JPH0541970A - Method for preventing deterioration of food - Google Patents

Abstract

PURPOSE:To simultaneously eliminate dissolved oxygen in food and drink and oxygen in a vacant space in a container and to prevent deterioration of food and drink by adding an ascorbic acid oxidase to the food and drink. CONSTITUTION:Food and drink is blended with an ascorbic acid oxidase, e.g. derived from cucumber or the enzyme with a substrate thereof or an edible deoxidant to remove oxygen dissolved in food or drink. Simultaneously, a deoxidant (e.g. tyrosine oxidase) is added to a vacant space in a container containing the food and drink to remove oxygen in the vacant space.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the removal of oxygen dissolved in foods and drinks by utilizing the consumption of oxygen by the reaction of an enzyme that oxidizes ascorbic acid or tyrosine or phenols with its substrate, and in the air. The present invention relates to a method for preserving foods and drinks by simultaneously removing oxygen in voids in a container with an oxygen scavenger capable of removing oxygen.

[0002]

2. Description of the Related Art It is generally known that oxygen is involved in the deterioration of food and drink, and a method of removing oxygen with an inorganic iron agent is known to prevent the deterioration of food and drink. However, with the inorganic iron agent, although oxygen in the voids in the container can be removed, it cannot be added directly to foods and drinks. Therefore, it takes a lot of time to completely remove oxygen dissolved in foods and drinks. there were.

There is also a method in which glucose oxidase or alcohol oxidase, which is an oxygen-consuming enzyme, is added directly to foods and drinks as a deoxidizing agent to replace the inorganic iron agent, and deoxidation is carried out. These enzymes react with their substrates. In that case, there is a drawback that hydrogen peroxide, which is a toxic substance, is generated, and although dissolved oxygen in food and drink can be removed, there is a drawback that it takes a lot of time to remove oxygen in voids. It should be noted that water is produced by the reaction of an enzyme that oxidizes ascorbic acid, tyrosine or phenols with its substrate, and there is no problem even if it is added to food and drink.

[0004]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention Even with an oxygen scavenger that efficiently removes oxygen in voids, it takes a long time to remove dissolved oxygen, and with an oxygen-consuming enzyme that efficiently removes dissolved oxygen, At the same time, it aims to solve the drawback that it takes a lot of time to remove oxygen in the voids.

[0005]

The present invention uses an oxygen scavenger capable of removing oxygen in the air and an enzyme agent capable of removing oxygen dissolved in foods and drinks in a container for the foods and drinks. The present invention provides a method for preventing deterioration of food and drink, which comprises removing oxygen in the voids and oxygen dissolved in food and drink at the same time.

[0006]

[Detailed Description] The method of the present invention is to remove oxygen in foods and drinks by the reaction of an enzyme that oxidizes ascorbic acid or tyrosine or phenols with its substrate, and to remove oxygen in foods and drinks in voids of foods and drinks by an oxygen absorber. It is characterized in that the removal of oxygen is performed at the same time. And the removal of oxygen in the voids of the container may be carried out by the reaction of an enzyme that oxidizes ascorbic acid or tyrosine or phenols with its substrate, or by another oxygen scavenger that cannot be introduced into food or drink. ..

[0007] In the removal of oxygen in foods and drinks using an enzyme that oxidizes ascorbic acid, foods and drinks that sufficiently contain ascorbic acid are added with an enzyme that oxidizes ascorbic acid or do not contain ascorbic acid or An enzyme that oxidizes ascorbic acid and a substrate thereof, for example, ascorbic acid or a salt thereof or isoascorbic acid or a salt thereof is added to foods and drinks that do not sufficiently contain it.
As a result, oxygen in the food and drink is consumed when a substrate such as ascorbic acid in the food and drink is oxidized by an enzyme that oxidizes ascorbic acid, and as a result, deterioration of the food and drink is prevented.

In this method, any of the enzymes of the present invention and the already known enzymes described above can be used. However, in acidic foods and drinks such as fruit juice, known enzymes do not sufficiently function, and therefore it is preferable to use the enzyme of the present invention having an optimum action pH in the acidic range. The amount of enzyme used varies depending on the type of food or drink, but is generally 0.00 per g or ml of food or drink.
1 unit or more, and preferably food or drink g or
0.02 units or more per ml. Even if the amount of enzyme is large, there is no disadvantage such as food deterioration, but even if the amount of enzyme is increased above a certain level, the deterioration preventing effect does not always increase accordingly.

