JP3226695B2 - Process for producing whey protein hydrolyzate excellent in emulsifiability and heat stability, and antiallergic formula using the whey protein hydrolyzate - Google Patents

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 flavored whey protein hydrolyzate having a low antigenicity, effective for the prevention and treatment of dietary allergies, and having excellent emulsifiability and heat stability. The present invention relates to an anti-allergic formula using a degraded protein.

[0002] In the present specification, percentages are values by weight unless otherwise specified, and proteins are values obtained by multiplying the amount of nitrogen by 6.38.

[0003]

2. Description of the Related Art The frequency of occurrence of food allergies in infants has tended to increase in recent years, and has become an important problem in child health. In general, antiallergic formula milk with reduced protein antigenicity is used for prevention and treatment of dietary allergy, and as a nitrogen source, hydrolyzed protein to reduce the antigenicity, especially degraded products A casein or a degradation product of whey protein, which is a milk protein, is used alone or in combination.

[0004] In order to improve the fat absorption of the formula, it is needless to say that the fat in the formula is desirably emulsified. However, when a milk-containing protein-degraded product having an antigen remaining activity of 10 ⁻⁴ or less in an ELISA test is generally used as a nitrogen source to produce a normal fat-containing milk, the emulsifying power of the protein-degraded product is reduced. However, it is difficult to maintain an emulsified state in which fat globules are completely dispersed because it is lower than the protein that has not been decomposed, and even if emulsification is performed with a normal homogenizer (for example, a homogenizer that passes through a homogenous valve), The fat globules easily aggregate to exhibit a fat separation state, and a phenomenon occurs in which fat separates into the upper layer of the liquid.

[0005] In the case of a milk formula with poor emulsification and separated fat, the flavor and composition differ between the upper layer and the lower layer of the liquid. It could change and was very inconvenient. Furthermore,
In order to ingest the required amount of nutrients, the whole amount must be ingested, and due to poor appearance, flavor and absorption, poorly emulsified milk from which fat is separated is not suitable as a commercial product.

[0006] Therefore, various anti-allergic formulas currently on the market are devised in terms of emulsification. The emulsifiers which are permitted to be added to the formula in Japan are lecithin, monoglyceride and polyglyceride. In the case of an antiallergic formula having an antigen residual activity of 10 ^-4 or less, nutritionally significant lecithin is used. It is difficult to maintain a sufficient emulsification by only using monoglyceride or polyglyceride in many cases. In addition, products that suppress the residual antigen activity of the protein hydrolyzate to about 10 ^-3 and maintain an emulsified state only with lecithin are commercially available, but in this case, the most important antiallergic function is sacrificed. Undesirable because.

[0007] In some cases, organic acid monoglycerides such as citric acid monoglyceride and stearic acid monoglyceride are used to improve the emulsified state, but it is not desirable to add unnecessary substances to infant products. or,
A production method for improving emulsifiability by blending starch or the like (preferably tapioca starch) is also commercially available, but in this case, it becomes impossible to approximate the components to the composition of human milk.

Further, in hospitals and clinics, in order to prepare a large amount of infant formula, an operation of sterilizing or sterilizing the formula has been adopted. This operation is extremely severe for the prepared milk, and not only destroys the emulsification but also significantly lowers the quality of the prepared milk, such as the formation of coagulates and precipitates. In conventionally known anti-allergic formula, handling under such harsh conditions is not considered at all,
No improvement was reported.

[0009] On the other hand, various methods have been disclosed in the past for producing enzymatically degraded products of milk proteins with low antigenicity. For example, a method for producing an oligopeptide having an antigen residual activity of 10 ^-4 or less by hydrolyzing whey protein with an endoprotease derived from Bacillus subtilus and enzymes of trypsin and chymotrypsin (Japanese Unexamined Patent Publication No. 248959), a method of producing an oligopeptide having an antigen residual activity of 10 ^-4 or less by decomposing whey protein with a heat-resistant enzyme while heat denaturing the same (Japanese Patent Laid-Open No. 4-127553). ing. However, in these methods, only the antigenicity, flavor and the like of the peptide are mentioned, and nothing is described about the emulsified state and the heat stability of the milk prepared using the peptide.

Also, a method for producing a low-antigenic peptide having good emulsifiability by enzymatically degrading whey protein having a reduced α-lactalbumin content (JP-A-4-3206)
No. 50) is also known, but in this method, the operation of reducing α-lactalbumin is complicated, and furthermore, the effect of the type of enzyme on emulsification and heat stability and the preparation of milk using the peptide are described. No mention is made of emulsion stability.

Various methods are also known for producing low-allergenic foods using low-antigenic proteolysates. For example, a method for producing a liquid dairy product and powdered milk characterized by degrading milk using an enzyme having a strong proteolytic action to make the passive cutaneous anaphylaxis reaction negative (for example, No. 45-31912), a partial hydrolyzate of whey protein and a low allergen containing the hydrolyzate.
A special nutritional dairy product (Japanese Patent Publication No. Hei 1-182745) is disclosed. However, none of these inventions describes the emulsion stability and heat stability of those products.

