JP2631202B2 - Peptide production method - 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 peptide having no bitter taste and a method for eliminating bitter taste from the peptide by using a complex enzyme containing a proteinase and a peptidase produced by a genus Rhizopus. Since the peptide prepared according to the present invention has no bitterness, it is very easy to eat and can fully utilize the nutritional value originally possessed by the peptide. Therefore, the present invention can be widely and advantageously used in various industries such as food, nutritional food, medical care, feed, and microbial industry.

[0002]

2. Description of the Related Art In a method of decomposing a protein into a peptide, a method using an enzyme is widely used industrially because it has advantages such as operation under mild conditions. However, peptides produced by such a method, that is, enzymatically degraded peptides are accompanied by bitterness,
Even if this is administered as a nutritional diet, in extreme cases, it may be exhaled depending on the infant or sick person, etc., resulting in the inability to administer, and the inability to utilize the nutritional and other properties inherent in the peptide. Often occurs.

For the purpose of improving this, for example, a method of screening for an enzyme that does not cause bitterness has been conventionally performed, for example, a method using a neutral and / or alkaline protease derived from the genus Penicillium such as Penicillium citrinum. Have been developed by the present inventors (JP-A-62-14796).

[0004] However, the technique of producing a peptide having a bitter taste eliminated by using an enzyme derived from the genus Rhizopus and a complex enzyme containing a proteinase and a peptidase as in the present invention has not been known at all, and a novel technique has been proposed. It is.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned state of the art, and develops a technique for removing bitterness, which is a conventional defect of enzymatically degraded peptides. It was done for the purpose.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made to achieve the above object, and as a result of investigations from various aspects, the reaction conditions are mild and the degree of decomposition can be controlled relatively easily. Therefore, the present inventors have paid attention to biological methods, particularly methods using microorganisms, because they have the advantage of being able to obtain the desired peptide freely. Accordingly, as a result of intensive screening of various microorganisms, it was found that certain Rhizopus bacteria have an action of producing a peptide having no bitterness (Table 1).

[0007]

[Table 1] Table 1 生成 Bacterial strain generation ──────────── ─────────────── Rhizopus pseudochinesis IAM 6042 − Rhizopus formosaensis No. 3540 − Rhizopus peka No. 3626 − Rhizopus hangchow No. 3545 − Rhizopus javanicus No. 3553 − Rhizopus niveus No. 3593 − Aspergillus oryzae IAM 2686 + Aspergillus niger IAM 2020 + Bacillus cereus IAM 1229 + Bacillus subtilis IAM 1163 + ───────────────────────────

[0008] The above-mentioned action involves not a single enzyme but a complex enzyme. Further, in addition to the action of directly producing a peptide having no bitterness from a protein, it has a bitter taste generated by another enzyme. A new and useful finding that it also has the action of removing bitterness from peptides was obtained.

As a result of further studies based on these new findings, the above enzyme is a complex enzyme containing proteinase and peptidase, and a microorganism capable of producing these enzymes and eliminating bitterness is Rhizops pseudokinesis (Rhizop pseudokinase).
us pseudochinesis), Rhizopus formosaensis (R. formosaensis), Rhizopus peka (R. peka),
R. hangchow, R. javanicus, R. niveus
niveus) and the like (Table 2).

[0010]

[Table 2] Table 2 生成 Bacterial strain generation ──────────── ─────────────── Rhizopus pseudochinesis IAM 6042 − Rhizopus formosaensis No. 3540 − Rhizopus peka No. 3626 − Rhizopus hangchow No. 3545 − Rhizopus javanicus No. 3553 − Rhizopus niveus No. 3593 − Rhizopus arrhizus IAM 6056 + Rhizopus delmer IAM 6062 + Rhizopus oryzae IAM 6060 + ───────────────────────────

The present invention is based on these new findings,
A complex enzyme (proteinase, peptidase) produced by Rhizopus sp.
And a system for removing bitterness from peptides.

To produce the complex enzyme preparation of the present invention,
Rhizopus bacteria may be cultured and the enzyme may be obtained from the culture. These treatments may be carried out by a conventional method. For example, for the cultivation of Rhizopus bacteria, a culture technique commonly used for filamentous fungi such as a solid culture method using bran or a liquid culture method is appropriately used.

