JPS62224245A - Production of easily soluble hydrolyzed milk protein free from unagreeable taste - Google Patents

Abstract

PURPOSE:To obtain the titled hydrolyzed product suitable as a raw material for food or drink, by hydrolyzing a milk protein (-containing material) in an alkaline solution with pancreatin and a microbial protease derived from a strain belonging to Bacillus genus and separating insoluble components from the hydrolyzed product. CONSTITUTION:A milk protein such as casein, lactoalbumin, etc., or a milk protein-containing material composed mainly of milk protein (e.g. whole milk, defatted milk, etc.) is dissolved in water and the pH of the solution is controlled to an alkaline region. The solution is made to simultaneously or successively react with pancreatin and a microbial protease (e.g. Bioplase: commercial name) originated from a microorganism belonging to Bacillus genus (e.g. Bacillus subtilis) to effect enzymatic hydrolysis of the protein. Insoluble components are separated and removed from the resultant enzymatic decomposition mixture to obtain an easily soluble hydriolyzed milk protein free from unagreeable taste. The hydrolyzed product is suitable as an transintestinal nutrient, nutrition drink, etc.

Description

[Detailed Description of the Invention] The present invention provides an easily soluble milk that has no unpleasant taste and is suitable for use as a food and drink material for enteral nutritional supplements, nutritional supplements, nutritional drinks, etc. The present invention relates to a method for producing a protein hydrolyzate.

In recent years, peptides obtained by enzymatic hydrolysis of proteins, especially low-molecular-weight peptides whose main components are G and T peptides, have been shown to have superior intestinal absorption compared to amino acid mixtures with the same composition. Because of this, it has been used in enteral nutrients, nutritional supplements, etc.

Therefore, the peptides obtained by hydrolyzing milk protein using enzymes according to conventional methods as a protein source often have a bitter taste, which may cause problems in feeding. Additionally, some of these peptides are insoluble, so they form precipitates when put into solution form.

Therefore, it has been difficult to use peptides obtained by enzymatically decomposing milk proteins according to conventional methods as a nitrogen source for oral and enteral nutrients, nutritional supplements, beverages, and the like.

The present invention was made in view of the problems in the use of peptides obtained by enzymatically decomposing milk proteins. The object of the present invention is to provide an enzymatic hydrolysis method for advantageously producing soluble low-molecular-weight peptides.

The present inventors achieved the above object by enzymatically hydrolyzing milk protein in a solution adjusted to an alkaline pH using a combination of pancreatin and a microbial protease derived from the genus Bacillus. This was successful and led to the present invention.

The present invention will be explained in detail below.

A feature of the present invention is that pancreatin and a microbial protease derived from the genus Bacillus are simultaneously or The purpose is to perform enzymatic hydrolysis by acting in stages, and to separate and remove insoluble matter from the resulting enzymatically decomposed mixture.

The milk proteins used here are casein and lactalbumin (
whey protein), and milk protein-containing substances include whole milk,
Examples include skimmed milk.

Steps for Solving the Problems In the present invention, the above-mentioned milk protein or milk protein-containing material is made into an aqueous solution with a milk protein concentration of 0.5% to 30%, preferably 5% to 15%. After heating and sterilizing the dissolved solution, the temperature is maintained at 50° C., and the pH thereof is adjusted to an alkaline range of 7 to 11, preferably 8.0 to 10.0, using an alkali. When using a milk protein that is difficult to dissolve in water, such as acid casein, it is preferable to add as little acid or alkali as possible to the water and dissolve it under heating.

In the present invention, the enzyme pancreatin used to act on the milk protein in the solution is obtained from pig pancreas, and it is preferable to use an enzyme similar to that listed in the Japanese Pharmacopoeia. In addition, the microbial proteases derived from the genus Bacillus that are also used in the present invention include "Bioplase" (manufactured by Nagase Seikagaku Kogyo Co., Ltd.), which is a protease derived from Bacillus subtilis, "Neutral Protease" (manufactured by the same company) and "
Preferred are those that act in the neutral and alkaline regions, such as "Protease N" (manufactured by Amano Pharmaceutical Co., Ltd.).

The amount of these enzymes used depends on the type of milk protein that is the substrate,
Although it varies depending on the concentration and digestion temperature and time, it is generally 0.05 to 10%, preferably 0.1% based on the substrate.
~5% by weight.

In addition, the reaction temperature for making these enzymes act is 30 to 60°C, which is the optimum temperature of the enzyme, preferably 40 to 60°C.
50℃, while the reaction time depends on the type of milk protein, concentration,
Although it varies depending on the amount of enzyme and reaction temperature, it is usually 3 to 48 hours, preferably 6 to 24 hours.