The present inventors have previously described oligopeptides having low antigenicity, a method for producing the same (JP-A-4-248959), and an antiallergic formula (JP-A-4-36544).
No. 4) and applied for a patent.

[0013]

According to the above-mentioned prior art, the antigenic activity is low, it is effective for the prevention and treatment of dietary allergies, and the antigen residual activity which is excellent in taste and nutrition is one.
With respect to the emulsifying property of the anti-allergic prepared milk of ^0-4 or less, only emulsifiers and combinations thereof are mainly studied, and the emulsifying property and heat stability of a milk protein decomposed product as a nitrogen source are used. No attempt has been made to improve the emulsifiability of the prepared milk in view of the above, and there is no known milk having an antigen remaining activity of 10 ^-4 or less and having good emulsifiability and heat stability.

[0014] Accordingly, antiallergic formulas having an antigen residual activity of 10 ^-4 or less, having excellent emulsifying properties and heat stability, and having a good flavor have been desired.

After the filing of the above patent application, the present inventors have studied the production conditions of milk protein hydrolyzate having further excellent emulsifiability and heat stability,
As a result of intensive studies on the emulsifiability and heat stability of milk prepared using the decomposed product, whey protein was converted to Bacillus
Hydrolysis using enzymes including three kinds of enzymes, subtilis-derived endo-type protease, trypsin and papain, to decompose a flavorful whey protein having an antigen remaining activity of 10 ^-4 or less and excellent emulsifying properties. Product, and the whey protein hydrolyzate 40 to 100 as a nitrogen source
%, A casein hydrolyzate having an antigen residual activity of 10 ^-4 or less as measured by an ELISA test using anti-casein serum is mixed with 60 to 0% of an antiallergic agent having excellent emulsifiability and heat stability. The present inventors have found that a milk formula can be produced, and have completed the present invention.

An object of the present invention is to provide a method for producing a flavorful whey protein hydrolyzate having low antigenicity, which is effective for prevention and treatment of dietary allergies, and which has excellent emulsifiability and heat stability. .

Another object of the present invention is to use the whey protein hydrolyzate, which has a low antigenicity, is effective for the prevention and treatment of dietary allergy, and has a good flavor with excellent emulsifiability and heat stability. It is to provide an antiallergic formula.

[0018]

According to a first aspect of the present invention, there is provided a whey protein having a purity of at least 30% (by weight) in a concentration of 5% to 20% by weight. And the pH of the solution was adjusted to 7.5 or more and 10.0 or less, and Bacillus subtilis (B) was added per 1 g of whey protein.
acillus subtilus) from the end-type protease 500
~ 2,000PUN units , trypsin 2,000 ~ 2
0,000 USP units and papain 1,000-5,000
A method for producing a whey protein hydrolyzate having excellent emulsifiability and heat stability, characterized in that the hydrolyzate is hydrolyzed by an enzyme containing three kinds of enzymes of 00 PUN units .

According to a second aspect of the present invention, which solves the above-mentioned problems, a whey protein having a purity of at least 30% (by weight) is obtained.
% Soluble in water at a concentration (by weight) more than 20% (by weight), the pH of the solution was adjusted to 7.5 to 10.0, milk
Kiyoshi protein 1g per Bacillus subtilis (Bacillus subt
ilus) -derived endoprotease 500-2,000
PUN unit , trypsin 2,000-20,000US
P unit and papain 1,000 to 5,000 PUN units Hydrolyzed by enzymes including three kinds of enzymes, and obtained using an anti-whey protein serum (ELISA: Enzyme
linked immunosolbent assay) obtained by hydrolyzing casein with a purity of at least 40% to 100% (by weight) and at least 80% (by weight) of a whey protein hydrolyzate having an antigen residual activity of 10 ^-4 or less as measured by an inhibition test method. A mixture of a casein hydrolyzate having a residual antigen activity (hereinafter referred to as "casein antigen residual activity") of ^10-4 or less as measured by an ELISA test using an anti-casein serum and 60 to 0% (by weight). Protein equivalent of nitrogen source consisting of 10-15
Per part, 15-40 parts fat, and 70-4 carbohydrates
The composition is an anti-allergic formula with excellent emulsifiability and heat stability, which has a composition of 0 parts.

Next, the present invention will be described in detail.

The whey protein used in the method of the present invention may be a commercially available product, or may be a whey protein concentrate separated and purified from whey by a conventional method (eg, ultrafiltration, ion exchange method, etc.). Product, whey protein isolate, or a mixture obtained by mixing these whey proteins and whey (eg, whey powder, desalted whey powder, etc.) and adjusting the whey protein purity to at least 30% or more. You may. If the purity of whey protein is less than 30%, the proportion of lactose in the whey protein hydrolyzate is increased, and the lactose content of the milk composition containing this as one component is undesirably high.