[0013] The enzyme produced by the culture can be obtained by a conventional method. In other words, when the enzyme is produced and accumulated in the cells, the cells are separated and destroyed,
In the case of an extracellular enzyme, the enzyme is extracted from the culture solution and purified by subjecting to salting out, ultrafiltration, reverse osmosis membrane, molecular sieve, ion exchange resin, etc. according to a conventional method.

The enzyme thus obtained is used as it is or after drying, as an enzyme preparation. In some cases, the enzyme may be used as a crude enzyme or a crude enzyme solution without purification, or as a crude enzyme. If necessary, another enzyme can be added, or a component acceptable in an enzyme preparation can be added and blended according to a conventional method. Conversely, each enzyme may be separated from each other, and these may be used in combination as needed.

The enzyme preparation of the present invention has a characteristic that it does not decompose proteins to produce bitterness, but in order to clarify the characteristics, proteinase and peptidase contained in the enzyme preparation are respectively purified and their properties are determined. Was examined. The peptidase of the enzyme powder used was proteinase 1
24.3 units were mixed per unit.

(1) Characteristics of Proteinase After dissolving the enzyme powder in acetate buffer (pH 4.0), the mixture was heated at 50 ° C. for 30 minutes to inactivate the peptidase, and then dialyzed. Phosphate buffer (pH 7.
Absorb to DEAE Sepharose buffered in 0),
After washing, the fraction was eluted with NaCl by a concentration gradient method, and the active fraction was collected, concentrated and purified by an ultrafiltration membrane having a molecular weight of 10,000. The contaminating peptidase of this sample was 0.016 units per 1 unit of proteinase, and the purity was increased about 1500 times.

The obtained sample is used to decompose acid casein, and a protease-derived commercially available enzyme agent having a high peptidase activity, "Protease P" (manufactured by Amano Pharmaceutical Co., Ltd.);
The molecular weight distribution of the decomposition products was compared. The results of the molecular weight distribution are shown in FIG. As is apparent from this drawing, the proteinase (B) contained in the enzyme preparation of the present invention containing no peptidase has a stronger effect of lowering the molecular weight of the protein than the commercially available enzyme preparation (A) containing more peptidase. .

(2) Characteristics of Peptidase The enzyme powder was dissolved in purified water, dialyzed, adsorbed on a DEAE Sepharose column buffered with a phosphate buffer (pH 8.0), washed, and concentrated with NaCl. Elution was performed by the method. The active fraction was collected and then heated at 50 ° C. for 30 minutes at pH 9.0 to inactivate the proteinase, and after dialysis, concentrated by an evaporator and purified. The purity of the contaminating peptidase of this sample was increased by about 3000 times in terms of 1 unit of proteinase.

With respect to the enzymatic properties of this peptidase (Rhizopus hansha No. 3545 peptidase), the optimum pH, pH stability, optimum concentration, and temperature stability were examined, and the results shown in FIG. 2 were obtained. As is clear from these results, this enzyme is excellent in pH and temperature stability, has a wide optimum pH and temperature range, is easy to use, and is suitable for industrial use.

Next, an experiment for eliminating bitterness was carried out using the obtained sample. "Protease A", a commercially available enzyme preparation
(Manufactured by Amano Pharmaceutical Co., Ltd.) "
After generating bitterness, add 4000 units of this sample and add 45 units.
C. for 3 hours. As a result, without adding peptidase, (+
When the present peptidase was added, the bitterness of 2) disappeared.

As described above, the proteinase contained in the enzyme preparation of the present invention has a strong action of lowering the molecular weight of a protein, and the peptidase contained at the same time has a strong action of eliminating bitterness. It is considered that even if the protein is highly degraded by the action of both, there is no generation of bitterness compared to the enzyme agent which has been conventionally studied.

When the complex enzyme preparation according to the present invention is applied to a protein, a protein degradation product having no bitterness, that is, a peptide can be directly obtained. Proteins to be processed include animal proteins such as casein and other milk proteins, fish proteins, and animal meat proteins; vegetable proteins such as soy protein, wheat protein, rice protein, peanut protein, rapeseed protein, sunflower protein; yeast proteins, etc. Microbial proteins and all other proteins can be used alone or in combination. These proteins may be denatured or undenatured, may be purified or crude, and may further contain impurities, such as pork broth.

