JPH0753106B2 - Enzyme preparation for peptide production - Google Patents

Description

TECHNICAL FIELD The present invention relates to an enzyme agent derived from Rhizopus for producing a bitterness-free peptide, a method for producing a bitterness-free peptide using the same, and a method for producing a bitterness using the same. It relates to an erasing method.

Since the peptide prepared according to the present invention has no bitterness, it becomes very easy to eat, and the nutritional value originally possessed by the peptide can be fully utilized.

Therefore, the present invention can be widely and advantageously utilized in various industries such as foods, nutritional foods, medical treatments, feeds, and microbial industries.

(Prior Art) In the method of degrading a protein into a peptide, the method using an enzyme is industrially widely used because it has an advantage that it can be operated under mild conditions. However, a peptide produced by such a method, that is, an enzyme-degraded peptide is accompanied by a bitter taste, and even if it is administered as a nutritional food, it may be exhaled by an infant or a sick person in an extreme case. As a result, they cannot be administered, and the nutritional properties and other properties inherent in the peptides are often unavailable.

For the purpose of improving this, for example, a method of screening an enzyme that does not cause bitterness has been conventionally performed,
For example, a method utilizing neutral and / or alkaline proteases derived from the genus Penicillium such as Penicillium citrinum has been developed by the present inventors (JP-A-62-14).
No. 796).

However, a technique for producing a peptide in which bitterness is eliminated by using an enzyme derived from a genus Rhizopus such as the present invention, and a complex enzyme containing proteinase and peptidase has never been known and is novel.

(Problems to be Solved by the Invention) The present invention has been made in view of the current state of the art described above, and develops a technology for removing bitterness which is a fatal defect of conventional enzyme-degrading peptides. It was made for the purpose.

(Means for Solving Problems) The present invention has been made in order to achieve the above-mentioned object, and as a result of studies from various aspects, the reaction conditions are mild, and the degree of decomposition can be controlled relatively easily. Since it has the advantage that it can be obtained and the desired peptide can be obtained freely, we focused on biological methods, particularly methods using microorganisms.

Then, as a result of intensive screening for various microorganisms, it was found that certain Rhizopus bacteria have an action of producing peptides having no bitterness (Table 1).

The above-mentioned action involves not only one enzyme but a complex enzyme, and in addition to the action of directly producing a bitter-taste peptide from a protein, the bitterness of a peptide having a bitterness produced by another enzyme We also obtained a novel and useful finding that it also has the action of removing

As a result of further investigation based on these new findings, the above enzyme is a complex enzyme containing proteinase and peptidase, and a microorganism capable of producing these enzymes to remove bitterness is Rhizopus pseudochisis.
nesis), R. formosaen
sis), R. peka, R. hangchow, R. jabonicas (R.ja)
vanicus) and Rhizopus nibeus (R. niveus) were also confirmed (Table 2).

The present invention is based on these new findings and has been completed as a result of further research, and relates to a system for removing bitterness from peptides by a complex enzyme (proteinase, peptidase) produced by Rhizopus.

In order to produce the complex enzyme preparation according to 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 culturing of Rhizopus, a culturing technique commonly used for filamentous fungi such as a solid culturing method or a liquid culturing method using a rice bran or the like is appropriately used.

The enzyme produced by the culture can be obtained by a conventional method. In other words, if the enzyme is produced and accumulated in the microbial cell, the microbial cell is separated and then destroyed, and in the case of an extracellular enzyme, the enzyme is extracted from the culture solution and salted out according to a conventional method. Purify by performing ultrafiltration, reverse osmosis membrane, molecular sieve, ion exchange resin, etc.

The enzyme thus obtained is used as it is or after being dried, as an enzyme preparation. In some cases, the crude enzyme or crude enzyme solution without purification may be used as an enzyme agent in a crude state. If necessary, other enzymes may be added, or components acceptable for the enzyme preparation may be added and blended according to a conventional method. On the contrary, each enzyme may be separated and used together if necessary.

The enzyme preparation of the present invention has a characteristic that it does not decompose a protein to produce a bitter taste, but in order to clarify the characteristic, the proteinase and peptidase contained in the enzyme preparation are each purified and their characteristics are examined. I did. The enzyme powder peptidase used was 24.3 units per unit of proteinase.

