method for producing a peptide mixture, the amount and composition of free amino acid contained in the peptide mixture are different for each production batch, and the quality of the peptide mixture is There was a fatal drawback that it was not constant and there was a strong demand for improvement.

Under these circumstances, the inventors of the present invention have conducted diligent research on a method for producing a peptide mixture with a constant quality in a stable state in view of the above-mentioned prior art. The amount of the specific amino acid released in the decomposition solution by the substance is measured over time and in a short time, and the ratio with the amount of the specific amino acid contained in the raw material protein is calculated. The present invention has been completed by finding that the desired peptide mixture can be obtained by stopping the hydrolysis as soon as it reaches within the range. An object of the present invention is to provide a novel method capable of easily producing a peptide mixture having a constant amount and composition of free amino acids and good quality.

[0014]

The present invention for solving the above-mentioned problems provides a method for producing a peptide mixture of constant quality, which comprises the steps of:
To an aqueous solution of a raw material protein consisting of two or more kinds of proteins, or an aqueous solution of a raw material protein which has been slightly hydrolyzed in advance,
One or more proteolytic enzymes are added to start the hydrolysis of the raw material protein or the raw material protein that has been slightly hydrolyzed in advance, and the amount of the specific amino acid released in the degradation solution by the hydrolysis can be measured with time. And measured in a short time, calculate the ratio of the amount of specific amino acid released to the total amount of specific amino acids contained in the raw material protein or raw protein slightly hydrolyzed in advance, the calculated value is a preset specific range The method for producing a peptide mixture is characterized in that the hydrolysis is stopped immediately when the content reaches the inside, and it is also a preferable embodiment that the specific amino acid is lysine, phenylalanine, leucine or arginine.

Next, the present invention will be described in detail. The raw material protein used as the starting material of the method of the present invention is animal protein (for example, animal milk-derived, egg-derived, fish meat-derived, meat-derived etc.), vegetable protein (cereal-derived, seaweed-derived, seed-derived, etc.) or It is an arbitrary mixture of these and is not particularly limited. Alternatively, a starting material may be a peptide mixture having a large molecular weight, which is a degradation product obtained by lightly hydrolyzing a protein in advance and which can be further degraded by a protease. The starting protein or the starting protein that has been slightly hydrolyzed in advance is dissolved in water at a concentration of about 10% in terms of protein, and the pH of the solution is adjusted to the optimum pH of the proteolytic enzyme by using an alkaline solution or an acid solution. Adjust to the vicinity and prepare the raw material aqueous solution.

Then, the raw material aqueous solution may be of animal origin (eg, pancreatin, pepsin, trypsin, etc.), plant origin (eg, papain, bromelain, etc.), microbial origin (eg, mold, actinomycete, bacterium, lactic acid bacterium, etc.). A proteolytic enzyme or a proteolytic enzyme in any combination thereof is appropriately selected according to the purpose, and a predetermined amount is added. For example, endopeptidase is added in an amount of 2000 to 50 per 1 g of the starting protein.
00 PUN unit, exopeptidase as raw material protein 1 g
Addition of 20 to 100 active units per unit can be exemplified as a desirable mode (PUN units and active units will be described later).

A raw material aqueous solution to which a predetermined amount of enzyme has been added is usually kept at the optimum temperature of the enzyme for a predetermined time to hydrolyze the protein by the enzyme, and it is necessary if the growth of microorganisms is concerned during the decomposition. Depending on the conditions, the temperature of the enzyme may be kept at a temperature higher or lower than the optimum temperature for a predetermined time to hydrolyze the protein by the enzyme.