Whey protein is dissolved in water or purified water at a concentration of 5% to 20%, preferably 10% to 15%,
The pH of the solution is adjusted to 7.5 or more and 10.0 or less. If the dissolution concentration is less than 5%, the production efficiency is poor, and if it exceeds 20%, the decomposition efficiency is reduced,
This is not desirable because viscosity rise during cooling occurs. or,
Since the optimal pH of the enzyme used in the present invention is from neutral to slightly alkaline, it is desirable to adjust the pH before decomposition to 7.5 or more and 10.0 or less. Further, as a pre-decomposition treatment step, heat treatment before or after pH adjustment, or both,
An ion exchange treatment or the like can be appropriately performed.

After adjusting the pH, Bacillus subtilis (Bacillu
s subtilus), the enzyme remaining in the whey protein is enzymatically decomposed to 10 ^-4 or less by a combination of enzymes including three types of enzymes, endo-type protease, trypsin and papain. The enzyme may be any commercially available product that is harmless in terms of food hygiene. Examples of the endoprotease derived from Bacillus subtilus include protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.), bioprase (manufactured by Nagase & Co., Ltd.), and Neutrase (Novorase). And trypsin such as PTN 6.0S (manufactured by Novo Nordisk), papain such as Papain W-40 (manufactured by Amano Pharmaceutical), and purified papain (manufactured by Asahi Breweries). Examples can be given.

In the present invention, the amount of enzyme added per whey protein is defined by an activity unit, and trypsin is used in USP.
Units (Units based on the United States Pharmacy Law. The United States Pharmaccopeia The Na
national formulary), pages 307 and 1431, United States Pharmacopial Convention, Inc.
harmacopeial Convention, Inc.), 1990], which is 1,000 to 30,000 USP units, preferably 2,000 to 20,000 USP units. For endopeptidases from Bacillus subtilis, PU
N units [milk casein (eg, Hammerstein (Hem
mersten). 3) When the enzyme is allowed to act on
The enzyme activity showing a color reaction of allylamino acid corresponding to 1 μg of tyrosine with the Folin reagent per minute at 0 ° C. is 1
250-3,000P
UN unit, preferably 500 to 2,000 PUN unit, and for papain, 500 to 6,000 PUN unit.
The unit is desirably 1,000 to 5,000 PUN units. If these three types of enzymes are added in predetermined amounts,
Other endo- or exo-type proteases
0 PUN unit or less, desirably 200 PUN unit or less,
Can be further added in the range of.

A predetermined amount of an enzyme is added to the whey protein solution according to the amount of the whey protein, and the enzyme is heated at a temperature of 30 to 60 ° C., preferably 40 to 55 ° C., which is the optimum temperature of the enzyme.
Hydrolyze for 24 hours, preferably 5 to 12 hours. The enzyme can be added simultaneously or at appropriate intervals,
Immobilized enzymes can also be used. In addition, the optimal enzyme
In order to maintain H, the pH of the solution can be adjusted appropriately during the decomposition.

When this decomposed product is used as a raw material for prepared milk, the pH of the decomposed liquid is preferably around neutral.
Therefore, if the pH of the solution after the decomposition is in the range of 7.0 ± 0.5, it is kept as it is, and if the pH is out of this range, it is appropriately 7.0 ± 0.
Adjust to 5.

When decomposed by batch processing, the decomposed liquid is heated by a conventional method to deactivate the enzyme. The heating temperature and the holding time can be set as appropriate under conditions that allow sufficient deactivation in consideration of the thermal stability of the enzyme used. After heat treatment,
It can be cooled by a conventional method, and can be obtained as it is or concentrated if necessary, and dried to obtain a powder product. The whey protein hydrolyzate thus obtained has excellent emulsifiability and heat stability, and has an antigen residual activity of 10 ^-4 or less.

Next, the production of an anti-allergic formula according to the second invention of the present invention will be described. The raw material casein of the casein hydrolyzate used in the formula of the present invention has a purity of at least 80%, and is separated from the acid casein such as casein lactate and casein hydrochloride by a conventional method.
Caseinates such as caseinate, potassium caseinate, or any mixture thereof can be used. Raw material casein is 5% or more and 15% or less, preferably 1%
It is dispersed in water at a concentration of 0% or more and 13% or less, and a dissolved salt such as sodium hydroxide, potassium hydroxide, or potassium carbonate is added, and the solution is adjusted to a pH of 6.6 to 10 and dissolved. If the concentration is less than 5%, the production efficiency is poor,
On the other hand, if it exceeds 15%, the viscosity increases and the production becomes difficult.

When the pH of the solution is less than 6.6, it is difficult to dissolve the acid casein. When the dissolved salt is added as the pH exceeds 10.0, the mineral content of the casein decomposition product becomes excessive. , Each undesirable.

The enzyme to be used is a commercially available enzyme whose optimum pH is neutral to alkaline and is harmless to food hygiene, and an enzyme derived from animals such as pancreatin, trypsin and chymotrypsin, and a plant derived enzyme such as papain and bromelain. , Bacteria, fungi, and other microorganism-derived enzymes. In addition, lactic acid bacteria cells or cell extracts can be added for the purpose of improving flavor.