The amount of ascorbic acid or its salt added to food and drink varies depending on the type of food and drink, the amount of ascorbic acid naturally contained in food and drink, and the like, but generally 0.01 to food and drink. ~ 1.0% by weight, preferably 0.01 ~
It is 0.5% by weight. As the salt of ascorbic acid, its sodium salt, potassium salt and the like can be used. Further, isoascorbic acid or a salt thereof can be used instead of ascorbic acid or a salt thereof.

When oxygen in the food and drink container is removed by the reaction between an enzyme that oxidizes ascorbic acid and its substrate,
An enzyme that oxidizes ascorbic acid and its substrate, such as ascorbic acid or a salt thereof, or isoascorbic acid or a salt thereof, is dissolved in an aqueous medium, for example, a buffer solution to prepare an oxygen consuming system, and this is contacted with food and drink. Seal in the same container without stress.

According to this method, oxygen in the container is consumed by the oxygen consuming system, and as a result, oxygen in food and drink is deoxygenated. In this embodiment, the optimum action of the enzyme used as an aqueous medium constituting the oxygen consuming system, for example, as a buffer solution.
Use one that has a pH value that is at or near pH. For example, since cucumber-derived ascorbate oxidase has an optimum action pH around 5.6, it is preferable to use a phosphate buffer of pH 5 to 7, and the enzyme produced by the strain of the genus Acremonium of the present invention is acidic. It is convenient to use, for example, an acetic acid buffer solution having a pH of 4 to 5 because it has an optimum working pH in the range.

In this case, the quantitative relationship between the enzyme that oxidizes ascorbic acid and its substrate and the food and drink is a sufficient amount for removing oxygen in the void of the container. In implementation,
The enzyme solution and the substrate solution can be prepared separately, mixed and then placed in a food and drink container, and the container can be sealed. However, in order to efficiently utilize the consumption of oxygen by the oxygen consuming system for deoxidation of food and drink, the enzyme and its substrate are placed in a food and drink storage container in a state where they do not react with each other, and the enzyme is sealed after sealing the container. It is preferable to start the reaction. For this purpose, for example, the enzyme solution and the substrate solution can be separately contained and placed in a storage container, and after sealing the storage container, one solution can be injected into the other solution.

Alternatively, the enzyme solution and the substrate solution may be accommodated with a partition wall therebetween, the storage container may be sealed, and then the partition wall may be broken to mix the two solutions. Alternatively, one of the enzyme and the substrate may be introduced as a solution and the other may be introduced as a powder into a storage container, and the storage container may be sealed and then the two may be mixed. In another embodiment, a mixture of the enzyme powder and the substrate powder and an enzyme reaction medium, for example, a buffer solution are separately introduced into a storage container, the storage container is sealed, and then the powder mixture and the reaction medium are mixed. be able to. In addition, the mixture of the enzyme powder and the substrate powder is wrapped in a membrane permeable to water and oxygen and introduced into a storage container so that it can be brought into contact with food and drink at the time of use.

The oxygen scavenger for removing oxygen in the voids of the container is prepared by adding the ascorbate oxidase and its substrate before use to a container having an opening or at least partly composed of a gas-permeable material. Is characterized in that they are housed so that they do not react with each other.
The oxygen scavenger container consumes air in the environment outside the oxygen scavenger container, for example, in the food and drink storage container, when the enzyme that oxidizes ascorbic acid and its substrate react therein. , Having an opening or a gas permeable portion. Various means can be used as means for preventing the enzyme and its substrate from reacting with each other before the use of the oxygen scavenger.

For example, the enzyme solution and the substrate solution may be put in separate containers, and these containers may be housed in the oxygen absorber container so that these solutions can be mixed at the time of use. For example, reverse the oxygen absorber container. Alternatively, an enzyme solution and a substrate solution may be stored in a deoxidizer container with a partition wall, and the partition wall may be destroyed during use. As a specific example, a bag made of a film or a sheet that can be easily broken as the partition wall is used, and when it is used, it can be broken by, for example, a needle-shaped member.

Alternatively, one of the enzyme and its substrate is used as a solution and the other is stored as a dry powder in an oxygen absorber container,
It can be mixed at the time of use. As another method, the enzyme and its substrate may be contained in a dry powder state in a deoxidizer container containing a reaction buffer solution, and the buffer solution and the dry powder may be mixed at the time of use. In the various embodiments described above, the powder can be placed in a bag made of, for example, a film or sheet, and the bag can be broken with a needle to bring the powder into contact with the buffer solution during use.

