JP3092870B2 - Method for producing milk whey protein hydrolyzate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for efficiently producing a milk whey protein hydrolyzate using a plant-derived proteolytic enzyme. According to the method of the present invention, β-lactoglobulin is selectively hydrolyzed, so that a hydrolyzate of milk whey protein with reduced allergen can be provided.

[0002]

2. Description of the Related Art Milk whey protein contains β-lactoglobulin as a main component, and proteins such as α-lactalbumin, immunoglobulin and bovine serum albumin. All of these proteins are allergens, albeit to varying degrees. In particular, β-lactoglobulin is not present in breast milk and is a strong allergen for allergic patients.

Hitherto, as a method for reducing the allergen of milk whey protein, a hydrolysis treatment using a proteolytic enzyme has been known. In particular, for the enzymatic hydrolysis and removal of β-lactoglobulin which is a strong allergen, a method of selectively decomposing by using a specific enzyme or a method of removing it by applying other treatment means is known.

[0004] For example, Food S of Hayashi et al.
ci. , 52, 1107 (1987), taking advantage of the difference in the susceptibility of β-lactoglobulin and α-lactalbumin to pressure denaturation under ultra-high pressure, and using the thermolysin (Daiwa) as a proteolytic enzyme under ultra-high pressure of 200 MPa. (Chemical Co., Ltd., Thermolysin)
A method for selectively decomposing and removing lactoglobulin is described.

Japanese Patent Application Laid-Open No. 2-265441 discloses bovine trypsin (Sigma, T-8003).
Bacillus subtilis-derived proteolytic enzyme (NOVO, Neut)
case) and Aspergillus oryzae ( Asperg)
illus oryzae ) (Amano Pharmaceutical Co., Ltd., Protease A).
A method for selectively decomposing β-lactoglobulin by treating at 7 to 9, 30 to 40 ° C. for 30 minutes to 20 hours is described.

[0006] Further, in Japanese Patent Application Laid-Open No. 3-19654,
A method is described which uses a strong base type anion exchanger to remove lactoglobulin from a starting material containing whey protein.

[0007] The selective decomposition or selective removal of β-lactoglobulin by the above-mentioned method can be performed, for example, by using β-lactoglobulin under high pressure.
-The selective degradation of lactoglobulin is difficult to process in large quantities, the proteolytic enzyme used is expensive, and a high-pressure device must be used, resulting in an increase in cost.

[0008] Bovine trypsin, a proteolytic enzyme derived from Bacillus subtilis, Aspergillus oryzae ( Asper)
selective degradation of β-lactoglobulin using a proteolytic enzyme derived from H. gillus oryzae increases the cost because the substrate concentration is as low as 1%, and degrades β-lactoglobulin due to treatment at a substrate concentration of 1%. Progresses, the decomposition of α-lactalbumin also proceeds, and as a result, β having a low α-lactalbumin content
-It becomes a selective degradation product of lactoglobulin. Furthermore, the degraded products obtained by using these enzymes do not have sufficiently reduced allergenicity.

Removal of lactoglobulin from a starting material containing whey protein using a strong base type anion exchanger results in a low yield and an increase in cost.

[0010]

SUMMARY OF THE INVENTION The present inventors selectively hydrolyze β-lactoglobulin contained in milk whey protein to reduce allergenicity and to increase the milk content of α-lactalbumin. Various studies were conducted on methods for producing whey protein hydrolyzate, and the concentration of milk whey protein was reduced to 5% by using bromelain and papain, which are plant-derived proteolytic enzymes.
It has been found that the hydrolysis can be increased to about 20% by weight, and the present invention has been accomplished.

Accordingly, an object of the present invention is to provide a method for efficiently producing a milk whey protein hydrolyzate using a plant-derived proteolytic enzyme.