The pH of the casein solution is adjusted, a predetermined amount of the enzyme is added according to the amount of casein in the casein solution, and the optimum temperature of the enzyme is 30 to 60 ° C., preferably 40 to 60 ° C.
Hydrolysis at a temperature of 55 ° C for 2 to 24 hours, preferably 5 to 12 hours. The type, combination and activity unit of the enzyme to be added can be appropriately determined within the range of the antigen remaining activity of 10 ^-4 or less. The enzymes can be added simultaneously or at appropriate intervals, and an immobilized enzyme can also be used.

Next, when the enzyme is decomposed by batch processing, the enzyme is inactivated or removed by heat treatment or ultrafiltration according to a conventional method. The heating temperature and the holding time can be set as appropriate under conditions that allow sufficient deactivation in consideration of the thermal stability of the enzyme used. After the heat treatment, the mixture is cooled by a conventional method, and the precipitate is removed by steps such as celite filtration, microfiltration, ultrafiltration, and centrifugal separation. Further, if necessary, it can be purified by steps such as resin adsorption and activated carbon filtration. . The thus obtained casein hydrolyzate having an antigen residual activity of 10 ^-4 or less can be used as it is or concentrated and dried to be used as a raw material of the antiallergic formula milk of the present invention.

The anti-allergic formula may be in liquid or powder form, but a particularly desirable method for producing a powder product is as follows.

The ratio between the whey protein hydrolyzate and the casein hydrolyzate produced as described above (whey protein hydrolyzate / casein hydrolyzate) is 100/0 to 40/60, and the total solids of the final product is Each hydrolyzate is weighed so that the protein equivalent occupies 10-15% of the total, and 5-15%
At a concentration of 40 to 6
The mixture was heated to 5 ° C., and 15 to 40 parts of fat, 40 to 70 parts of saccharides mainly composed of dextrin and lactose, and a predetermined amount of vitamins were added and mixed with 10 to 15 parts of protein equivalent. Emulsified, heat sterilized by a conventional method,
It can be concentrated and spray-dried to obtain an anti-allergic formula.

The liquid antiallergic formula also includes
It can be manufactured similarly to powdered products. However, since the concentration of the final product is 5 to 40% of a liquid substance, it is 12 to prevent decay.
It is necessary to heat sterilize at a temperature of 0 ° C. or more, preferably 130 ° C. or more.

The anti-allergic formula of the present invention obtained as described above, as apparent from the test described below,
Excellent emulsifiability and heat stability, good flavor, effective in preventing and treating allergies due to reduced antigenicity, and also has excellent properties in amino acid balance and protein efficiency I have.

Next, the present invention will be described in detail with reference to test examples. Test Example 1 This test was performed to examine a combination of enzymes for obtaining a whey protein hydrolyzate having an antigenicity of 10 ⁻⁴ or less and having excellent emulsifiability and heat stability.

(1) Preparation of Sample A commercially available whey protein concentrate (hereinafter sometimes referred to as WPC; manufactured by Mirai) was dissolved in water at a concentration of 5%, and sodium hydroxide was added thereto to prepare a solution. The pH was adjusted to 9.0, and 5,000 units of the enzyme were added per gram of protein.
Decompose at 0 ° C for 5 hours, adjust the pH to 7.0 after decomposition,
Subsequently, the enzyme was inactivated by heating at 85 ° C. for 10 minutes, and cooled to obtain a sample.

When only one kind of enzyme was added, the amount of the enzyme was 5,000 units, when two kinds of enzymes were added, 2,500 units each, and when three kinds of enzymes were added, 1,667 units each.
Examples of the enzyme include papain (manufactured by Amano Pharmaceutical Co., Ltd.), bromelain (manufactured by Amano Pharmaceutical Co., Ltd.), protease N [Bacillus.
Derived from Bacillus subtilis. Amano Pharmaceutical Co., Ltd.]
Protease A [Aspergillus oryzae
soyzae). Amano Pharmaceutical Co., Ltd.] and bioprase [from Bacillus subtilis]. From Nagase & Co., Ltd., trypsin (from Novo), Alcalase [from Bacillus licheniformis]. Novo], actinase [Streptomyces
From Griseus (Streptomyces griseus). Kaken Pharma Inc.], PD enzyme [Penicilium Dupont (Peni
cillium duponti). Seishin Pharmaceutical Co., Ltd.] was used.

(2) Test method 1) Measurement of the amount of precipitate generated The sample was centrifuged at 1,000 G for 10 minutes, and the amount of precipitate (volume / volume) generated was measured.

2) Thermal stability test A sample was diluted to a concentration of 2%, dispensed into a test tube, and treated at 120 ° C. for 10 minutes using an autoclave.
The mixture was centrifuged at G for 10 minutes, and the amount of the resulting precipitate (volume / volume) was measured.