After the hydrolysis is started, the amount of the specific amino acid released in the decomposition solution is measured with time and in a short time. Specifically, for example, HPLC, Biotech analyzer (manufactured by Asahi Kasei Corporation), perfusion chromatography (manufactured by Perceptive Biosystems. BioCA.
D) etc. can be used. Since the amount of released amino acid varies depending on the type of raw material protein and enzyme used,
It is desirable to select an amino acid that is released in a large amount as the specific amino acid. As a particularly desirable mode, online measurement of the amount of the specific amino acid released in the decomposition solution can be exemplified. Further, as the specific amino acid, lysine, phenylalanine, leucine, arginine and the like are exemplified as particularly preferable ones. With these, the amount of the specific amino acid released in the decomposition solution was measured over time and in a short time, and the ratio of the amount of the specific amino acid released to the total amount of the specific amino acid contained in the protein as the starting material was preset. When the specific range is reached, the enzyme in the reaction solution is immediately inactivated or removed to stop the hydrolysis. The specific range varies depending on the target peptide mixture, the starting material used, the enzyme used, etc., but for example, the whey protein is hydrolyzed while measuring the amount of free lysine over time and in a short time. In this case, the range of the amount of free lysine is 5 to 4
0% can be exemplified.

In the present invention, the method of inactivating or removing the enzyme in the reaction solution to stop the hydrolysis is not particularly limited, and an appropriate method can be used, but the hydrolysis is stopped. May be accompanied by a time lag (for example, until a certain amount of decomposition solution is heated to inactivate the enzyme,
It may take 30 to 60 minutes). In this case, hydrolysis may proceed, so a preliminary test is performed in advance, and the degree of progress of the decomposition (for example, production of a specific free amino acid is performed under predetermined conditions). It is desirable that the specific range is determined and set in consideration of the time required to inactivate or remove the enzyme by measuring the (rate).

The decomposed solution containing the peptide mixture after the decomposition can be concentrated by a known method to obtain a concentrated solution, and the concentrated solution is dried by a known method.
It can also be made into powder. Further, the liquid containing the peptide mixture can be purified by a known method such as ultrafiltration and gel filtration, and concentrated by a known method to give a concentrated solution, and the concentrated solution is dried by a known method. However, it can also be made into powder.

The shape of the reaction vessel for hydrolysis (eg tank type, tube type, column type, etc.), decomposition treatment method (eg batch type, continuous type, sequential type, etc.), enzyme deactivation, separation Alternatively, known methods and devices can be used for the removal method, the peptide purification method, etc., and are not particularly limited. As described above, a peptide mixture in which the amount and composition of free amino acids are always constant and the quality does not change can be produced.

The PUN unit and the activity unit are as follows. The PUN unit of endopeptidase is casein [Hammerstein]. Manufactured by Merck & Co., Inc.] is treated with endopeptidase, and 1 μm per minute at 30 ° C.
The enzyme activity showing the color reaction of the allyl amino acid corresponding to g of tyrosine with the Folin reagent is 1 PUN unit.

The activity unit of exopeptidase is
It was measured by the following method. A powder containing exopeptidase is dispersed or dissolved in a 0.1 mol phosphate buffer (pH 7.0) at a ratio of 0.2 g / 100 ml to prepare an enzyme solution. On the other hand, leucyl paranitroanilide (manufactured by Kokusan Kagaku KK, hereinafter referred to as Leu-pNA) is dissolved in 0.1 mol of a phosphate buffer (pH 7.0) to prepare a 2 mM substrate solution. Add 1 ml of substrate solution to 1 ml of enzyme solution 3
After reacting at 7 ° C. for 5 minutes, 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 μm per minute
The amount of enzyme required to decompose Leu-pNA of ol is 1
It is an activity unit and calculated by the following formula. Activity unit (per 1 g of powder) = 20 × (A / B) (where A and B are the absorbance of the sample at a wavelength of 410 nm and the absorbance of 0.25 mM paranitroaniline, respectively)

Next, the present invention will be described in detail by showing test examples. In the present invention, the following test method was adopted. (1) Measuring method of 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, it was 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 with an amino acid analyzer (manufactured by Hitachi Ltd., model 835) to measure the mass of amino acids.

(2) 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) to measure the mass of the free amino acid. Then, the percentage of the free amino acid mass to the mass of each amino acid obtained by the analysis of the amino acid composition was calculated.

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

(4) HPLC Inertsil PREP-ODS (GL Science Co., Ltd. 6.5 × 25)
0mm) column is attached to HPLC (manufactured by Shimadzu Corporation),
A concentration gradient in which 0.1 ml of the decomposition solution is supplied and the ratio of the eluent B (0.1% trifluoroacetic acid-acetonitrile solution) to the eluent A (0.1% trifluoroacetic acid solution) becomes 50% in 100 minutes. Elution was carried out at a flow rate of 1.5 ml / min.

