JPS61233A - Production of water-soluble hydrolyzate of whey protein - Google Patents

Abstract

PURPOSE:To obtain a high-molecular, protein-free, water-soluble hydrolyzate of whey protein easily in high yields, by hydrolyzing an aqueous whey protein solution with a neutral protease, acidifying the solution with an acid, heating it and removing the formed precipitate. CONSTITUTION:Whey protein is prepared by condensing and drying whey formed by removing casein from milk in a process for producing a milk product such as cheese. An aqueous solution of this protein is hydrolyzed with a neutral protease (e.g., fungous protease from the genus Aspergillus) at a pH of 5-11. The pH of the resulting aqueous hydrolyzate solution is adjusted to 2-4 by the addition of an acid, and the formed precipitate is removed to obtain the purpose water-soluble hydrolyzate of whey protein. The obtaned hydrolyzate is a low- molecular one containing at least 50% di- and tri-peptides and can be suitably used as a nutrient for tubal feeding or the like.

Description

Detailed Description of the Invention ■1 Background of the Invention, Technical Field The present invention removes undegraded proteins and high molecular moieties in the hydrolyzate from an enzymatic hydrolyzate of whey protein, and at the same time removes the aggregation of the low molecular weight hydrolyzate. suppresses insolubilization and precipitate formation due to
An improved method for producing a water-soluble hydrolyzate of whey protein in high yield.

Whey protein is a protein mainly containing lactoglobulin and lactalbumin, and although it has a slightly low level of phenylalanine as an essential amino acid, it contains a good balance of other essential amino acids and has high nutritional value.

Whey protein is obtained as a residue after recovering casein from milk during the manufacturing process of dairy products such as cheese, and is mainly used as livestock feed and pasture fertilizer.

Recently, attempts have also been made to use whey protein hydrolyzate for tube nutrition and general nutritional supplements, and various hydrolysis methods have been reported (Japanese Patent Application Laid-open Nos. 55-124458 and 57-1).
94753).

Prior art When a hydrolyzate of whey protein is used as a tube nutrient or as a nutritional supplement for cases of maldigestion and absorption, it is necessary to use hydrolyzate that is easily hydrolyzed to amino acids or di- or trihaptides in which two or three of them are bonded. It is advantageous for the protein to be fully digested and absorbed by the body. Therefore, in order to obtain such a low-molecular hydrolyzate, the degree of hydrolysis is appropriately controlled, but with this operation alone, a considerable amount of undegraded protein and high molecular weight portions of the hydrolyzate are lost. be caught. Removal of these polymeric substances has conventionally been carried out by heating the enzyme ice-thawing mixture as it is to denature it and remove the resulting precipitate. However, in the case of whey protein,
In conventional methods, high molecular moieties and insoluble proteins form colloids in the purified solution, which cannot be completely removed, and a portion of the desired low molecular weight hydrolyzate may undergo denaturation such as aggregation upon heating, resulting in insolubilization and There is a phenomenon in which the recovery rate decreases due to the formation of precipitates.

■0 Purpose of the Invention The present invention efficiently removes high-molecular proteins from whey protein enzymatic hydrolyzate with a simple operation, while suppressing insolubilization or precipitate formation due to aggregation of low-molecular-weight hydrolyzates, resulting in a high yield. An object of the present invention is to provide a method for producing a whey protein hydrolyzate.

■0 Detailed Description of the Invention The present invention involves hydrolyzing a whey protein aqueous solution at pH 5 to 11 using a neutral protease, adding an acid to the resulting hydrolyzate aqueous solution to adjust the pH to 2 to 4, and then converting the solution to a pH of 2 to 4. The present invention provides a method for producing a water-soluble whey protein hydrolyzate, which comprises heating and removing the generated precipitate.

The present invention also provides a method for producing a water-soluble whey protein hydrolyzate, wherein the neutral protease is a fungal protease belonging to the genus Rugillus.

Furthermore, the present invention provides a method for producing a water-soluble whey protein hydrolyzate, wherein the water-soluble whey protein hydrolyzate is a low molecular weight hydrolyzate containing at least 50 qb of di- and tripeptides.

The whey protein used as a raw material in the method of the present invention is obtained by concentrating and drying the so-called milk whey after casein is removed from milk in the manufacturing process of dairy products such as cheese. Crude protein powder containing albumin and lactoglobulin as main components. The aqueous solution of this product is a pale yellow-white colloid with extremely high emulsifying properties, making it difficult to extract lipids using chloroform or methanol solutions or to separate proteins using TCA or sulfosalicylic acid.