3) Measurement of residual antigen activity Elisa test method (Japanese Journal of Pediatric Allergy, Vol. 1,
(Page 36, 1987). A 96-well plate (manufactured by Nunc) is coated with whey protein, washed, and a mixture of rabbit anti-whey protein serum and a sample is supplied to the plate to react, and after washing,
An alkaline phosphatase-labeled goat anti-rabbit IgG antibody (Zeimed Laboratories) was reacted, washed, and added with sodium p-nitrophenyl phosphate.
After 30 minutes, 5N sodium hydroxide was added to stop the reaction, and the reaction product was measured with a microplate reader.

4) Emulsifiability test 100 g of vegetable fat (a mixture of coconut oil and palm oil; manufactured by Taiyo Yushi Co., Ltd.) was added to 1,000 ml of a sample, and 120 kgf / cm was passed through a homogenizer through a homogeneous valve.
^The emulsion was emulsified at a pressure of ² and the diameter of fat globules in the obtained emulsion was measured with a microscope to obtain an index of emulsifying property. Further, the emulsion was instantaneously heated at 100 ° C., and thereafter, the presence or absence of separation of the fat layer was visually observed to test the stability of the emulsion.

(3) Test Results The results of this test are as shown in Table 1. As is clear from Table 1, a combination of enzymes for obtaining a whey protein hydrolyzate which does not form a precipitate upon heating inactivation, has an antigenicity of 10 ^-4 or less, and is excellent in heat stability and emulsifiability. Is trypsin,
It has been found that three enzymes, papain and endoprotease derived from Bacillus subtilis (bioprase and protease N), are essential. Although not described in Table 1, in this test, all the above conditions could not be satisfied with a combination of enzymes other than the combination of trypsin, papain, and endo-type protease derived from Bacillus subtilis. The same test was conducted for other combinations of enzymes, but no combination of enzymes that satisfied all the above conditions was found.

[0045]

[Table 1] Test Example 2 This test was performed to determine the amount of enzyme in the combination of enzymes determined in Test Example 1.

(1) Preparation of Sample WPC was dissolved in the same manner as in Test Example 1, and the amounts of trypsin, papain, and bioprase (derived from Bacillus subtilis) were changed as shown in Table 2. The decomposition product was produced in the same manner as in Test Example 1 to obtain a sample.

(2) Test method By the same method as in Test example 1, the amount of precipitate formed, the thermal stability,
Antigen remaining activity and emulsifiability were tested.

(3) Test Results The results of this test are as shown in Table 2. As is clear from Table 2, trypsin, papain, and the like are not produced when heating is inactivated, do not form a precipitate, have an antigenicity of 10 ^-4 or less, and have excellent heat stability and emulsifiability. The range of the amount of the enzyme of bioprase is as follows. When the amount of papain is 1,000 units and the amount of trypsin is 2,000 units, the minimum amount of bioperase is 250 units or more, preferably 500 units or more. 50
When 0 units and trypsin are 2,000 units, papain is 500 units or more, preferably 1,000 units.
If the unit is more than 500 units and bioaplase is 500 units and papain is 1,000 units, trypsin is 1,000 units.
It turned out to be more than units, preferably more than 2,000 units.

The upper limit of the amount added per gram of protein is 5,000 units for papain and 2 units for trypsin.
In the case of 000 units, the amount of bioaplase is 3,000 units or less, desirably 2,000 units or less, the amount of bioprase is 2,000 units, and the amount of trypsin is 20,000.
In the case of a unit, papain is 6,000 units or less, desirably 5,000 units or less, bioaplase is 2,000 units, and papain is 5,000 units, trypsin is 30,000 units or less, desirably 20 units. 000
It was found to be less than the unit.

Therefore, in the method of the present invention, the amount of trypsin per gram of whey protein is 1,000 units or more, and
2,000 units or less, desirably 2,000 units or more,
000 units or less, papain 500 units or more, 6,000
Unit or less, preferably 1,000 to 5,000 units, and the endoprotease derived from Bacillus subtilis 250 to 3,000 units, preferably 5 to 3,000 units.
It has been found that the addition may be performed in a range of from 00 units to 2,000 units.

[0051]

[Table 2] Test Example 3 In this test, in addition to the combination and amount of the enzymes determined in Test Example 1 and Test Example 2, the amount of the enzyme to be added when other enzymes were added was examined.

(1) Preparation of Sample WPC was dissolved in the same manner as in Test Example 1, and 20,000 units of trypsin (manufactured by Novo), 5,000 units of papain (manufactured by Amano Pharmaceutical), and bioprase (manufactured by Nagase & Co., Ltd.) )
Added to 2,000 units, and further from protease A [from Aspergillus oryzae]. Amano Pharmaceutical] and bromelain (Amano Pharmaceutical) are added at 50 ° C.
For 5 hours, and then heated at 85 ° C. for 10 minutes to inactivate the enzyme, and then cooled to obtain a sample.

(2) Test method By the same method as in Test example 1, the amount of precipitate formed, the thermal stability,
Antigen remaining activity and emulsifiability were tested.