In the present invention, pancreatin and a microbial protease derived from the genus Bacillus are used together.
They may be added to the milk protein aqueous solution at the same time to act, or they may be added in stages to act, and when they are added in stages, the order of addition of both enzymes does not matter.

The p++ of the reaction solution when both enzymes are allowed to act on the milk protein as a standard should be adjusted especially after the pH of the raw material aqueous solution before addition of the enzyme is adjusted to the alkaline range as described above. Allow the reaction to proceed. Therefore, the pH during the reaction suddenly decreases to around neutrality (pH 6).
, 6 to 7.0), but the hydrolysis reaction proceeds with this ρ11 maintained until the end of the reaction.

In addition, when acid casein is used as the milk protein in the above reaction, the viscosity increases when the concentration in the solution exceeds 10%, so it is preferable to proceed with the reaction while performing appropriate stirring.

After the reaction, the obtained milk protein hydrolyzate is heated at a temperature of 85°C or higher for 5 minutes or more to inactivate the enzyme, then cooled and left at a temperature of 5°C or lower for 16 hours or more to make it insoluble. The insoluble matter is separated and removed. This insoluble matter is
Although it is thought to consist of denatured peptides and poorly soluble peptides, these can be efficiently separated and removed by the following method.The first means of separating and removing insoluble matter is the application of continuous high-speed centrifugation. This makes it possible to remove insoluble matter with a relatively high specific gravity. The second method is to apply a filtration method, which includes a method using filter cotton,
The method using Zeta Plus filter media and the method using Zeta Plus filter media each separately,
Or they can be used in combination.

By applying these two means, it is possible to completely separate and remove insoluble matter and clarify the milk protein hydrolyzate solution.

Incidentally, filter cotton is cotton-like cellulose, and is used as a filter by dispersing it in water and layering it in a filter cylinder.

Moreover, Zeta Plus filter medium is a filter medium made of cellulose and diatomaceous earth, and is formed into a filter.

This filter can filter with high filtration efficiency due to the synergistic effect of the adsorption effect of zeta potential and the physical filtration effect of ordinary porous materials, and can effectively filter even fine colloidal impurities. Can be separated.

Furthermore, when a mixture of the above Zeta Plus filter medium and Celite (manufactured by Wako Pure Chemical Industries, Ltd., Hyfloth Purcel) in a ratio of 1=4 is used as a filter aid, the above impurities can be filtered out with a very high filtration efficiency. be able to.

The milk protein hydrolyzate obtained by removing insoluble matter as described above is dried by freeze drying or spray drying, which is a usual drying method, to form a powder into a product.

The milk protein hydrolyzate product obtained in this way is a low peptide containing amino acids and dipeptides and tripeptides as main components, has good solubility in water, and has a concentration of 4/-% in aqueous solution. The pH is neutral between 6.0 and 7.0, and there is almost no bitterness.

Therefore, the product obtained according to the present invention can be effectively used as a nitrogen source material for enteral nutrients, nutritional supplement foods and drinks, or as a pharmaceutical material.

As mentioned above, the present invention hydrolyzes milk protein using a combination of pancreatin and a microbial protease derived from the genus Bacillus to adjust the pH during the enzymatic decomposition reaction and to neutralize the reaction solution after the reaction. This makes it possible to advantageously produce a low-molecular-weight peptide that is good as the nitrogen source without performing operations such as mixing and desalting. EXAMPLES The present invention will be explained in more detail with reference to Examples below.

The yield, average peptide chain length, free amino acid content, and molecular weight distribution of the low-molecular-weight peptides shown in Examples were measured as follows.

■Peptide yield Total nitrogen content in raw protein ■Average peptide chain length of peptide (APL) Quantification of amino groups was performed by the TNBS (linitrobenzenesulfonic acid) method, and complete hydrolysis was performed in 6N IIcI at 110%
℃ for 24 hours.

■For solutions with a concentration that allows quantitative measurement of free amino acids, Hitachi 835
It was quantified using an automatic amino acid analyzer.

(2) Molecular weight distribution Gel filtration was performed using a 5 ephadex G-15 column (1.5 x 150 cm) using a 1% acetic acid solution as the equilibrium eluent. For detection, each fraction was completely hydrolyzed and the amount of amino acids was quantified by the ninhydrin method. Furthermore, the AP of each fraction
L was measured by the method described in (2) above to determine the molecular weight distribution.

Example 1 1 kg of acid casein (made in New Zealand, raftic casein) was mixed with 20 g of KzCOz and 1Ja2c032
A solution prepared by adding 0 g of water 10Z and dissolving it while heating it to 60°C was heat sterilized at a temperature of 90°C or higher for 15 minutes.

Next, a 4N aqueous solution of caustic soda was added to this solution at a temperature of 50°C. was added to adjust the pH to 8.0 to 9.0.