Further, the enzyme agent according to the present invention can remove bitter taste from the enzyme agent by acting it on a peptide having bitter taste produced by another enzyme. There are no particular restrictions on the conditions under which the enzyme agent acts, and usually,
The protein or peptide concentration is about 1 to 30% and the temperature is 3
The temperature is suitably about 0 to 58 ° C., and the enzyme concentration may be appropriately selected depending on the enzyme titer, the substrate concentration and the like.

[0024]

The present invention will be described in more detail with reference to Test Examples, Production Examples and Examples.

Test Example 1 (Comparative experiment of bitterness generation in partial decomposition of protein) 5%
A commercially available enzyme preparation (manufactured by Amano Pharmaceutical Co., Ltd.) and a crude enzyme obtained from the bacterial strain of the present invention were added to an acid casein solution (pH 7.0), and the mixture was reacted at 45 ° C. for 1 hour. The solution was obtained and subjected to a sensory test for bitterness to obtain the results shown in Table 3.
The decomposition rate was adjusted to pH 4.6 with acetic acid, and the soluble fraction was subjected to Kjeldahl quantification, and expressed as a comparison with the whole.
In addition, the determination of bitterness is based on the bitterness due to caffeine as an index.
Caffeine concentration 0.10% bitterness (+5), 0.08
% As (+4), 0.06% as (+2), 0.02% as (+1) and no bitterness as (0).

As is clear from the results of the bitterness test of the partially decomposed product of acid casein in Table 3, the acid casein decomposed solution with a commercially available enzyme preparation had a bitter taste, but the enzyme produced by the strain of the present invention did not. The decomposition solution had no bitterness.

[0027]

[Table 3] Table 3 酵 Enzyme Bitterness ─────────── ──────────────── Protease A +2 Protease P +3 Protease S +2 Protease N +1 Proleather +1 Pancreatin F +3 Trypsin +3 Bromelain +2 Papain W-40 +1 (strain of the present invention) Rhizopus pseudochinesis 0 Rhizopus formosaensis 0 Rhizopus peka 0 Rhizopus hangchow 0 Rhizopus javanicus 0 Rhizopus niveus 0 ───────────────────────────

Test Example 2 (Comparative test of bitterness generation in advanced protein degradation) 5%
A commercially available enzyme preparation and the crude enzyme obtained according to the present invention in 1 g of protein were added to acid casein and a 5% soybean protein solution (pH 7.0).
Then, 300 units were added per proteinase activity and reacted at 45 ° C. for 16 hours. Table 4 shows the results of the sensory test for bitterness.

As is clear from the results of the comparative experiments of bitterness generation in the advanced degradation of various proteins shown in Table 4, the degradation solution by the commercially available enzyme preparation had a bitter taste, whereas the degradation solution by the enzyme of the present invention had a bitter taste. There was neither casein nor soy protein.

[0030]

[Table 4] Table 4-Enzyme agent Milk protein (acid casein) Soybean Protein ───────────────────────────────── Protease A + 1 + 1 Protease P + 2 + 2 Protease S + 4 + 2 Protease N + 4 + 2 Proleather +4 +2 Pancreatin F +2 +4 Trypsin +4 +4 Bromelain +4 +4 Papain W-40 +4 +4 (strain of the present invention) 0 ─────────────────────────────────

Test Example 3 (Elimination test of bitterness) 5% acid casein (pH 7.0) was added to "Protease A", "Protease P", "Protease S", "Proleather" and "Pancreatin F".
200 units of each (manufactured by Amano Pharmaceutical Co., Ltd.) were added at a proteinase activity per 1 g of acid casein, and reacted at 45 ° C. for 16 hours. As a result of a sensory test of the bitterness of the reaction solution, the bitterness was from (+5) to (+2). The enzyme of the present invention was added to each digested solution as a peptidase activity, and the enzyme was added in an amount of 20 / g of acid casein.
00 units, 4000 units, 8000 units added, 45 ° C
As a result, a significant elimination of bitterness was observed as shown in Table 5.