In the method of the present invention, it is necessary to enzymatically hydrolyze whey proteins under conditions of pH 5 to 11, and therefore, as the hydrolyzing enzyme, a neutral protease having activity at the pH level Q is used.

As the neutral protease, a fungal protease belonging to the genus Rugilus is most preferred, but enzymes derived from animals or plants such as pancreatin and papain can also be used.

Enzymatic hydrolysis is carried out by methods known per se.

For example, in a whey protein aqueous solution, whey protein and enzyme 2 to
8 chloride was added and the reaction was carried out at pH 5 to 11.40 to 60°C for about 1 to 10 hours. After the ice has been thawed, an acid is added to the reaction solution to adjust the pH to 2 to 4, followed by heating at 80 to 100°C for 1 minute to 1 hour. Phosphoric acid, hydrochloric acid, sulfuric acid, etc. are suitable as the acid used for pH adjustment, but phosphoric acid It is advantageous to use , sulfuric acid, etc. The above heating inactivates the enzyme and precipitates undegraded proteins and high molecular parts in the hydrolyzate. Also, the above heating deactivates the low molecules produced by hydrolysis. Insolubilization and precipitate formation due to aggregation of hydrolyzate can be suppressed.

After heating, it is desirable to adjust J)H to neutrality by adding a base such as calcium hydroxide, sulfuric acid, or sodium hydroxide to the aqueous solution. The precipitate that has formed up to this point is removed, for example, by centrifugation or filtration.

Furthermore, a water-soluble protein is obtained from the aqueous solution obtained here by a conventional method (for example, by concentration drying).

The protein thus obtained is a completely digested water-soluble whey protein low-molecular hydrolyzate with a high content of di- and tripeptides, and is highly digestible and absorbable in the intestine. In the present invention, the term "aqueous" or "aqueous solution" includes both true solutions and colloidal solutions.

Next, the method of the present invention will be explained in more detail with reference to Examples.

Example 1 Whey protein Kitamine A (manufactured by Wakodo Co., Ltd.) 5? was dissolved in 80 ml of water, and a solution of 0.3 t of Amano A (Aspergillus fungal protease, manufactured by Amano Pharmaceutical Co., Ltd.) dissolved in 20 ml of water was added and incubated at 50°C for 6 hours.

The pI was adjusted to 5 with phosphoric acid while cooling on ice. After heating in a boiling water bath for 1 hour, it was allowed to cool to room temperature. This solution was neutralized to pH 7 with calcium hydroxide and heated at 3000 r, p, m,
The mixture was centrifuged for 15 minutes to obtain a clear supernatant.

The turbidity (absorption at 660 am) of this supernatant was 0 after dilution 5 times.
．． It was 14. The average molecular weight of the hydrolyzate in this supernatant was 207, and the recovery rate was 92.7%.

Therefore, the centrifuged semen obtained by the above procedure was found to be a water-soluble whey protein low molecular weight hydrolyzate solution.

Example 2 Orientase IDN (derived from Bacillus subtilis, manufactured by Ueda Chemical Industry Co., Ltd.) 0.05P was used instead of Amano A (Aspergillus fungal protease, manufactured by Amano Pharmaceutical Co., Ltd.) used in Example 1 as a protease. , the same operation as in Example 1 was performed.

Turbidity of the resulting centrifuged supernatant (absorbance at 66 onm)
was 17 at a 5-fold dilution. The average molecular weight of the hydrolyzate in this supernatant was 983, and the recovery rate was 93.0%.

Example 3 As a protease, Amano A (Aspergillus fungal protease, manufactured by Ohno M Pharmaceutical Co., Ltd.) used in Example 1 was added to vancreatin (porcine pancreatic juice, manufactured by Soiko Pure Chemical Industries, Ltd.) 0
．． The same operation as in Example 1 was performed using No. 59. The turbidity (absorbance at 660 nm) of the resulting centrifuged supernatant was 5
The dilution was 0.08. The average molecular weight of the hydrolyzate in this supernatant was 650, and the recovery rate was 71.9%.

Example 4 As a protease, 0.5 L of Proleather (As is a fungal protease of the genus Rugillus, manufactured by Amano Pharmaceutical Co., Ltd.) was used instead of Ama/A (fungal protease of the genus Aspergillus, manufactured by Amano Pharmaceutical Co., Ltd.) used in Example 1. The same operation as in Example 1 was performed using . The turbidity of the resulting centrifuged supernatant (6
The absorbance at 6° nm was 0.16 at 5-fold dilution.

The average molecular weight of the hydrolyzate in this supernatant was 529, and the recovery rate was 8B.