[0012]

That is, the present invention relates to a method of adding β-protein in milk whey protein by adding a plant-derived proteolytic enzyme to an aqueous solution of milk whey protein adjusted to a milk whey protein concentration of 5 to 20% by weight. The present invention relates to a method for selectively decomposing lactoglobulin to produce an enzyme hydrolyzate of milk whey protein having low allergenicity. More specifically, a 5-20% milk whey protein aqueous solution is
Adjust the pH to 7-9 with OH, KOH or Ca (OH) ₂ ,
20-500 u / g-W of plant-derived proteolytic enzyme
PC was added and treated at 40 to 60 ° C. for 30 minutes to 12 hours.
After completion of the hydrolysis, a method in which the enzyme is inactivated by heating with a plate heat exchanger and dried with a freeze dryer or a spray dryer to obtain a milk whey protein enzyme hydrolyzate having low allergenicity and a high concentration of hydrolyzate. About.

The enzyme in the present invention is a plant-derived proteolytic enzyme, and examples of such enzymes include bromelain (Amano Pharmaceutical Co., Ltd., Bromelin F) and papain (Amano Pharmaceutical Co., Ltd., PapainW). Can be. One or two or more enzymes can be used, and when multiple enzymes are used, a combination of a plant-derived proteolytic enzyme, bromelain or papain, and another proteolytic enzyme derived from a source other than a plant is used. Is valid.

The enzymatic reaction is carried out by adjusting the aqueous solution containing 5 to 20% by weight of milk whey protein to pH 7 to 9 as described above, adding the proteolytic enzyme thereto, and
For 30 minutes to 12 hours.

In the conventional method, proteolytic enzyme treatment was performed with a solution containing 1% by weight of milk protein. However, in the present invention, the use of the above-mentioned proteolytic enzyme enables efficient and high concentration of milk protein. An enzymatic degradation product can be obtained. This is one of the major features of the present invention.

Inactivation of the enzyme after the completion of the enzymatic decomposition can be carried out for enzymes such as bromelain and papain by heating at 125 ° C. for 5 seconds using a plate type heat exchanger without heating α-lactalbumin. As a result, the enzyme is deactivated, and a decomposition product can be obtained. The resulting solution can be used as it is, or can be used as a raw material for allergen-reduced food after being concentrated or dried and powdered. According to the method of the present invention, β-lactoglobulin is selectively decomposed, and a milk whey protein hydrolyzate in which α-lactalbumin accounts for 50% or more of the starting material can be obtained. The milk whey protein obtained by the method of the present invention uses the fact that the ratio of α-lactalbumin and β-lactoglobulin has a difference in their molecular weights to determine their molecular weight distribution by Swergo.
ld et al. [Analytical Biochem.
issue, 131, 295 (1983)].

The molecular weight distribution can be measured under the following conditions. (1) Sample concentration: 0.05% (2) Injection volume: 20 μl (3) Column: TSKgel G3000PW _XL (4) Solvent: 55 containing 0.1% trifluoroacetic acid
% Acetonitrile solution (5) Solvent speed: 0.30 ml / min (6) Detection wavelength: 210 nm (7) Analysis temperature: 20-30 ° C

The allergenicity of the whey protein hydrolyzate can be determined by the Inhibition ELISA test [Journal of the Japanese Society of Pediatric Allergy, 1, 36 (1987)] or M
Ota et al. Antigen-antibody reaction by PCA (passive skin anaphylaxis) [LifeScience 8 813
(1969)].

Inh using β-lactoglobulin as an index
The solution and the like used in the ibition ELISA test can be prepared as follows.

(1) Coating of β-lactoglobulin: 0.05M NaHCO ₃ -Buffer 11m
1 μl of β-lactoglobulin (1 mg / ml)
Dissolve and dispense (100 μl / well). (2) Sample: 200 ml of sample is 1 ml in PBS
Dissolve in (3) Goat whole serum dilution: 34 μl of goat whole serum in PBS
Dissolve in 5.1 ml of tween20. (4) Anti-β-lactoglobulin (β-Lg) rabbit serum diluent: 34 μl of anti-β-Lg rabbit serum was added to PBS-tw
Dissolve in 5.1 ml of een20. (5) Peroxidase-labeled anti-rabbit immunoglobulin (IgG) goat IgG: 3 μl of peroxidase-labeled anti-rabbit IgG goat IgG was added to PBS-tween20.
Dissolve in 10 ml. (6) ABTS solution: ABTS (2,2'-Azino
・ Bis-3-ethyl benzthiazoli
nesulfonic acid) was dissolved in 5 ml of deionized water and 0.006% H ₂ O ₂ −0.2
M Na citrate buffer, pH 4.0 (Buf for ABTS)
fer) 5 ml.