[0032]

[Table 5] Table 5-The amount of the peptidase of the present invention not added 2000 Unit 4000 units 8000 units プ ロ テ ア ー ゼ Protease A +2 +10 0 Protease P +4 +3 +10 Protease S +5 +4 +3 +2 Pro Leather +5 +4 +3 +2 Pancreatin F +2 +1 +1 0 ─────────────────────────────── ──

[Production Example 1] 150 g of water was added to 180 g of bran.
After adding and sterilizing, add Rhizopus pseudochinesis IAM 6042
Was inoculated and cultured at 25 ° C. for 4 days. 20 mM, pH 7, phosphate buffer is added to the koji obtained after the culturing, 500 m
l, and the mixture was extracted overnight in a low-temperature room to obtain 300 ml of a crude enzyme solution. After centrifugation, freeze-drying and enzyme powder 3.
6 g were obtained. The enzymatic activity of this powder is proteinase 12
5 u / g and peptidase 37500 u / g.

[Production Example 2] 0.5% glucose, 2.0% soybean protein, 0.2% polypeptone, 0.2% yeast extract
%, Dipotassium phosphate 0.1%, magnesium sulfate 0.
Rhizopus javanicus N is added to 100 ml of a medium having a composition of 05%.
o.3553 spores were inoculated and shake-cultured at 30 ° C. for 3 days. The cells were separated by centrifugation to obtain an enzyme solution.
The enzyme activity of this solution is 2.4 u / ml of proteinase,
The peptidase was 20.4 u / ml.

Production Example 3 Water was added to 1800 g of bran with 15
After sterilization by adding 00 ml, seed koji of Rhizopus hangchow No. 3545 was inoculated and cultured at 30 ° C. for 3 days. 2 in the obtained koji
10 liters of 0 mM, pH 7 phosphate buffer was added, and the mixture was extracted overnight in a low temperature room to obtain 7350 ml of a crude enzyme solution. After centrifugation, hollow fiber (Asahi Kasei Corporation AI
L-1010), followed by lyophilization to obtain 15.5 g of enzyme powder. The enzymatic activity of this powder was 2100 u / g of proteinase, and the peptidase was 9
It was 3500 u / g. The activity yield was 68
%, 61%.

[Example 1] "Decomposition of acid casein" 5% acid casein solution (pH 7) 9
640 mg of the enzyme powder obtained in "Production Example 3"
An enzyme solution dissolved in ml of purified water is added (acid casein 1).
(300 units of proteinase activity per gram) and allowed to stand at 45 ° C. for 16 hours. After heating at 100 ° C. for 10 minutes after the reaction, centrifugation was performed to obtain a clear solution. When the bitterness of this solution was measured by a sensory test, no bitterness was found. Pharmacia FPLC system (column:
The result of analyzing the molecular weight distribution using Superose 12) is shown in FIG. 3, and almost all peptides and amino acids having a molecular weight of 1,000 or less were obtained.

[Example 2] "Decomposition of soybean protein" 5% soybean protein solution (pH 7) 100
To 0 ml, 7.1 g of the enzyme powder obtained in "Production Example 3" was added, and the mixture was reacted while stirring at 45 ° C for 17 hours. After reaction 1
After heating at 00 ° C. for 10 minutes, the solution was centrifuged to obtain a clear solution, and then freeze-dried to obtain 42.5 g of a slightly yellow powder. As a result of a sensory test of the bitterness of the powder, no bitterness was found. FIG. 4 shows the result of analyzing the molecular weight distribution in the same manner as in “Example 1”.
0 or less peptides and amino acids.

Example 3 "Decomposition of meat broth" Boiled pork (protein concentration 10%) 10
480 m of the enzyme powder obtained in "Production Example 3"
After adding g, the mixture was reacted at 55 ° C. for 4 hours. After the reaction, the mixture was heated at 100 ° C. for 10 minutes for the purpose of deactivating and sterilizing the enzyme, and a sensory test showed no bitterness.

[Example 4] "Elimination of bitterness" 90 ml of 5% acid casein solution (pH 7)
"Protease P-1" (manufactured by Amano Pharmaceutical Co., Ltd.) 90 m
g was dissolved in 10 ml of purified water (200 units of proteinase activity per gram of acid casein), and
The reaction was allowed to stand at 16 ° C. for 16 hours. After the reaction, 190 mg of the enzyme powder obtained in "Production Example 3" was added (4000 units as peptidase activity per 1 g of acid casein), and the mixture was added at 45 ° C for 5 minutes.
The time further reacted. After the heat treatment after the reaction, the bitterness was subjected to a sensory test. As a result, the solution of the present invention before the addition of the enzyme had a bitterness of +4, but no bitterness after the enzyme addition treatment.