To the obtained acid casein solution, 7 g of pancreatin (manufactured by Amano Pharmaceutical Co., Ltd., listed in the Japanese Pharmacopoeia) dissolved or suspended in an appropriate amount of water and Bacillus subtilis (Bacillus subtilis) were added.
Protease N derived from S. illus 5ubttlis)
(manufactured by Amano Pharmaceutical Co., Ltd.) were sequentially added as separate solutions, and the mixture was reacted for 16 hours while being maintained at 50°C. After the reaction was completed, the enzyme was deactivated by heating at 85°C for 10 minutes or more, and then
It was left at 5° C. or lower for 16 hours. After standing, impurities were removed using a high-speed centrifuge without returning to room temperature, and the reaction solution was further treated by cotton filtration and Zeta Plus filter filtration to clarify the reaction solution. Spending this night on a rotary evaporator? Shrink it and freeze-dry it to make 860g of peptide powder.
was obtained (yield 86%).

This preparation had an average peptide chain length of 2.0 and a di- and tripeptide content of 30% or less.

Moreover, the molecular weight distribution (gel filtration bagun) of the above sample obtained by gel filtration was as shown in the attached figure.

Next, the taste of the milk protein hydrolyzate obtained as described above was tested by a panel test, and the results are shown in the table below. For comparison, a milk protein hydrolyzate prepared without following the method of the present invention was also tested in the same manner, and the results are also shown in the table.

Preparation of samples: ■Sample A (invention) A solution in which the peptide powder obtained in Example 1 was dissolved in water to a concentration of 4 W/W%.

■Sample B (comparative example) Peptide powder prepared by the same procedure as described in Example 1 except that only pancreatin was used without using protease N was prepared at a concentration of 4-/-%. solution dissolved in water.

■Sample C (comparative example) Peptide powder prepared by the same procedure as described in Example 1 except that only protease N was used without using pancreatin was prepared at a concentration of 4-/-%. solution dissolved in water.

■Sample D (comparative example) In Example 1, except that trypsin LOg was used instead of pancreatin and protease N as enzymes,
A solution in which tA-produced peptide powder was dissolved in water to a concentration of 4-8% according to the same procedure as described in Example 1.

Panel test: For each sample listed, one panelist aged between 10 and 50
A sensory test was conducted on 0 people (total of 50 people), and the average value of the evaluations was shown.

As seen in the above table, it can be seen that the milk protein hydrolyzate (Sample A) prepared according to the method of the present invention has no unpleasant taste compared to that prepared by the conventional method.

Example 2 The same procedure as described in Example 1 was followed except that 50 g of pancreatin and LOg of protease NLO were added and allowed to react at 50° C. for 8 hours.
g peptide was obtained. This preparation has an average peptide chain length of 1
．． 8. The free amino acid content was 32%, and the molecular weight distribution by gel filtration showed almost the same pattern as the peptide in Example 1.

Example 3 In Example 1, "Bioprase Conc (150,000P) manufactured by Nagase Seikagaku Kogyo was used instead of Protease N.
850 g according to the same procedure as described in Example 1 except that 15 g of IN/g) was added and allowed to act at 50° C. for 16 hours.
peptide powder was obtained. The average peptide chain length of this product was 2.0, and the free amino acid content was 30%. Moreover, the molecular weight distribution showed almost the same pattern as the peptide in Example 1.

Example 4 Same as in Example 1 except that 15 g of protease N was first allowed to act at 50°C for 3 hours, then 7 g of pancreatin was added and allowed to act for a further 12 hours, without adding enzymes at the same time. Following the same procedure as above, 890 g of peptide was obtained. This preparation had an average peptide chain length of 2.0, a free amino acid content of 30%, and a molecular weight distribution determined by gel filtration that was almost the same as in Example 1.

Example 5 In Example 1, the substrate was replaced with acid casein by dissolving it in water 101 using lactalbumin, which is a milk whey protein (Biopro: Hkg manufactured by Bioisolate), and heating it at 90"C for 15 minutes and then heating it at p11. 4N Koj
810 g of a peptide composition was prepared according to the same procedure as described in Example 1, except that the pH was adjusted to 9.3 with i. The average peptide chain length of the obtained peptide sample was 2.3, the free amino acid was 27%, and the molecular weight distribution was almost the same as in Example 1.

Claims (2)

[Claims] (1) Milk protein or a milk protein-containing material whose main component is milk protein, in a solution whose pH is adjusted to an alkaline region,
An unpleasant taste-free method characterized by enzymatically hydrolyzing pancreatin and a microbial protease derived from the genus Bacillus by acting simultaneously or in stages, and separating and removing insoluble matter from the resulting enzymatically decomposed mixture. A method for producing a readily soluble milk protein hydrolyzate. (2) Adjust the pH of the solution of milk protein or its contents to 7 or more.
11, preferably 8.0 to 10.0.