(3) Test Results The results of this test are as shown in Table 3. As is evident from Table 3, in addition to the predetermined amounts of trypsin, papain and bioprase, when adding other enzymes, the amount of addition of the enzymes is adjusted to 300 units or less, preferably 200 units or less. It became clear that heat stability and emulsifiability could be maintained. The same tests were conducted for other enzymes, but almost the same results were obtained.

Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples.

[0056]

【Example】

Example 1 1 kg of commercially available WPC (protein content: 76%, manufactured by Mirai)
Was dissolved in 9 kg of purified water, and 75
At 15 ° C. for 15 seconds, then adjust the pH of the solution to 9.0 by adding sodium hydroxide, 500 PUN units of biopulase (manufactured by Nagase Sangyo), 5,000 USP units of trypsin (manufactured by Novo) and 1 g of protein. Papain (manufactured by Amano Pharmaceutical Co., Ltd.) is added at a rate of 1,000 PUN units, and 50
Decomposed at ℃ for 5 hours. After the decomposition, the pH of the decomposition solution is 7.
Since it was 0, the enzyme was inactivated by directly heating at 90 ° C. for 20 minutes. Then, it was concentrated and dried by a conventional method to obtain about 1 kg of a powdery whey protein hydrolyzate.

The residual antigen activity of the obtained whey protein hydrolyzate was tested by the same method as in Test Example 1. As a result, it was 10 ^-4 and both heat stability and emulsifiability were good.

Example 2 3 kg of commercially available WPC (protein content 85%, manufactured by Danish Protein) was dissolved in 17 kg of purified water, sterilized with a plate-type sterilizer at 75 ° C. for 15 seconds, and then potassium hydroxide was added. The pH of the solution was adjusted to 8.0 with protein 1
Biopulase (made by Nagase & Co., Ltd.) 1,000 g per g
N units, 10,000 USP units of trypsin (manufactured by Novo), 2,000 PUN units of papain (manufactured by Amano Pharmaceutical) and 200 PUN of protease A Amano (manufactured by Amano Pharmaceutical)
It was added in a unit ratio and decomposed at 50 ° C. for 12 hours. Since the pH of the decomposed solution after the decomposition was 6.4, the pH was adjusted to 7.3 by adding sodium hydroxide, and then 2 hours at 130 ° C. and 85 ° C. for 5 minutes using a plate-type sterilizer. After heating for 2 seconds to deactivate the enzyme, it was concentrated by a conventional method and dried to obtain about 3 kg of a powdery whey protein hydrolyzate.

The residual antigen activity of the obtained whey protein hydrolyzate was tested by the same method as in Test Example 1. As a result, it was 10 ^-5 and both heat stability and emulsifiability were good.

Example 3 Commercially available WPC (protein content: 76%, manufactured by Mirai) 1.5
was dissolved in 8.5 kg of purified water, and potassium-type cation exchange resin (Diaion SK-1B, manufactured by Mitsubishi Kasei)
The solution was passed through 1 liter and lyophilized to prepare about 1.4 kg of ion-exchanged WPC powder. 1 kg of the obtained ion-exchange WPC powder and 1 kg of desalted whey powder (protein content: 14%, manufactured by Domo) are dissolved in 13 kg of purified water, sterilized at 80 ° C. for 10 minutes, and added with potassium carbonate to adjust the pH to 7. , And added at a ratio of 500 PUN units of protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.) per 1 g of protein, decomposed at 45 ° C. for 1 hour, and then trypsin (manufactured by Novo) 10.0,0
00USP units were added and digested for an additional 2 hours. Since the pH of the obtained decomposition solution was 6.5, the pH was adjusted to 7.5 by adding potassium hydroxide, and then 4,000 PUN units of papain (manufactured by Amano Pharmaceutical Co., Ltd.) were added.
Decomposed at ℃ for 3 hours. Since the pH of the decomposed solution after the decomposition was 6.8, the enzyme was inactivated by heating at 85 ° C. for 5 minutes and at 130 ° C. for 2 seconds using a plate-type sterilizer without adjusting the pH. . Subsequently, it was concentrated by a conventional method to obtain about 15 kg of a liquid whey protein hydrolyzate.

The residual antigen activity of the obtained whey protein hydrolyzate was tested by the same method as in Test Example 1. As a result, it was 10 ^-5 and both heat stability and emulsifiability were good.

Example 4 1 kg of commercially available casein lactate (purity: 85%, manufactured by New Zealand Daily Board) was dispersed in 19 kg of purified water.
The pH was adjusted to 7.0 by adding potassium hydroxide and 80
C. for 10 minutes to completely dissolve. Set the solution temperature to 5
After cooling to 0 ° C., the pH was adjusted to 10.0 by adding sodium hydroxide, and 1,500 PUN units of protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.), 500 PUN units of bioprase (manufactured by Nagase Sangyo Co., Ltd.) and 1 g of protein were added. Trypsin (manufactured by Novo) at a rate of 3,000 USP units and 5
The enzyme was decomposed at 0 ° C. for 12 hours, and then heated at 80 ° C. for 30 minutes to inactivate the enzyme. Then, the precipitate was removed by filtration through Celite using Hyflo Super Cell (manufactured by Manville), concentrated by a conventional method, dried, and powdered casein hydrolyzate (about 8) was obtained.
00 g was obtained. The residual antigen activity of the obtained casein hydrolyzate was tested by the same method as in Test Example 1, and the result was 10 ^-5 .