The preparation of antiserum in the antigen-antibody reaction by PCA and the determination by PCA can be performed as follows.

(1) Preparation of antiserum: Al (OH) ₃
Disperse 0mg into 200ml PBS and sterilize 20m
The reaction solution was diluted with PBS so that 1 and the protein equivalent would be 10 mg / ml.
20 ml of the solution, which was made up to 1 l with shaking and mixing 4
00 μl (containing 20 μg as an antigen) was intraperitoneally administered to BALB / c mice (5-week-old male) bred for 11 days in five divided doses at weekly intervals over 4 weeks. Five days after the fifth administration, the base of the thigh was cut and whole blood was collected.
Stored at 0 ° C.

(2) Judgment by PCA: 1/2 dilution of mouse anti-α-lactalbumin serum, mouse anti-β-lactoglobulin serum, and mouse anti-peptide serum obtained by preparing the above antiserum using physiological saline. Column (1/10, 1/2
0, 1/40, 1/80, 1/100), and 50 μl of each diluted serum is injected subcutaneously into the back of a shaved SD rat (10-week-old male). Twenty-four hours later, 1.0 ml of a 0.6% Evans blue solution containing each peptide solution (containing 1 mg as an antigen) was injected from the tail vein, and sacrificed 30 minutes later. For the determination, the maximum dilution ratio at which a positive reaction was observed was defined as the antibody titer.

Now, the present invention will be described in further detail with reference to Examples.

Example 1 100 g of milk whey protein and 800 g of water
The solution was adjusted to pH 8 with Ca (OH) ₂ , and a 10% solution was obtained by adding 100 u / g-WPC to bromelain, the pH was kept constant at 8 during the reaction time, and the enzyme treatment was carried out at 45 ° C. for 4 hours. After 4 hours, 125 in a plate heat exchanger
The enzyme was inactivated by heating to 5 ° C. for 5 seconds and freeze-dried to obtain a milk whey protein hydrolyzate. TSKgelG30
The molecular weight distribution of this decomposed product was measured using a 00P _XL column (Tosoh Corporation) and a 55% acetonitrile solution containing 0.1% trifluoroacetic acid. As shown in FIG. 1, undegraded whey protein was observed. Β-lactoglobulin was decomposed as compared with that of Example 1, and α-lactalbumin was able to obtain a decomposition product of 50% or more of the starting material.

[0025]

Example 2 100 g of milk whey protein and 800 g of water
, Adjusted to pH 8 with Ca (OH) ₂ , added 100 u / g-WPC of papain dissolved in water, further added water to make a 10% solution, and kept the pH at 8 during the reaction time, 45 ° C. For 4 hours. After 4 hours, the enzyme was inactivated by heating at 125 ° C. for 5 seconds using a plate heat exchanger, and the mixture was freeze-dried to obtain a milk whey protein hydrolyzate. When the molecular weight distribution of the decomposition product was measured, β-lactoglobulin was decomposed as compared with the undecomposed whey protein as shown in FIG. I got it.

[0026]

[Comparative Example 1] 100 g of milk whey protein and 800 g of water
And adjusted to pH 7 with Ca (OH) ₂ , and dissolved in water Neutrase (NOVO, Neutrase)
l. 5MG) was added at 100 u / g-WPC, and water was further added to make a 10% solution, followed by enzyme treatment at 40 ° C. for 4 hours.
After 4 hours, the enzyme was inactivated by heating at 125 ° C. for 5 seconds in a plate heat exchanger, followed by freeze-drying to obtain a milk whey protein hydrolyzate. When the molecular weight distribution of the decomposed product was measured, β-lactoglobulin and α-lactalbumin were not decomposed compared to the undecomposed whey protein, and α-lactalbumin was used as the starting material 5 as shown in FIG.
0% or more of the decomposition product could not be obtained.