(5) Degradation rate Degradation rate (%) is a percentage of the amount of formol nitrogen in the degradation solution per the total amount of nitrogen in the raw material protein solution, and was determined by the following method. Mix 4 ml of proteolytic solution with 30 ml of distilled water and p with 0.2N sodium hydroxide solution or hydrochloric acid solution.
Adjust H to 6.8. This solution was adjusted to pH 8.0 with 0.2N sodium hydroxide solution, formalin solution 5
ml is added and titrated with 0.1 N sodium hydroxide solution until the pH reaches 7.9. The titer at this time is Am
l, the factor of 0.1N sodium hydroxide solution is F,
The decomposition rate was calculated from the following equation, with the protein concentration of the raw material protein solution as B (%). Decomposition rate (%) = 22.3 x A x F / B

Test Example 1 This test consists of a conventionally used method for producing a peptide mixture, that is, a method of stopping the enzymatic decomposition using the reaction time as an index and a method of stopping the enzymatic decomposition using the decomposition rate as an index. The peptide mixture was produced by the method of the present invention, and the effects on the quality of the obtained peptide mixture (change in free amino acid composition and free amino acid amount) were compared and examined.

1) Preparation of sample In the above method, the enzymatic decomposition was stopped in 4 hours,
In the above method, enzymatic decomposition was stopped when the decomposition rate reached 23%, and in the above method of the present invention, the concentration of free lysine in the decomposition solution was measured over time and in a short time,
Hydrolysis of the whey protein solution was repeated 5 times in the same manner as in Example 1 except that the enzymatic degradation was stopped when the release rate of lysine reached 15%, and a total of 15 types were obtained. Samples were prepared.

2) Test Method The free amino acid amount and free amino acid composition of each sample were measured by the above-mentioned method, and the average value and standard deviation of the results of 5 times were calculated.

3) Test Results The test results are shown in Table 1. As is clear from Table 1, although there were some differences in the average value of each free amino acid amount and the total amount of free amino acids in the peptide mixture produced by each method, the standard deviation and the free amount of each free amino acid were different. The standard deviation of the total amount of amino acids was found to be the smallest and stable for the method of the present invention, and the least for the following method, and for the method, the largest variation and unstable production quality were observed. It was

[0033]

[Table 1]

Test Example 2 This test was carried out in order to compare each method in the case where hydrolysis was carried out under different starting protein and decomposition conditions from Test Example 1. 1) Preparation of sample and test method Milk casein (trade name ALACID.
90% protein content. 1 kg of New Zealand Daily Board) was suspended in 9 kg of deionized water, and the pH was adjusted to 7.0 with a 20% caustic soda solution and dissolved.
In the above method, the enzymatic decomposition was stopped in 9 hours,
In the above method, the enzymatic decomposition was stopped when the decomposition rate reached 30%, and in the above method of the present invention, the concentration of free lysine in the decomposition solution was measured over time and in a short time. Hydrolysis was repeated 5 times in the same manner as in Test Example 1 except that the enzymatic decomposition was stopped when the liberation rate of was reached 32%, and a total of 15 types of samples were prepared.

2) Test Results The test results are shown in Table 2. As is clear from Table 2, although there were some differences in the average value of each free amino acid amount and the total amount of free amino acids in the peptide mixture produced by each method, the standard deviation and the free amount of each free amino acid were found. The standard deviation of the total amount of amino acids was found to be the smallest and stable in the method of the present invention, and the second method, and it was recognized that the method had the largest fluctuation and the production quality was unstable.

[0036]

[Table 2]

Test Example 3 This test was carried out in order to compare the respective methods when hydrolysis was carried out under different starting protein and decomposition conditions from Test Example 1 and Test Example 2. 1) Preparation of sample and test method Soybean protein (trade name SUPRO. Protein content 90%, manufactured by Fuji Oil Co., Ltd.) was used as a protein raw material, and in the above method, enzymatic decomposition was stopped in 6 hours. In the method, the enzymatic decomposition was stopped when the decomposition rate reached 13%, and in the above-mentioned method of the present invention, the concentration of free lysine in the decomposition solution was measured with time and in a short time, and Hydrolysis was repeated 5 times in the same manner as in Test Example 1 except that the enzymatic decomposition was stopped when the liberation rate reached 21%, and a total of 15 types of samples were prepared.

