JPS6214796A - Process for enzymatic decomposition of protein - Google Patents

Abstract

PURPOSE:To obtain an enzymatic decomposition product having extremely low bitterness, by treating a vegetable protein, etc., with a neutral and/or alkaline protease originated from Penicillium genus strain. CONSTITUTION:A 5-20wt% solution of a vegetable protein (e.g. soybean protein) is added with an enzyme composed of an alkaline protease originated from Penicillium citrinum. The ratio of the enzyme (in terms of 5,000U/g) to the protein is 1/(1,000-50). The components are made to react with each other at 5-10pH and 30-55 deg.C to decompose the protein to peptides having a molecular weight of several thousands.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for enzymatically decomposing proteins that produces extremely little bitterness. More specifically, the present invention provides an enzymatically decomposed protein that exhibits extremely little bitterness using neutral and/or alkaline protease derived from Penicillium flexi.

(Prior art) In general, protein-degrading enzymes derived from animals, plants, and microorganisms are used. Among these, those derived from microorganisms include those of the genus Aspergillus (such as Aspergillus oryzae) and the genus Bacillus (such as Bacillus subtilis). , Pediococcus , and Pseudomonas .

By the way, the generation of bitterness due to enzymatic decomposition of proteins is a major problem that needs to be solved, and the solutions include selection of +al enzyme, selection and combination of fbl substrates (combination of different substrates, etc.)
, (C) Improvements in decomposition methods (two-stage decomposition, etc.), (di removal of bitter components, (e+) and other methods have been studied and disclosed.

For example, (with respect to the selection of al enzymes, Aspergillus
Production of a non-bitter proteolytic enzyme belonging to a strain of P. oryzae (Japanese Patent Application No. 13748/1982), and an enzyme derived from Pseudomonas fluorescens that decomposes peptides that cause bitterness in foods ('1HIII 51/19763).
No.) etc. are known.

However, there is no known method for producing proteins with extremely low bitterness using proteases derived from the genus Penicillium, particularly neutral and/or alkaline proteases, as in the present invention.

(Problems to be Solved by the Invention) The present inventors aimed to solve the problem of bitter taste caused by enzymatic decomposition of proteins.

(Means for Solving the Problem) In order to solve the above problem, the present inventors investigated the types of proteins, various enzymes, ice melting conditions, etc., and found that the neutral and/or We have found that not only is there no or very little bitterness produced when hydrolysis is carried out using alkaline protease, but also that bitterness is reduced when proteins that have been thawed with other enzymes and have a bitter taste are similarly hydrolysed. As a result, the present invention has been completed. That is, the present invention is a method for enzymatically decomposing proteins using neutral and/or alkaline protease derived from the genus Penicillium.

The proteins used in the present invention include animal proteins (milk proteins such as casein, animal meat proteins, fish meat proteins, etc.), vegetable proteins, microbial proteins (
Although all proteins such as yeast protein, etc. can be used, vegetable proteins are particularly preferred because they are extremely unlikely to produce bitter taste when the enzyme of the present invention is used. Particularly preferred vegetable proteins include oilseed proteins such as soybean protein, peanut protein, rapeseed protein, and sunflower protein, and cereal proteins such as wheat protein and rice protein.

Note that the protein may be unheated or heat-treated. In other words, it may be denatured or undenatured.

The enzyme used in the present invention is a neutral and/or enzyme derived from the genus Penicillium.
Or alkaline protease is suitable. For example, especially Pen, citrinum+ Pen, casei,
Pen.

cory 1oph i to 1des + Pen
, cyaneo-fu lyum, Pen, me
Neutral and/or alkaline proteases from P. leagrinum, Pen, rubrum are suitable;
ea, chrysogenum+Pen, coryl
Ophilum, Pen, notatum, Pen
．． Also preferred are neutral and/or alkaline proteases derived from E. steckii and the like.

The protease used in the present invention is preferably a neutral and/or alkaline protease, and the optimum pH is in the neutral to alkaline range, that is, about pH 5 or higher (pH about 5 to 12).

In the present invention, proteins enzymatically decomposed using neutral and/or alkaline proteases derived from the genus Penicillium produce a bitter taste compared to when proteins are hydrolyzed using enzymes derived from other molds (genus Aspergillus, genus Mucor, etc.). are extremely rare.