[0027]

[Comparative Example 2] 100 g of milk whey protein and 800 g of water
, Adjusted to pH 7.5 with Ca (OH) ₂ , and dissolved in water with Protease A (Amano Pharmaceutical Co., Ltd., Prote
aseA) was added at 100 u / g-WPC, and water was further added to make a 10% solution, followed by enzyme treatment at 40 ° C. for 4 hours.
After 4 hours, the enzyme was inactivated by heating at 125 ° C. for 5 seconds in a plate heat exchanger, followed by lyophilization to obtain a decomposed product. When the molecular weight distribution of the decomposed product was measured, β-lactoglobulin was decomposed as compared with the undecomposed whey protein as shown in FIG. 4, but α-lactalbumin contained 50% or more of the decomposed product of the starting material. Could not get.

The degradation products obtained by the above (Example 1), (Example 2), (Comparative Example 1) and (Comparative Example 2) were evaluated using β-lactoglobulin as an index.
n ELISA tests were performed. The result is shown in FIG.
This figure shows that the greater the distance from the β-lactoglobulin curve, the lower the antigenicity of the degradation product. As shown in the figure, bromelain and papain showed a remarkable decrease in antigenicity as compared with Neutrase and Protease A.

When the degradation products obtained by (Example 1), (Example 2) and (Comparative Example 2) were determined by PCA, undegraded whey protein was determined as shown in FIGS. In comparison, protease A remained allergenic, while bromelain and papain exhibited reduced allergenicity.

Further, (Example 1) and (Example 2)
The raw material cost including the enzyme was low, the yield was almost 100%, and the sensory evaluation showed little bitterness.

[0031]

Industrial Applicability According to the present invention, β-lactoglobulin contained in milk whey protein in a large amount is selectively hydrolyzed by a plant-derived protease, and α-lactalbumin is enriched in milk whey protein. A hydrolyzate can be obtained. The resulting decomposed product can be effectively used as a low-allergen food.

[Brief description of the drawings]

FIG. 1 shows the molecular weight distribution of a milk whey protein hydrolyzate by bromelain of Example 1.

FIG. 2 shows the molecular weight distribution of the milk whey protein hydrolyzate from papain of Example 2.

FIG. 3 shows the molecular weight distribution of a milk whey protein hydrolyzate by the neutrase of Comparative Example 1.

FIG. 4 shows the molecular weight distribution of a milk whey protein hydrolyzate by protease A of Comparative Example 2.

FIG. 5 shows Inhibi of Examples 1 and 2 and Comparative Examples 1 and 2.
3 shows the results of the Tion ELISA test.

FIG. 6 shows the results of an immunogenicity evaluation by PCA in Example 1.

FIG. 7 shows the results of immunogenicity evaluation using PCA in Example 2.

FIG. 8 shows the results of immunogenicity evaluation of Comparative Example 1 by PCA.

FIG. 9 shows the results of immunogenicity evaluation of Comparative Example 2 by PCA.

────────────────────────────────────────────────── ─── Continuing on the front page (72) Tetsuro Nakamura, Inventor 580-5 Shimofujisawa, Iruma City, Saitama Prefecture (72) Nobuaki Takahashi 5-11-3, Shinjuku-cho, Kawagoe-shi, Saitama ) Inventor Masaharu Shimatani 3-1-2, Shinsayama, Niiyama, Sayama, Saitama 303 (72) Inventor Kenichi Hirano 212-11, Inari, Inaricho, Iwakura, Aichi, Japan 221 Inaricho (56) References JP-A-2-265441 (JP, A) JP-A-4-117275 (JP, A) JP-A-5-17368 (JP, A) Tokuyo Sho 62-501472 (JP, A) A) (58) Field surveyed (Int. Cl. ⁷ , DB name) A23J 3/08-3/34 A23L 1/305 C12P 21/06 BIOSIS (DIALOG)

Claims (2)

(57) [Claims] To 1. A milk whey protein aqueous solution adjusted to a concentration 5 to 20 wt%, whey protein degrading enzymes derived from plants
-Add 20-500u per 1g of protein and process at 40-60 ° C
And sense, alpha-lactalbumin, characterized in that selectively hydrolyze β- lactoglobulin in said protein
Producing how milk whey protein hydrolyzate residual ratio is 50% or more. 2. A method for producing a proteolytic enzyme derived from plant, milk Hoetanpa click hydrolyzate according to claim 1 which is bromelain and / or papain.