In the present specification, the measurement of the enzyme activity and the molecular weight distribution were performed as follows. Enzyme activity measurement method Reaction was performed at 37 ° C. using proteinase 0.75% milk casein (manufactured by Merck) as a substrate, 0.4 mol of trichloroacetic acid was added in the same amount as the reaction solution, and the mixture was filtered through filter paper (Toyo Filter Paper No. 131). Color was developed with Foreign reagent. 6 per ml of filtrate
The amount of color development equivalent to 100 micrograms of tyrosine per 0 minute was defined as 1 unit. 2. Peptidase was reacted at 30 ° C. using 0.125 mM leucine / glycyl / glycine as a substrate (pH 7.0), and the reaction was carried out by the ninhydrin method. One micromol of amino acid produced per minute was defined as one unit.

Measurement method of molecular weight distribution A "Sparose 12" gel filtration column manufactured by Pharmacia was used. As the molecular weight standards, 25000 is chymotrypsinogen, 12300 is cytochrome C,
6500 used trypsin inhibitor and 1450 used bacitracin.

[0042]

Industrial Applicability The present invention has been successfully achieved for the first time by using an enzymatic agent containing proteinase and peptidase produced by Rhizopus sp. As active ingredients. According to the present invention, a peptide having no bitterness is directly produced from various proteins. In addition to removing bitterness from peptides having bitterness produced by other enzymes.
As described above, according to the present invention, a peptide having no bitterness can be efficiently prepared, so that even a peptide having nutritional properties or other usefulness has a bitter taste and cannot be used freely because of its bitterness. Products, oral preparations, nutrients, feeds, etc., and the use of peptides is dramatically expanded.

Specific examples thereof include medical food, enteral nutrition, infant food, hypoallergenic infant formula, balanced nutrition food,
In addition to human nutrition sources such as health foods and functional foods,
Non-human nutrients such as feed, feed, and media peptones. Therefore, according to the present invention, even if a peptide is administered, infants and the disabled will not exhale due to bitterness, and the present invention greatly contributes to human health and medical care.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating the molecular weight distribution of an acid casein hydrolyzate, wherein FIG.
(B) illustrates the molecular weight distribution of the degradation product of the proteinase of the present invention.

FIG. 2 illustrates the enzymatic properties of a peptidase from Rhizopus hangchow No. 3545.

FIG. 3 illustrates the molecular weight distribution of the acid casein hydrolyzate.

FIG. 4 illustrates the molecular weight distribution of a soybean protein degradation product.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location // A23L 1/313 A23L 1/313 (C12N 9/48 C12R 1: 845) (72) Inventor Yukitaka Sukunobe 6083-8 Furuya, Kawagoe-shi, Saitama B2-205 (56) References JP-A-48-52967 (JP, A)

Claims (2)

(57) [Claims] 1. An enzyme preparation for peptide production comprising a complex enzyme containing a proteinase and a peptidase having the following physicochemical properties as an active ingredient, which can be produced by a Rhizopus bacterium and can produce a peptide having no bitterness. And producing a peptide having no bitterness. Physicochemical properties of peptidase Produces a non-bitter peptide. 2. The optimum pH is 6-8. 3. The optimum temperature at pH 7.0 for 60 minutes is 35 to 47 ° C. 4. Shows 80% or more residual activity at 37 ° C., 60 minutes, pH 5 or more. 5. It shows a residual activity of 90% or more at pH 7.0 for 60 minutes at 50 ° C. or less. 2. An enzyme preparation for producing a peptide, comprising a complex enzyme containing a proteinase and a peptidase having the following physicochemical properties as an active ingredient, which is capable of producing a peptide having no bitterness, which is produced by a Rhizopus bacterium. A method for producing a peptide having no bitterness, wherein the peptide has no bitterness. Physicochemical properties of peptidase Produces a non-bitter peptide. 2. The optimum pH is 6-8. 3. The optimum temperature at pH 7.0 for 60 minutes is 35 to 47 ° C. 4. Shows 80% or more residual activity at 37 ° C., 60 minutes, pH 5 or more. 5. It shows a residual activity of 90% or more at pH 7.0 for 60 minutes at 50 ° C. or less.