This is because the hydrolysis mechanism by the enzyme of the present invention is different from the hydrolysis mechanism by other enzymes, so it is presumed that bitterness is less likely to occur. That is, compared to other enzymes that hydrolyze randomly and the molecular weight distribution of the hydrolyzed protein shows a broad distribution ranging from several hundred to several hundred, for example, the hydrolyzed protein of soybean protein by the enzyme of the present invention is The molecular weight distribution shows a relatively sharp peak at several thousand points, and since there are very few proteins with a molecular weight of several hundred, the hydrolysis mechanism is different from other enzymes, and this difference may be responsible for the ice-melting mechanism that produces extremely little bitterness. It is assumed that they are involved in the form of In other words, the present invention is a method in which proteins can be enzymatically decomposed into peptides mainly having a molecular weight of several thousand using neutral and/or alkaline protease derived from the genus Penicillium.

Known methods for culturing Penicillium and neutral and/or alkaline protease purification methods can be used.

Normally, protease produced by liquid culture or solid culture (mainly extracellular protease) can be extracted and filtered and used as it is, and if necessary, salting out and [IF, R
It can be purified using O1 molecular sieve, ion exchange resin, etc.

The conditions for enzymatically decomposing proteins are not particularly limited, but usually the protein concentration is about 5 to 20% by weight and the enzyme (500% by weight).
(0 tl/g conversion), the enzyme/protein ratio is approximately 1/1000
-1)50. pl+ is near the neutral to alkaline region (approximately p
H5-10), the temperature is suitably about 30-55°C.

For example, when soybean protein is hydrolyzed with an enzyme other than the enzyme used in the present invention to a decomposition rate of about 15-22%, a bitter taste occurs;
In the decomposition method of the present invention, no bitter taste occurs even when decomposed to the same degree of decomposition. However, in the enzymatic decomposition method of the present invention, unlike ice-melting mechanisms using other enzymes, it is possible to melt ice almost uniformly up to peptides with a molecular weight of approximately several thousand, so it is difficult to simply discuss the degree of decomposition based on the decomposition rate described below. Can not. In other words, even if the degradation rate is the same as that of other enzymes, the degradation method of the present invention can actually degrade the peptides into several thousand smaller peptides, and the generation of low-molecular-weight peptide fractions including bitter peptides is extremely low. There are few.

As a rough guide to the degree of decomposition, the decomposition rate was expressed as the ratio (percentage) of 0.2M TCA (1-lichloroacetic acid) soluble nitrogen to total nitrogen.

Furthermore, even if the protein has already been enzymatically decomposed and a bitter taste has been generated, the enzymatic decomposition of the present invention has the effect of reducing the bitterness. Therefore, the decomposition rate can be increased.

That is, in the past, when the decomposition rate was increased, the generation of bitterness increased, and for example, a protein could be decomposed only up to a certain decomposition rate. Combining decomposition has the effect of further increasing the decomposition rate and decreasing bitterness. Any known enzyme can be used, such as enzymes derived from molds such as Aspergillus spp. and Mucor, bacteria such as Bacillus, so-called microbial enzymes such as lactic acid bacteria, and enzymes derived from animals such as trypsin, pepsin, and renin. and plant-derived enzymes such as papain.

This is because the enzymatic decomposition mechanism of the present invention is different from that of other enzymes. It is unclear whether the mechanism by which bitterness is reduced is by decomposing bitter components, conversely synthesizing them to eliminate bitter components, or by some other mechanism. At least, the mechanism by which the broad molecular weight distribution of molecular weights ranging from 100 to 100,000 as described above when ice is decomposed by other enzymes, changes to a relatively sharp molecular weight distribution of approximately several thousand molecular weights by the enzymatic decomposition method of the present invention. seems to be involved.

Furthermore, in the protein decomposition method of the present invention, since proteins are enzymatically decomposed in a neutral to alkaline range, neutralization is not necessary, or even in the case of neutralization, the consumption of acid is relatively small due to isothermal generation. You can avoid food as much as possible and prefer it.

The protease activity was measured by using 0.75% Merck milk casein (pH 8,0) as a substrate, reacting at 37°C, and then measuring the soluble fraction of 0.2 M) dichloroacetic acid by the Folin method. The activity of releasing 400 μg of tyrosine in 60 minutes was defined as 1 unit (IU).

(Example) Embodiments of the present invention will be described below with reference to Examples.

Example 1 (Preparation of protein) Add 10 times the amount of water to defatted soybeans, stir and extract, centrifuge to remove okara, obtain soy milk with a protein concentration of 6.5%, adjust to pl + 4.5 with hydrochloric acid, etc. The electroprecipitated soybean protein was neutralized with caustic soda to obtain a 12% soybean protein solution.

(Protease no Kan! 1) Among the strains of Penicillium genus and Mucor genus listed in Table 1, Pencilillin citrinum IFO6
The enzyme preparation method will be described below using 026 as an example.

The inoculum was inoculated into a medium consisting of 500 g of wheat gluten that had been sterilized at 120°C for 30 minutes and 400g of water, and cultured at 30°C for 4 days.