2) Test Results The test results are shown in Table 3. As is clear from Table 3, although there were some differences in the average value of each free amino acid amount and the total amount of free amino acids of the peptide mixture produced by each method, the standard deviation and the free amount of each free amino acid were The standard deviation of the total amount of amino acids was found to be the smallest and stable in the method of the present invention, and the second method, and it was recognized that the method had the largest fluctuation and the production quality was unstable. In addition, protein raw material is used for livestock meat, fish meat,
The same test was carried out by changing to eggs and the like, and in each case, the free amino acid composition and the amount of free amino acids were found to be the least varied and stable in the method of the present invention.

[0039]

[Table 3]

Test Example 4 This test was conducted to investigate the method for measuring a specific free amino acid. 1) Preparation of sample and test method Except that the amount of free lysine in the decomposition solution was measured over time and in a short time by using HPLC (manufactured by Shimadzu Corporation) instead of Biotech Analyzer (manufactured by Asahi Kasei Corporation). ,
A peptide mixture was prepared from whey protein by the same method as in Test Example 1 above.

2) Test Results The test results are shown in Table 4. As is clear from Table 4, the average value of the free amount of each amino acid and the average value of the total amount of free amino acids were measured regardless of whether the amount of free lysine in the degradation solution was measured by a Biotech analyzer or HPLC. Although different, it was recognized that the standard deviation of the free amount of each amino acid and the standard deviation of the total amount of free amino acids were not significantly different between the two. Therefore, it was found that any of these measuring methods for the amount of free amino acid can be used in the present invention. It should be noted that, although the test was carried out by changing the kind of protein and the hydrolysis condition, almost the same result was obtained.

[0042]

[Table 4]

Test Example 5 This test was conducted to examine the type of specific free amino acid to be measured. 1) Preparation of sample and test method Using HPLC (Shimadzu Corporation), the amount of free phenylalanine in the decomposition solution was measured over time and in a short time. When the amount of free phenylalanine reached 25%, enzymatic decomposition was performed. A peptide mixture was prepared from casein by the same method as in Test Example 2 except that it was stopped.

2) Test Results The test results are shown in Table 5. As is clear from Table 5, even when the amount of free phenylalanine in the degradation solution was measured, the standard deviation of the free amount of each amino acid and the standard deviation of the total free amino acid amount were small, and a peptide mixture of constant quality was obtained from casein. , Was confirmed to be obtained. It should be noted that, although the test was carried out by changing the kind of protein and the hydrolysis condition, almost the same result was obtained.

[0045]

[Table 5]

Test Example 6 This test was carried out in the same manner as in Test Example 5 except that the type of specific free amino acid measured was changed. 1) Preparation of sample and test method Using HPLC (manufactured by Shimadzu Corporation), the amount of free leucine in the decomposed solution was measured over time and in a short time, and enzymatic decomposition was performed when the amount of free leucine reached 10%. A peptide mixture was prepared from soybean protein by the same method as in Test Example 3 except that it was stopped.

2) Test Results The test results are shown in Table 6. As is clear from Table 6, even when the amount of free leucine in the decomposition solution was measured, the standard deviation of the free amount of each amino acid and the standard deviation of the total amount of free amino acids were small, and the peptide mixture of soybean protein with a constant quality was obtained. Was found to be obtained. Also, when the amount of free arginine was measured, almost the same result was obtained. Tests were also performed when the type of the specific amino acid to be measured was changed to an amino acid other than leucine and arginine, or when the type of protein and the hydrolysis conditions were changed, but almost the same results were obtained.

[0048]

[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 5 of filtrate A and filtrate B
Glucose 5 parts to 00 parts, monopotassium phosphate 2.5 parts,
2.5 parts of dipotassium phosphate and 5 parts of sodium acetate were added, pH was adjusted to 6.4 with 30% sodium hydroxide, and water was added to adjust to 1000 parts.