The enzyme for oxidizing ascorbic acid used in the above method is of plant origin, such as cucumber-derived ascorbate oxidase, microorganism-derived ascorbate oxidase such as acremonium sp. Ascorbate oxidase from ( Acremonium sp .) HI-25 is used. In addition, this Acremonium sp
HI-25-derived ascorbate oxidase is a novel enzyme, and its production method and properties are described in Reference Examples.

In the removal of oxygen in foods and drinks using an enzyme that oxidizes tyrosine or phenols, an enzyme that oxidizes tyrosine or phenols is added to foods and drinks that sufficiently contain tyrosine or phenols, or tyrosine Alternatively, an enzyme that oxidizes tyrosine or phenols and a substrate thereof, such as tyrosin or catechin or other phenols, is added to foods and drinks that do not contain or do not sufficiently contain phenols. As a result, oxygen in the food or drink is consumed when the substrate such as tyrosine or phenols in the food or drink is oxidized by the enzyme that oxidizes tyrosine or phenols, and as a result, deterioration of the food or drink is prevented. The removal of oxygen in the voids of the container using these oxygens can be performed in the same manner as the oxidation of ascorbic acid.

As the oxygen scavenger other than the enzyme for removing oxygen in the voids of the food and drink container, one utilizing the oxidizing effect of iron, such as Ageless FX- sold by Mitsubishi Gas Chemical Co., Inc. 20, etc. can be used. Next, the method of the present invention will be described more specifically by way of examples.

Example 1. Deoxygenation of buffer solution (1) When sodium ascorbate and ascorbate oxidase are added to the buffer solution In a container that can be kept at a constant temperature and a constant temperature, 27 ml of acetic acid buffer solution (pH 4.0) containing 0.1% sodium ascorbate and Acremoni
um sp .HI-25-derived ascorbate oxidase 27
Unit (1 unit is the amount required to oxidize 1 μmol ascorbic acid per minute at 30 ° C) or 0.1
Phosphate buffer (pH 7.0) containing ascorbic acid 27%
ml and 27 units of ascorbate oxidase derived from cucumber, and sealed while leaving 20 ml of air in the container.

The oxygen concentration in the void in the container is measured by Beckman OXY
GEN ANALYZER 39550 OXYGEN ELECTRODE was inserted and measured, and the dissolved oxygen of the buffer solution was measured by DO-METER MODEL TD-100 manufactured by Toko Kagaku Kenkyusho Co., Ltd. The temperature of the container was controlled at 25 ± 0.1 ° C. The results are shown in Table 1.
Although the dissolved oxygen was able to 0ppm within 1 hour, 9.8% in the case of oxygen of the air gap portion containing the ascorbate oxidase from cucumber in the 12 hour hold, Ac
When ascorbate oxidase derived from remonium sp . HI-25 was added, 15.75% oxygen remained.

Table 1 Putting the enzyme and its substrate in the buffer
When O _2: oxygen concentration of the air gap portion (%) DO: buffer dissolved oxygen (ppm) C-ASOD: cucumber origin ascorbate oxidase A-ASOD:. (. Acremonium sp) Acremonium sp HI-25 origin of ascorbic Acid oxidase

(2) When an oxygen absorber is added to the voids 27 ml of an acetic acid buffer solution (pH 4.0) containing 0.1% sodium ascorbate is placed in a container that can be kept at a constant temperature and sealed, and 20 ml of air is placed in the container. And seal. An oxygen absorber is enclosed in the space inside the container so that it does not come into contact with the liquid. The following three types of deoxidizers were used.

Mitsubishi Gas Chemical Co., Ltd. Ageless FX-
20. 3x with oxygen-permeable membrane dincome (made by Unitika)
A 3 cm square pouch is made, and 2 ml of an acetic acid buffer solution (pH 4.0) containing 20% sodium ascorbate and Acremonium sp. ( Acremonium sp .) Encapsulating 300 units of ascorbate oxidase derived from HI-25. 3 x 3 with oxygen-permeable membrane dincome (made by Unitika)
A small square bag with a size of 10 cm is filled with 2 ml of phosphate buffer (pH 7.0) containing 20% sodium ascorbate and 300 units of cucumber-derived ascorbate oxidase. Keep the temperature of the container at 25 ± 0.1 ℃,
The oxygen concentration in the void and the dissolved oxygen in the buffer solution were measured. The results are shown in Table 2.