Add 500 ml of purified water, leave to extract overnight at 4°C, filter through cotton, salt out the resulting enzyme solution with 80% saturated ammonium sulfate, dialyze with a cellophane tube, and use a membrane filter (manufactured by Touki Roshi). Filtered to 108 U/mjl! Enzyme solution 50
m1 was obtained. Next, this was freeze-dried to enzyme powder (6,
3U/mg) 680 mg was obtained.

(Degradation and sensory test) That is, 1000 (units) of enzyme purified from the strains shown in Table 1.
10 g of a 12% soybean protein solution was enzymatically decomposed at 50°C for a predetermined period of time. The same table also shows the results of the sensory test when the sample was decomposed to the decomposition rate shown in Table 1.

Table-1 Bacterial strain Degradation rate Sensory test results Pen, c
itrin+ua IFO602622,3-Pen
, casei IAM7280 17.1
-Pen, chrysogenum No. 3007
22.1 ±Pen, corylophiloi
des IAM709418.9 -Pen, cya
neo-fulium IAM769418.9
-Pen, meleagrinum IAM7161
17.8 -Pen, corylophilun+
IAM7694 16.9 ±Pen, not
atum No, 3050 19.0 ±P
en. steckii 16M7051 22.0
±Pen, rubrum No., 3107
15.8 -Mucor petrinsul
ans No, 4023 15.8 +However, - means no bitterness, 2 means slightly bitterness, + means bitterness. Same below.

The decomposition rate was expressed as a percentage of the final 0.2M TC^ (trichloroacetic acid) soluble nitrogen divided by the total nitrogen.

From the above, it was found that the decomposition of soybean protein by enzymes produced by Penicillium genus does not produce bitter taste or is extremely unlikely to produce bitter taste.

Comparative Example 1 Same as Example 1.1! Soybean protein solution made by a 200
Commercially available protease (Samoase Ni-Yamato Kasei) was added to 1 (pH 7, 0M 8%) at 8, 16, and 32 m, respectively.
g (0,05,0,1,0,2%/soybean protein) was added, enzymatically decomposed at 70°C for 30 minutes, and heat deactivated at 90°C for 15 minutes to obtain enzymatically decomposed soybean protein liquid P1. The results are shown in Table-2.

Table-2 PI Samoase addition % 0.05% 0.1% 0.2% Decomposition rate % 12.41 15.96 24.53 Flavor
-11+4 Bitterness occurred when the decomposition rate exceeded 14%.

Example 2 Pen, citrinum IF in the same manner as Example 1
Culture O6026 and make enzyme solution powder (6,30/mg)
(hereinafter referred to as RP) was obtained.

Enzyme-digested protein solution Loom cleaved in the same manner as Comparative Example 1
Using N as a substrate and using 10 mg of the enzyme powder, 50'
The soybean protein solution P2 was obtained by enzymatically decomposing the protein C for 30 minutes and inactivating it by heating at 80° C. for 20 minutes.

The results are shown in Table-3.

Table-3P2 Samoase addition % 0.05% 0.1% 0.2
%RP O,125% 0.125%
0.125% Decomposition rate% 27.19 30.14
34.87 Flavor -± +3 No bitterness was felt even after enzymatic decomposition to 30%.

Experimental Example 1 The properties of the Penicillium-derived enzyme obtained in the same manner as in Example 1 were investigated.

Optimal pt+ was determined by a conventional method using 0.01M sulfate buffer, lactate buffer, phosphate buffer, and Atkirs-Pontin buffer as enzyme solutions. Optimum pH is 5-12
This suggests that there are multiple enzymes present.

Comparative Example 2 Casein (10% 5oln, 20g/200ml
) as a substrate, ProLeather (manufactured by Ohno Pharmaceutical Co., Ltd.) was used as a substrate.
0.01,0.02.0.04g (0,05,0,
1,0,2%/casein) was added, hydrolyzed at 50°C for 30 minutes, and then heated to inactivate at 80°C for 20 minutes to obtain enzymatically decomposed casein P.
I got 3.

The decomposition rate and sensory test results are shown in Table 4, and the taste was felt to be bitter.

Table-4P3 Proleather addition % 0.1% 0.2% 0.4%
Decomposition rate % 25.24 32.15 40.2
7 Flavor +4 +〇 x Example 3 Casein (10% 5oln 20g / 200m
l) in the same manner as in Example 2, adding 0.02 RP of the product a.
,0.05. O, lOg (0, 1, 0, 25, 0,
5%/casein) and hydrolyzed at 50°C for 30 minutes, then at 80°C2.
The enzymatically decomposed casein P4 was obtained by heat inactivation for 0 minutes. The decomposition rate and sensory test values are shown in Table-5.