(1) Properties of proteinase The enzyme powder was dissolved in acetate buffer (pH 4.0), heated at 50 ° C for 30 minutes to inactivate the peptidase, and then dialyzed. Adsorb to DEAE Sepharose buffered with phosphate buffer (pH 7.0), wash and elute with NaCl by concentration gradient method, and then fractionate the active fraction into an ultrafiltration membrane with a molecular weight fraction of 10,000. It was concentrated and purified.

Contaminant peptidase of this preparation was 0.016 units per proteinase unit, and the purity was increased about 1500 times.

The acid casein was decomposed using the obtained preparation, and a comparison of the molecular weight distribution of the decomposed product with “Protease P” (manufactured by Amano Pharmaceutical Co., Ltd.), which is a commercially available enzyme agent of mold origin containing a large amount of peptidase activity. I did. The result of the molecular weight distribution is shown in FIG. As is clear from this figure, the proteinase (B) contained in the enzyme agent of the present invention containing no peptidase has a stronger action of lowering the molecular weight of protein than the commercially available enzyme agent (A) containing a large amount of peptidase. .

(2) Characteristics of peptidase The enzyme powder was dissolved in purified water and dialyzed, and then adsorbed on a DEAE sepharose column buffered with a phosphate buffer (pH 8.0), washed and eluted with a concentration gradient method with NaCl. Was done.
The active fraction was collected, and thereafter, in order to inactivate the proteinase, it was heated at 50 ° C for 30 minutes at pH 9.0, and after dialysis, it was concentrated and purified by an evaporator.

Contaminant proteinase of this standard was about 3000 times higher in purity per unit of peptidase.

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

Next, a bitterness elimination experiment was carried out using the obtained preparation.

The acid casein was decomposed by using "Protease A" (manufactured by Amano Pharmaceutical Co., Ltd.), which is a commercially available enzyme preparation, to generate bitterness, and then 4000 units of this standard was added and reacted at 45 ° C for 3 hours. The bitterness was (+2) when peptidase was not added, but the bitterness disappeared when this peptidase was added.

As described above, the proteinase contained in the enzyme preparation of the present invention has a strong action of lowering the molecular weight of the protein, and the peptidase contained at the same time has the characteristic of having a strong action of eliminating bitterness. It is considered that this is the reason why even if the protein is highly decomposed with, the bitterness is not generated as compared with the enzyme agents that have been conventionally studied.

By acting the complex enzyme preparation according to the present invention on a protein, a proteolytic product without bitterness, that is, a peptide can be directly obtained. Proteins to be treated include animal proteins such as casein and other milk proteins, fish meat proteins, animal meat proteins; soy proteins, wheat proteins, rice proteins, peanut proteins, rapeseed proteins, sunflower proteins, etc .; yeast proteins, etc. Microbial proteins and all other proteins can be used alone or in combination.

These proteins may be denatured or undenatured, purified or crude, and may be those containing a large amount of impurities, such as pork broth.

In addition, the enzyme agent according to the present invention can remove the bitterness from this by acting it on a peptide having a bitterness produced by another enzyme.

There are no particular restrictions on the conditions under which the enzyme agent acts,
Usually, a protein or peptide concentration of about 1 to 30% and a temperature of about 30 to 58 ° C are suitable, and the enzyme concentration may be appropriately selected depending on the enzyme titer, substrate concentration and the like.

Hereinafter, the present invention will be described in more detail with reference to test examples and examples.

Test Example 1 (Comparative Experiment of Bitterness Generation in Partial Degradation of Protein) A commercially available enzyme preparation (manufactured by Amano Pharmaceutical Co., Ltd.) and a crude enzyme obtained from the strain of the present invention were added to a 5% acid casein solution (pH 7.0). Then, a partially decomposed solution having a decomposition rate of 15% was obtained by reacting at 45 ° C. for 1 hour, and a sensory test for bitterness was performed 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 quantified by Kjeldahl. or,
The bitterness is determined using the bitterness of caffeine as an index (+5) for a caffeine concentration of 0.10% and (+5) for 0.08%.
4), 0.06% (+2), 0.02% (+1) and no bitterness (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 decomposition solution with the commercial enzyme preparation had bitterness, but the decomposition solution with the enzyme produced by the strain of the present invention Had no bitterness.