Lactobacillus helveticus was cultivated in 10 l of the sterilized medium having the above-mentioned composition, and the obtained culture solution was centrifuged to recover the lactic acid bacterium cells, which were suspended in sterilized water and centrifuged to remove the lactic acid bacterium. The operation of collecting the body is repeated twice to wash the cells, and then the cells are suspended in sterilized water at a concentration of 20%, and then ultrasonically disrupted (manufactured by Branson. SONIFIER mod
El 250), the cells were crushed and freeze-dried to obtain about 25 g of lactic acid bacterium-derived exopeptidase powder.

[0051]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. Example 1 1 kg of a commercial whey protein powder having a whey protein content of 75% (manufactured by California Protein Co.) and 9 kg of deionized water
And sterilized by holding at 70 ° C. for 5 minutes to adjust the pH to 9.
Adjusted to 0, commercially available Protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 1.5 million PUN units (200 per 1 g of whey protein)
0 PUN unit) and 40,000 active units of Lactobacillus helveticus cell disrupted product (60 active units per 1 g of whey protein) prepared by the same method as in Reference Example 1 were added, and 5
When hydrolysis was started by maintaining at 0 ° C, the amount of free lysine was measured with a biotech analyzer (manufactured by Asahi Kasei Kogyo Co., Ltd.) over time and in a short time, and when the amount of free lysine reached 14%. Then, heat at 80 ° C for 6 minutes to inactivate the enzyme, stop the enzymatic decomposition, and then freeze-dry by a conventional method,
About 950 g of a peptide mixture was obtained from whey protein. The peptide mixture obtained by repeating the above production method twice was tested by the above test method, and as a result, almost no difference was observed in the amount of each free amino acid and the total amount of the free amino acids.

Example 2 1 kg of commercially available wheat protein powder (manufactured by Riken Vitamin Co., Emmasoft EX-100) having a protein content of 80% was mixed with 9 parts of deionized water.
It was dissolved in kg, the pH was adjusted to 7.0, and the mixture was kept at 70 ° C. for 5 minutes for sterilization. Commercially available pancreatin (manufactured by Amano Pharmaceutical Co., Ltd.) was added to this wheat protein solution in the amount of 2 million PUN units (2,500 PUN units per 1 g of wheat protein) and the Lactobacillus helveticus cell disruption product 6 prepared in the same manner as in Reference Example 1 above. 10,000 activity units (75 g / g wheat protein)
Activity unit), and the mixture is kept at 50 ° C. to start hydrolysis, and the amount of free lysine is measured with a biotech analyzer (Asahi Kasei Kogyo Co., Ltd.) over time and in a short time.
When the amount of free lysine reached 34%, the enzyme was inactivated by heating at 80 ° C for 10 minutes to stop the enzymatic decomposition, and then freeze-dried, and about 950 g of a peptide mixture from wheat protein was obtained.
I got The peptide mixture obtained by repeating the above production method twice was tested by the above test method, and as a result, almost no difference was observed in the amount of each free amino acid and the total amount of the free amino acids.

Example 3 500 g of a commercial wheat protein powder having a protein content of 80% (manufactured by Riken Vitamin Co., Emmasoft EX-100) and a commercially available soybean protein powder having a protein content of 90% (manufactured by Fuji Oil Co., trade name SU.
500 g of PRO) was dissolved in 9 kg of deionized water, the pH was adjusted to 7.0, and the mixture was kept at 70 ° C. for 5 minutes for sterilization. In this mixed protein solution, commercially available pancreatin (manufactured by Amano Pharmaceutical Co., Ltd.) 850,000 PUN units (1000 PU per 1 g of protein)
N units), Protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 17
Add 50,000 PUN units (2,000 PUN units per 1 g of protein) and 40,000 active units (47 active units per 1 g of protein) of Lactobacillus helveticus cell disruption product prepared by the same method as in Reference Example 1 above, and add at 50 ° C. To start hydrolysis and measure the amount of free arginine with HPLC (manufactured by Shimadzu Corporation) over time and in a short time,
When the amount of free arginine reaches 21%, 10 at 80 ° C
The mixture was heated for a minute to inactivate the enzyme, stop the enzymatic decomposition, and then freeze-dried to obtain about 950 g of a peptide mixture. The peptide mixture obtained by repeating the above production method twice was tested by the above test method, and as a result, almost no difference was observed in the amount of each free amino acid and the total amount of the free amino acids.