Table-5P4 RP addition % 0.1% 0.25% 0.5% Decomposition rate % 7.21 15.03 31.55 Flavor
+2+4 Casein had a tendency to produce bitterness more easily than soybean protein.

Example 4 Casein (10% 5oln, 20g/200ml
) to 0.0025, 0.005.0.0
1,0.02,0.04g <0.0125.0.0
25.0.05, 0.1, 0.2%/casein) added 5
After melting the ice at 0℃ for 30 minutes and inactivating it by heating at 80℃ for 20 minutes, RP0
．． 125% was added and hydrolyzed at 50°C for 30 minutes, followed by heat inactivation at 80°C for 20 minutes to obtain enzymatically decomposed casein P5. The results are shown in Table-6.

Table-6P5 Pro Leather 0.05% 0.1% 0.2%R
P 0.125% 0.125% 0.125%
Decomposition rate % 30.05 40.15 40.87 Flavor +2 +4 +6 Bitterness decreased compared to Comparative Example 2.

Comparative Example 3 Gluten (10% 5o1n, 20g/200mj2
, pH 10) as a substrate and protin at 0.02, 0.0
4. Add 0.16g (0.1゜0.2,0.8%/gluten), hydrolyze at 50℃ for 30 minutes, neutralize to pn7,
C. for 20 minutes to obtain enzymatically decomposed gluten P6. The results are shown in Table-7.

(Left below) Table 7P6 Protin addition % 0.1% 0.2% 0.8% Decomposition rate % 14.18 22.84 37.71 Flavor
+3 Bitterness occurrence was relatively low.

Example 5 Gluten (10% 5oln, 20g/200mj?
, pH 10) with protin 0.02, 0.04, 0.16
g (0, 1, 0, 2, 0, 8%/glutin) was added, hydrolyzed at 50°C for 30 minutes, neutralized to pl+7, and then inactivated by heating at 8°C for 20 minutes, resulting in RP of 0.125%/glutin. Gluten was added, hydrolyzed for 30 minutes at 50°C, and inactivated by heating at 80°C for 20 minutes to obtain enzymatically decomposed gluten P7. The results are shown in Table-8.

(Left below) Table 8P7 Protin 0.1% 0.2% 0.8%RP
O,125% 0.125% 0.1
25% Decomposition rate% 19.1) 25.0
40.26 flavor +1
Experimental Example 2 Soybean protein was hydrolyzed in the same manner as in Example 1 except that RP was used as the enzyme (degradation rate 20%, no bitterness) and Alcalase 0.6L (manufactured by Novo) was used to hydrolyze the soybean protein in the same manner as in Example 1. HPLC of soy protein (decomposition rate 20%, bitter taste)
(LC-4A type manufactured by Shimabara Seisakusho, carrier is Toyo Soda idTs)
The molecular weight distribution (mw of 200,000 or less) was completely lowered using the Kgel G2000SW.

The results are shown in Figures 1 and 2.

It can be seen that the hydrolysis mode by RP shows a unique degradation mode compared to other enzymes. That is, the protein exhibits a sharp peak around the molecular weight of several thousand, undergoes uniform enzymatic decomposition, and can obtain proteins with uniform molecular weights.

In comparison, ice thawing using other enzymes shows broad peaks ranging from hundreds to hundreds, and only proteins with uneven molecular weights are obtained.

It is inferred that the above-mentioned differences in the ice-melting mechanism are involved in some way in the bitterness generation mechanism.

(Effects) As detailed above, the present invention enables an enzymatic decomposition method that does not generate bitterness or is extremely unlikely to generate bitterness, and provides one solution to a long-standing problem. ,
It greatly contributes to the development of industry.

[Brief explanation of the drawing]

FIG. 1 shows the molecular weight distribution according to IIPLc of soybean protein hydrolyzate using protease derived from Pen and citrinum. FIG. 2 shows the molecular weight distribution according to IIPLc of soy protein hydrolyzate using a commercially available protease (Alcalase 0.6). Figure 1

Claims (4)

[Claims] (1) A method of enzymatically decomposing proteins using neutral and/or alkaline protease derived from the genus Penicillium. (2) The method according to claim (1), wherein the protein has already been enzymatically degraded. (3) Claim No. (1) in which the protein is a vegetable protein
The method described in section. (4) Penicillium strains include Pen. citrinum, Pen. casei, and Pen. corylophiloides.
, Penicillium cyaneo
-fulyum), Penicillium melagrinum (Pe
n. meleagrinum), Pen. rubrum, Pen. chrysogenum, Pen. corylophilum
m), Penicillium notatu (Pen. notatu)
m), Pen.steck
The method according to claim (1), wherein the bacterial species selected from ii) is used.