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

As is clear from the results of comparative experiments of bitterness generation in advanced decomposition of various proteins shown in Table 4, the decomposition solutions with commercially available enzyme preparations had bitterness, but the decomposition solutions with enzymes of the present invention had acid casein and soybeans. There was no protein.

Test Example 3 (Bitterness elimination experiment) 5% acid casein (pH 7.0) with "Protease A", "Protease P", "Protease S", "Pro Leather"
And "Pancreatin" (manufactured by Amano Pharmaceutical Co., Ltd.) were added at a proteinase activity of 200 units per 1 g of acid casein.
The reaction was carried out at 5 ° C for 16 hours. As a result of a sensory test of the bitterness of the reaction solution, the bitterness was (+5) to (+2). As the peptidase activity of the enzyme of the present invention, 2000 units, 4000 units, and 8000 units were added to each decomposition solution per 1 g of acid casein, and the reaction was carried out at 45 ° C. for 3 hours. As a result, as shown in Table 5, remarkable bitterness was obtained. Was erased.

(Example 1) ▲ 180 g of fukuro was added with 150 ml of water and sterilized, and then Rhizopus pseudo
The seed koji of chinesis IMA 6042 was inoculated and cultured at 25 ° C for 4 days. To the koji obtained after the culturing, 500 ml of 20 mM, pH 7, and phosphate buffer was added, and the mixture was extracted overnight in a low temperature room to obtain 300 ml of a crude enzyme solution. After centrifugation, freeze-dry and enzyme powder 3.6g
Got

The enzyme activity of this powder was 125 u / g of proteinase and 37500 u / g of peptidase.

(Example 2) Glucose 0.5%, soybean protein 2.0%, polypeptone 0.2
%, Yeast extract 0.2%, dipotassium phosphate 0.1%, magnesium sulfate 0.05% Rhizopus javani in 100 ml medium
The spores of cus No.3553 were inoculated and shake culture was carried out at 30 ° C for 3 days. The bacterial cells were separated by centrifugation to obtain an enzyme solution. The enzyme activity of this solution was proteinase 2.4 u / ml and peptidase 20.4 u / ml.

(Example 3) 1800 g of fu * 1500 ml of water was added to sterilize 1800 g, and then Rhizopus hang
Chow No. 3545 seed koji was inoculated and cultured at 30 ° C. for 3 days. To the obtained koji, 10 mM of 20 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 (AIL-101, Asahi Kasei Corporation)
0) and then freeze-dried to prepare enzyme powder 1
Obtained 5.5 g. The enzyme activity of this powder is Proteinase 2100
u / g and peptidase was 93500 u / g. Also,
The activity yields were 68% and 61%, respectively.

(Example 4) "Decomposition of acid casein" To 90 ml of a 5% acid casein solution (pH 7) was added the enzyme solution prepared by dissolving 640 mg of the enzyme powder obtained in "Example 3" in 10 ml of purified water (per 1 g of acid casein). The proteinase activity was 300 units), and the reaction was allowed to stand at 45 ° C for 16 hours. 10 at 100 ℃ after reaction
After heating for a minute, centrifugation was performed to obtain a clear solution. When the bitterness of this solution was measured by a sensory test, there was no bitterness. In addition, the results of analysis of the molecular weight distribution using an FPLC system (column; Superose 12) manufactured by Pharmacia Co. are shown in Fig. 3. Almost all the peptides and amino acids had a molecular weight of 1000 or less.

(Example 5) "Degradation of soybean protein" 7.1 g of the enzyme powder obtained in "Example 3" was added to 1000 ml of 5% soybean protein solution (pH 7), and the mixture was reacted at 45 ° C for 17 hours with stirring. After the reaction, the mixture was heated at 100 ° C. for 10 minutes and then centrifuged to obtain a clear liquid, and then freeze-dried to obtain 42.5 g of a slightly yellow powder. As a result of a sensory test for bitterness of this powder, no bitterness was found. Further, the result of analyzing the molecular weight distribution as in "Example 4" is shown in Fig. 4, and almost all the peptides and amino acids had a molecular weight of 1000 or less.

(Example 6) "Decomposition of meat broth" To 1000 ml of pork broth (protein concentration 10%), 480 mg of the enzyme powder obtained in "Example 3" was added, and then reacted at 55 ° C for 4 hours. After the reaction, the product was heated at 100 ° C. for 10 minutes for the purpose of deactivating the enzyme and sterilizing it, and a sensory test revealed that no bitterness was felt.