Example 4 500 g of a commercial whey protein powder (manufactured by California Protein Co.) having a whey protein content of 75% and a protein content of 90
% Commercial soy protein powder (manufactured by Fuji Oil Co., Ltd., trade name SUPR
O) 500 g is dissolved in 9 kg of deionized water and the mixture is heated to 70 ° C. for 5 times.
It was kept for minutes to be sterilized, the pH was adjusted to 9.0, and commercially available protease A Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 2.5 million PUN units (4848 PUN units per 1 g of protein) and the same method as in Reference Example 1 were used. Lactobacillus helveticus cell disrupted product 40,000 activity units (48 per 1 g protein)
Activity unit), and the mixture is kept at 50 ° C. to start hydrolysis, and the amount of free lysine is measured with a biotech analyzer (manufactured by Asahi Kasei Kogyo Co., Ltd.) over time and in a short time.
When the amount of free lysine reached 18%, the mixture was heated at 80 ° C. for 6 minutes to inactivate the enzyme, stop the enzymatic decomposition, and freeze-dry to obtain about 950 g of a peptide mixture. The peptide mixture obtained by repeating the above production method twice was tested by the above test method, and as a result, almost no difference was observed in the amount of each free amino acid and the total amount of the free amino acids.

[0055]

As described above in detail, the present invention is a novel method for producing a peptide mixture, and the effects of the present invention are as follows. 1) A peptide mixture is obtained in which the amount of each free amino acid and the total amount of free amino acids are almost constant. 2) The desired peptide mixture is always stably obtained. 3) A peptide mixture of constant quality can be easily produced.

[Procedure amendment]

[Submission date] December 13, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

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

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0051

[Correction method] Change

[Correction content]

[0051]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. Example 1 1 kg of a commercial whey protein powder having a whey protein content of 75% (manufactured by California Protein Co.) and 9 kg of deionized water
And sterilized by holding at 70 ° C. for 5 minutes to adjust the pH to 9.
Adjusted to 0, commercially available Protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 1.5 million PUN units (200 per 1 g of whey protein)
0 PUN unit) and 40,000 active units of Lactobacillus helveticus cell disruption product (60 active units per 1 g of whey protein) prepared by the same method as in Reference Example 1 were added, and
When hydrolysis was started by maintaining at 0 ° C, the amount of free lysine was measured with a biotech analyzer (manufactured by Asahi Kasei Kogyo Co., Ltd.) over time and in a short time, and when the amount of free lysine reached 14%. Then, heat at 80 ° C for 6 minutes to inactivate the enzyme, stop the enzymatic decomposition, and then freeze-dry by a conventional method,
About 950 g of a peptide mixture was obtained from whey protein. The peptide mixture obtained by repeating the above production method twice was tested by the above test method, and as a result, almost no difference was observed in the amount of each free amino acid and the total amount of the free amino acids.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0052

[Correction method] Change

[Correction content]

Example 2 1 kg of commercially available wheat protein powder (manufactured by Riken Vitamin Co., Emmasoft EX-100) having a protein content of 80% was mixed with 9 parts of deionized water.
It was dissolved in kg, the pH was adjusted to 7.0, and the mixture was kept at 70 ° C. for 5 minutes for sterilization. Commercially available pancreatin (manufactured by Amano Pharmaceutical Co., Ltd.) was added to this wheat protein solution for 2 million PUN units (2,500 PUN units per 1 g of wheat protein) and 60,000 crushed Lactobacillus helveticus cells prepared in the same manner as in Reference Example 1. Activity unit (75 per 1 g wheat protein)
Activity unit), and the mixture is kept at 50 ° C. to start hydrolysis, and the amount of free lysine is measured with a biotech analyzer (Asahi Kasei Kogyo Co., Ltd.) over time and in a short time.
When the amount of free lysine reached 34%, the enzyme was inactivated by heating at 80 ° C for 10 minutes to stop the enzymatic decomposition, and then freeze-dried, and about 950 g of a peptide mixture from wheat protein was obtained.
I got The peptide mixture obtained by repeating the above production method twice was tested by the above-mentioned test method, and as a result, almost no difference was observed in the amount of each free amino acid and the total amount of the free amino acids.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0053