(Comparative example) Whey protein Kitamine A (manufactured by Wakodo Co., Ltd.) 25? After dissolving it in 1 t of water, divide it into 250-4 equal parts, each with 80
0 units of Amano A (as is a fungal protease belonging to the genus Rugilus, manufactured by Amano Pharmaceutical), Orientase 1ON (protease derived from Bacillus subtilis, manufactured by Ueda Chemical Industries), Proleather (as is fungal protease belonging to the genus Rugilus, manufactured by Amano Pharmaceutical) , Nocuncreatin (derived from pig pancreatic juice, manufactured by Wako Tsumugi Pharmaceutical)
was added and incubated at 50°C for 6 hours. Dispense each reaction solution into 10-unit portions while cooling each reaction solution with ice.
The pH was adjusted to 2, 3, 4 and 6 (unadjusted) with phosphoric acid. After heating in a boiling water bath for 20 minutes, 3000 r,
The mixture was centrifuged for 15 minutes at P and M, and the turbidity (absorbance at 660 nm) of the supernatant was measured. Although some differences were observed depending on the type of enzyme, insoluble colloid-like substances were removed by heating at pH 2 to 4. Furthermore, Table 1 shows the average molecular weight and recovery rate of the melted ice in the pH 1 supernatant liquid showing the lowest turbidity value for each enzyme. As shown in Table 1, the lowest average molecular weight was obtained with Amano A (as is a fungal protease belonging to the genus Rugilus, manufactured by Ohno Pharmaceutical Co., Ltd.), and the recovery rate at this time was also high.

Table 1 Amano A 3 207 92.7 Orientase ION 4 854 68.1 Proleather 2 430 75.9 Pancreatin 2 409 90.0 Hydrolyzed with Amano A (Aspergillus fungal protease, manufactured by Daikei Pharmaceutical Co., Ltd.) At this time, the centrifuged supernatant after heat treatment in p) 13 and the centrifuged semen after heat treatment without pH adjustment,
Sephadex G-2 equilibrated with 6M guanidine hydrochloride
The figure shows the absorbance at 280 nm when gel filtration was performed with 5 columns. As shown in the figure, < s pH
Compared to the centrifuged supernatant obtained by heat treatment without preparation, the pH
In the centrifuged supernatant obtained by heat treatment in step 3, a marked decrease in the high molecular weight fraction and an increase in the low molecular weight fraction were observed.

0 Effects of the Invention As detailed above, according to the method of the present invention, high molecular weight proteins and their insoluble colloid-like substances can be removed efficiently (high molecular weight proteins and their insoluble colloid-like substances) from whey protein enzyme hydrolyzate with simple operations. On the other hand, it is possible to prevent insolubilization and precipitate formation due to aggregation of low-molecular-weight hydrolysates and recover them, and to obtain a water-soluble whey protein hydrolyzate at a high yield.In other words, a whey protein aqueous solution can be neutralized. pi using protease (p by adding acid to the aqueous solution of the hydrolyzate obtained by hydrolysis in steps 5 to 11)]
2 to 4, heating the solution, and removing the generated precipitate, it is possible to obtain a water-soluble whey protein hydrolyzate with extremely low content of undegraded proteins and high molecular weight peptides at a high yield. can.

Further, in the present invention, when a fungal proteacea belonging to the genus Aspergillus is used as the neutral protease, a water-soluble whey protein hydrolyzate having an extremely high content of free amino acids or di- and tripeptides can be obtained.

Therefore, the water-soluble whey protein low-molecular-weight hydrolyzate obtained by the method of the present invention is well absorbed from the intestinal tract and is excellent as a tube nutritional supplement or a nutritional supplement for cases of poor digestion and absorption.

[Brief explanation of the drawing]

The figure shows the gel filtration pattern of the centrifuged supernatant of an aqueous hydrolyzate solution. In the figure, the solid line indicates pH 5, and the dotted line indicates the gel filtration pattern of the number of supernatants obtained by heating and centrifuging the hydrolyzate aqueous solution without pH adjustment.

Claims (3)

[Claims] (1) Whey protein aqueous solution is purified using neutral protease.
A water-soluble whey protein hydrolyzate characterized by hydrolyzing with H5-11, adding an acid to the resulting aqueous solution of the hydrolyzate to adjust the pH to 2-4, heating the solution, and removing the generated precipitate. Production method. (2) The method for producing a water-soluble whey protein hydrolyzate according to claim 1, wherein the neutral protease is a fungal protease belonging to the genus Aspergillus. (3) The method for producing a water-soluble whey protein hydrolyzate according to claim 1, wherein the water-soluble whey protein hydrolyzate is a low molecular weight hydrolyzate containing at least 50% of di- and tripeptides.