0.48 kg of the obtained casein hydrolyzate,
Whey protein hydrolyzate obtained by the same method as in Example 1.
2 kg was dissolved in 10 kg of purified water, a predetermined amount of minerals dissolved in 250 g of purified water was added, the mixture was heated to 60 ° C., and 2.5 kg of vegetable fat (manufactured by Taiyo Yushi Co., Ltd.) was added. 3. 1.0 kg of malt dextrin (manufactured by Showa Sangyo KK) dissolved in water at a concentration of 20% and lactose (manufactured by Domo KK)
2 kg of the mixture is added, and then a predetermined amount of vitamins are mixed. The mixture is emulsified by a high-pressure homogenizer, sterilized by a conventional method, concentrated, spray-dried, and powdered antiallergic. About 9 kg of prepared milk was obtained.

The antigen-remaining activity of the obtained anti-allergic formula was tested by the same method as in Test Example 1.
^-5 , the thermal stability and the emulsifiability are good, the ratio of whey protein hydrolyzate to protein equivalent is 70% and the ratio of casein hydrolyzate is 30%. It was as follows.

Protein 14.5 (%) Fat 26.5 Carbohydrate 53.5 (of which lactose 45.0) Ash 2.5 Moisture 3.0

Example 5 1 kg of commercially available sodium caseinate (purity 90%, manufactured by Unyle) was dissolved in 9 kg of purified water, heated at 75 ° C. for 15 seconds in a plate-type sterilizer, and then sodium hydroxide was added. To adjust the pH of the solution to 8.5, and the protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.)
000 PUN unit, trypsin (Novo Corporation) 5,000
USP unit and lactic acid bacterium crushed product (Morinaga Milk Industry Co., Ltd.) 5 mg were added, decomposed at 50 ° C. for 12 hours, cooled to 4 ° C., and ultrafiltration membrane having a molecular weight cutoff of 3,000 (manufactured by Asahi Kasei Corporation)
And ultrafiltration is performed using a plate-type sterilizer.
The mixture was heated at 5 ° C. for 5 minutes and at 130 ° C. for 2 seconds, then concentrated by a conventional method, dried, and powdered casein decomposition product (about 700 g)
Obtained. The antigen remaining activity of the obtained casein hydrolyzate was tested in the same manner as in Test Example 1 except that anti-whey protein serum was changed to anti-casein serum.
It was 0 ^-6 .

0.5 kg of the casein hydrolyzate obtained was combined with the whey protein hydrolyzate 1.0 obtained by the same method as in Example 2.
was dissolved in 50 kg of purified water, a predetermined amount of minerals dissolved in 1 kg of purified water was added, the mixture was heated to 60 ° C., and 2.5 kg of vegetable fat (manufactured by Taiyo Yushi Co., Ltd.) was added. A mixed solution prepared by dissolving 0.5 kg of dextrin (manufactured by Showa Sangyo) and 4.5 kg of lactose (manufactured by Domo) in 50 kg of purified water is added, and then a predetermined amount of vitamins are mixed.
This mixed solution was emulsified by a high-pressure homogenizer, sterilized by a plate sterilizer at 140 ° C. for 3 seconds, and aseptically filled in 200 ml portions to obtain 500 liquid anti-allergic prepared milk.

The antigen-remaining activity of the obtained anti-allergic formula was tested by the same method as in Test Example 1.
^-5.5 , heat stability and emulsifiability are good, the ratio of whey protein hydrolyzate to protein equivalent is 65% and the ratio of casein hydrolyzate is 35%. It was as follows.

Protein 1.05 (%) Fat 2.5 Carbohydrate 4.7 (Lactose 4.0) Ash 0.3 Moisture 82.0

Example 6 1 kg of commercially available casein lactate (purity: 85%, manufactured by New Zealand Daily Board) was dispersed in 9 kg of purified water.
The pH was adjusted to 7.0 by adding sodium hydroxide,
Heating at 0 ° C. for 10 minutes completely dissolved. The temperature of the solution was cooled to 50 ° C., and the ratio of pancreatin (manufactured by Amano Pharmaceutical Co., Ltd.) 3,000 PUN units, protease A Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 100 PUN units, and crushed lactic acid bacteria (manufactured by Morinaga Milk Industry Co., Ltd.) 5 mg / g of protein At 50 ° C. for 20
After decomposing for a time, the enzyme was inactivated by heating at 95 ° C. for 10 minutes. Then, the mixture was cooled to 4 ° C., 50 g of activated carbon (trade name “Shirasagi”, manufactured by Takeda Pharmaceutical Co., Ltd.) was added, and the mixture was allowed to stand for 12 hours. And concentrated by a conventional method, and dried to obtain a powdered casein hydrolyzate of about 700 g.
I got The antigen remaining activity of the obtained casein hydrolyzate was tested in the same manner as in Test Example 1 except that anti-whey protein serum was changed to anti-casein serum.
It was 0 ^-4 .