[Correction method] Change

[Correction content]

Example 3 500 g of a commercial wheat protein powder having a protein content of 80% (manufactured by Riken Vitamin Co., Emmasoft EX-100) and a commercially available soybean protein powder having a protein content of 90% (manufactured by Fuji Oil Co., trade name SU.
500 g of PRO) was dissolved in 9 kg of deionized water, the pH was adjusted to 7.0, and the mixture was kept at 70 ° C. for 5 minutes for sterilization. In this mixed protein solution, commercially available pancreatin (manufactured by Amano Pharmaceutical Co., Ltd.) 850,000 PUN units (1000 PU per 1 g of protein)
N units), Protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 17
100,000 PUN units (2000 PUN units per gram of protein) and lactobacco prepared by the same method as in Reference Example 1 above.
Was added Shirasu helveticus disrupted cells 40,000 activity units (protein 1g per 47 activity units), and held at 50 ° C. to initiate hydrolysis, over time using HPLC (manufactured by Shimadzu Corporation), and Measure the amount of free arginine in a short time,
When the amount of free arginine reaches 21%, 10 at 80 ° C
The mixture was heated for a minute to inactivate the enzyme, stop the enzymatic decomposition, and then freeze-dried to obtain about 950 g of a peptide mixture. The peptide mixture obtained by repeating the above production method twice was tested by the above test method, and as a result, almost no difference was observed in the amount of each free amino acid and the total amount of the free amino acids.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0054

[Correction method] Change

[Correction content]

Example 4 500 g of a commercial whey protein powder (manufactured by California Protein Co.) having a whey protein content of 75% and a protein content of 90
% Commercial soy protein powder (manufactured by Fuji Oil Co., Ltd., trade name SUPR
O) 500 g is dissolved in 9 kg of deionized water and the mixture is heated to 70 ° C. for 5
It was kept for minutes to sterilize, the pH was adjusted to 9.0, and commercially available protease A Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 2.5 million PUN units (4848 PUN units per 1 g of protein) and the same method as in Reference Example 1 were used. Lactobacillus helveticus cell disruption product 40,000 active units (48 per 1 g protein)
Activity unit), and the mixture is kept at 50 ° C. to start hydrolysis, and the amount of free lysine is measured with a biotech analyzer (Asahi Kasei Kogyo Co., Ltd.) over time and in a short time.
When the amount of free lysine reached 18%, the mixture was heated at 80 ° C. for 6 minutes to inactivate the enzyme, stop the enzymatic decomposition, and freeze-dry to obtain about 950 g of a peptide mixture. The peptide mixture obtained by repeating the above production method twice was tested by the above test method, and as a result, almost no difference was observed in the amount of each free amino acid and the total amount of the free amino acids.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication location // A23L 1/305 (72) Inventor Hitoshi Saito 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Institute of Nutrition Science Co., Ltd. (72) Inventor Yasushi Kawaguchi 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Co., Ltd. (72) Inventor Yoko 5-1-5-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry (72) Inventor Hiroshi Ochi 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Co., Ltd.

Claims (2)

[Claims] 1. A method for producing a peptide mixture of constant quality, which comprises one or more aqueous solutions of a raw material protein consisting of one or more proteins or an aqueous solution of a raw protein which has been slightly hydrolyzed in advance. The proteolytic enzyme is added to start the hydrolysis of the raw material protein or the raw material protein that has been slightly hydrolyzed beforehand, and the amount of the specific amino acid released in the degradation solution due to the hydrolysis is measured with time and in a short time. ,
Calculate the ratio of the amount of the specific amino acid released to the total amount of the specific amino acid contained in the raw material protein or the raw material protein that has been slightly hydrolyzed in advance, and immediately hydrolyze when the calculated value reaches the preset specific range. A method for producing a peptide mixture, which comprises stopping the decomposition. 2. The released specific amino acid is lysine, phenylalanine, leucine or arginine.
The method for producing the peptide mixture according to 1.