(Example 7) "Elimination of bitterness" To 90 ml of 5% acid casein solution (pH 7), "Protease P-"
1 "(manufactured by Amano Pharmaceutical Co., Ltd.) was added to a solution prepared by dissolving 90 mg in 10 ml of purified water (200 units of proteinase activity per 1 g of acid casein), and the reaction was allowed to stand at 45 ° C for 16 hours. After the reaction, 190 mg of the enzyme powder obtained in "Example 3" was added (4000 units as peptidase activity per 1 g of acid casein), and 45 ° C.
And reacted further for 5 hours. After heat treatment after the reaction,
The bitterness was sensory tested. As a result, the solution of the present invention before the enzyme addition had a bitterness of +4, but did not have the bitterness after the enzyme addition treatment.

In this specification, the enzyme activity and the molecular weight distribution were measured as follows.

Enzyme activity assay 1. Proteinase 0.75% milk casein (Merck) as substrate at 37 ℃
The reaction mixture was added with 0.4 mol of trichloroacetic acid in the same amount as the reaction solution, filtered through filter paper (Toyo Filter Paper No. 131), and the filtrate was developed with a Folin reagent. The amount of color development corresponding to 100 micrograms of tyrosine per 1 ml of the filtrate for 60 minutes was defined as 1 unit.

2. Peptidase 0.125mM Leucine / glycyl / glycine as substrate (pH
7.0), and reacted at 30 ° C. by the ninhydrin method.
The amount of 1 micromol of amino acid produced per minute was defined as 1 unit.

Method for measuring molecular weight distribution A "Sparose 12" gel filtration column manufactured by Pharmacia was used. As the molecular weight standard, 25000 was chymotrypsinogen, 12300 was cytochrome C, 6500 was trypsin inhibitor, and 1450 was bacitracin.

(Effects of the Invention) The present invention was the first to succeed in developing an enzyme preparation containing proteinases and peptidases produced by Rhizopus as active ingredients, and directly produces peptides without bitterness from various proteins according to the present invention. In addition to being manufactured, the bitterness can be removed from the bittering peptides produced by other enzymes.

As described above, according to the present invention, a peptide having no bitterness can be efficiently prepared. Therefore, even a peptide having nutritional or other usefulness has a bitterness and therefore cannot be used freely. , Orally administered drug, nutritional supplement,
It can be widely used as a feed and the like, and the use of peptides is dramatically expanded.

Specific examples are medical food, enteral nutrition, infant food,
In addition to human nutrition sources such as hypoallergenic infant formula, balanced nutrition food, health food, functional food, feed, feed,
Non-human nutritional sources such as culture medium peptone are included. Therefore, according to the present invention, even if a peptide is administered, infants and sick people do not spit it out due to bitterness, and the present invention greatly contributes to human health and medical care.

[Brief description of drawings]

FIG. 1 shows the molecular weight distribution of the acid casein degradation product. (A) shows the degradation product by protease P,
(B) is a diagram showing the molecular weight distribution of the degradation product of the proteinase of the present invention. Figure 2 shows Rhizopus hangcho
w This is a diagram showing the enzymatic properties of peptidase derived from No. 3545. FIG. 3 and FIG. 4 show the molecular weight distributions of acid casein degradation products and soybean protein degradation products, respectively.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 9/58 C12R 1: 845) (C12P 21/06 C12R 1: 845) (72) Inventor Sukunobe Yukitaka Furuyagami 6083-8, B2-205 (56) Kawagoe City, Saitama Prefecture References JP-A-48-52967 (JP, A)

Claims (1)

[Claims] 1. An enzyme preparation for peptide production, which comprises a complex enzyme containing a proteinase and a peptidase having the following physicochemical properties, which can be produced by a bacterium of the genus Rhizopus and can produce a peptide having no bitter taste, as an active ingredient. Physicochemical properties of peptidases 1. Produce peptides without bitterness. 2. The optimum pH is 6-8. 3. pH 7.0, optimum temperature for 60 minutes is 35-47 ℃. 4. Remains 80% or more of residual activity at 37 ° C for 60 minutes at pH 5 or higher. 5. It shows a residual activity of 90% or more at pH 7.0, 60 minutes and 50 ° C or less.