0.65 kg of the obtained casein hydrolyzate,
Whey protein hydrolyzate obtained by the same method as in Example 1.
0 kg was dissolved in 20 kg of purified water, a predetermined amount of minerals dissolved in 1 kg of purified water was added, the mixture was heated to 60 ° C., and 2.5 kg of vegetable fat (manufactured by Taiyo Yushi Co., Ltd.) was added.
Further, 1.0 kg of malt dextrin (manufactured by Showa Sangyo KK) dissolved at a concentration of 20% and 4.2 kg of lactose (manufactured by Domo KK)
And then a predetermined amount of vitamins are mixed, and the mixture is emulsified by a high-pressure homogenizer, sterilized by a conventional method, concentrated, spray-dried, and powdered anti-allergic formula. Was obtained in an amount of about 9 kg.

The antigen-remaining activity of the obtained anti-allergic formula was tested by the same method as in Test Example 1.
^-4.5 , good thermal stability and good emulsifiability, the ratio of whey protein hydrolyzate to protein equivalent is 60% and the ratio of casein hydrolyzate is 40%. It was as follows.

Protein 13.5 (%) Fat 26.5 Carbohydrate 55.5 (of which lactose 45.0) Ash 2.5 Moisture 2.0

[0074]

The effects of the present invention are as follows. 1) A whey protein hydrolyzate for producing an anti-allergic prepared milk with significantly reduced antigen residual activity is obtained. 2) A whey protein hydrolyzate for the production of an antiallergic milk formula having extremely good heat stability and emulsifying properties is obtained. 3) An anti-allergic formula with significantly reduced antigen residual activity is obtained. 4) An anti-allergic milk formula having extremely good heat stability and emulsifying properties can be obtained.

[Table 3]

Continuation of the front page (51) Int.Cl. ⁷ Identification code FI A23L 1/305 A23L 1/305 (72) Inventor Yasushi Kawaguchi 118 Minami-Kihogaoka, Asahi-ku, Yokohama-shi, Kanagawa 118 Mori Nagamogaoka dormitory (72) Inventor Yoko Akasemi 2-2-5-504, Sabudai, Sagamihara-shi, Kanagawa Prefecture (72) Inventor Hiroshi Ochi 118 Minami-Kihogaoka dormitory, Asahi-ku, Yokohama-shi, Kanagawa Morinaga Noborigaoka dormitory (72) Inventor Shigefumi Kitagawa 5-19-5, Minamidai, Sagamihara-shi, Kanagawa-do Me Odakyu Sagamihara 312 (56) References JP-A-5-209000 (JP, A) JP-A-4-248959 (JP, A) JP-A-4-365444 (JP, A) (58) Fields studied (Int) .Cl. ⁷ , DB name) A23C 9/15 A23C 21/00 A23J 3/08-3/34 A23L 1/305 JICST file (JOIS) JAFIC file (JOIS)

Claims (2)

(57) [Claims] 1. A whey protein having a purity of at least 30% (by weight) is dissolved in water at a concentration of 5% to 20% by weight, and the pH of the solution is 7.5 to 10.0. adjusted to, whey protein 1g per Bacillus subtilis (Bacil
lus subtilus) -derived endoprotease 500-
2,000 PUN units , trypsin 2,000-20,
000 USP units and papain 1,000-5,000
ELISA with anti-whey protein sera, which comprises hydrolyzing by including enzymes three enzymes PUN units (ELISA: Enzyme linked immunosolbent assay) antigen was determined by inhibition test method residual activity ^10-4 A method for producing a whey protein hydrolyzate having excellent emulsifiability and heat stability as follows. 2. A whey protein having a purity of at least 30% (by weight) is dissolved in water at a concentration of 5% to 20% by weight, and the pH of the solution is 7.5 to 10.0. adjusted to, whey protein 1g per Bacillus subtilis (Bacil
lus subtilus) -derived endoprotease 500-
2,000 PUN units , trypsin 2,000-20,
000 USP units and papain 1,000-5,000
An ELISA using an anti-whey protein serum obtained by hydrolysis with an enzyme containing three kinds of enzymes of PUN units (ELI
SA: Enzyme linked immunosolbent assay) 40 to 100% (by weight) and at least 8 whey protein hydrolysates having a residual antigen activity of 10 ^-4 or less as measured by the inhibition test method.
Obtained by hydrolyzing casein of 0% (weight) purity,
The amount of fat per 10 to 15 parts of the nitrogen source protein consisting of a mixture of casein hydrolyzate having a residual antigen activity of 10 ^-4 or less and 60 to 0% (weight) measured by the ELISA test using anti-casein serum. An anti-allergic formula having excellent emulsifiability and heat stability comprising a composition of 15 to 40 parts and 70 to 40 parts of carbohydrate.