Table 2 Oxygen concentration in the void and the dissolved oxygen in the buffer when the oxygen absorber is enclosed in the void ─────────────────────── ────── No. 1 2 3 Void area A-ASOD C-ASOD O ₂ DO O ₂ DO O ₂ DO First time 20.3 5.0 20.9 5.4 20.7 6.1 30 minutes 9.8 3.3 17.8 4.8 14.5 4.8 1 hour 3.5 3.2 15.3 4.5 9.7 4.3 1.5 hours 0.0 2.9 13.2 4.1 6.5 4.0 2 hours 0.0 2.7 11.4 3.8 4.4 3.6 4 hours 0.0 2.1 6.5 2.7 1.4 2.4 6 hours 0.0 2.0 4.0 2.3 1.0 1.7 9 hours 0.0 1.8 2.7 1.4 0.8 1.2 12 hours 0.0 1.7 2.1 1.3 0.8 1.0 ──────────────────────────── O ₂ : Oxygen concentration in the void (%) DO: Dissolved oxygen in the buffer (ppm) C-ASOD: Ascorbate oxidase derived from cucumber A-ASOD: Ascorbate oxidase derived from Acremonium sp .

(3) When sodium ascorbate and ascorbate oxidase are put in the buffer solution and an oxygen scavenger is put in the voids. An acetic acid buffer solution (pH 4) containing 0.1% sodium ascorbate in a container that can be kept at a constant temperature and closed. 0.0) 27 ml and Acremonium sp. 27 units of ascorbate oxidase derived from ( Acremonium sp .) HI-25 are placed and sealed while leaving 20 ml of air in the container. An oxygen absorber is enclosed in the space inside the container so that it does not come into contact with the liquid. The following two types of enclosed oxygen scavengers were used.

Mitsubishi Gas Chemical Co., Ltd. Ageless FX-
20. 3x with oxygen-permeable membrane dincome (made by Unitika)
A 3 cm square pouch is made, and 2 ml of an acetic acid buffer solution (pH 4.0) containing 20% sodium ascorbate and Acremonium sp. ( Acremonium sp .) Encapsulating 300 units of ascorbate oxidase derived from HI-25. The temperature of the container was kept at 25 ± 0.1 ° C., and the oxygen concentration in the void and the dissolved oxygen in the buffer solution were measured.

The results are shown in Table 3. When ascorbic acid oxidase was added to the voids as a buffer solution containing ageless, the dissolved oxygen was within 1 hour and the oxygen concentration in the voids was 1.
It could be removed within 5 hours. Further, when an oxygen absorber using ascorbate oxidase was added to the voids ascorbate oxidase in the buffer solution, dissolved oxygen could be removed within 30 minutes and the oxygen concentration in the voids could be removed within 6 hours. ..

From the above, (3) compared with (1) and (2)
It is clear that this is more effective for deoxidation. O ₂ : Oxygen concentration in the void (%) DO: Dissolved oxygen in the buffer (ppm) A-ASOD: Acremonium sp . HI-25-derived ascorbate oxidase

Example 2. Deoxidation of Foods and Drinks Instead of the buffer solution of Example 1, juice (Suntory honey lemon), black tea (black tea in the afternoon of giraffe), and milk were used for deoxygenation. "Suntory honey lemon" is Ageless in the void and Acremonium sp. ( Ac
remonium sp .) HI-25-derived ascorbate oxidase was added, and dissolved oxygen and oxygen concentration in the void were measured.

"Kirin Afternoon Black Tea" is a 3 x 3 cm square pouch made of oxygen-permeable membrane dynecom (made by Unitika) in the void, and 2 ml of acetic acid buffer solution (pH 4.0) containing 20% sodium ascorbate and Acre. Monium sp. ( Acremoni
um sp .) HI-25-derived ascorbate oxidase 300 units were encapsulated, and Acremonium sp. ( Acremonium sp .) HI-25-derived ascorbate oxidase was added to measure the dissolved oxygen and the oxygen concentration in the voids.

Milk is an acetic acid buffer solution (pH 4.0) containing 20% sodium ascorbate in a 3 × 3 cm square pouch made of an oxygen permeable membrane dincome (made by Unitika) in the void.
2 ml and Acremonium sp. ( Acremonium sp .) HI-
Cucumber-derived ascorbate oxidase was put in the liquid part of the one in which 300 units of 25-derived ascorbate oxidase were enclosed, and the dissolved oxygen and the oxygen concentration in the void were measured. In the case of milk, 0.05% sodium ascorbate (vitamin C) was added. The results are shown in Table 4. In each case, dissolved oxygen was within 30 minutes, and oxygen in the voids was ageless for 2 hours. Acremonium sp. ( Ac
Remonium sp .) HI-25-derived ascorbate oxidase could be removed in 6 to 9 hours with an oxygen scavenger.

[0033] of Table 4 food and drink deoxygenated ──────────────────────────── No. 1 2 3 food and drink honey lemon afternoon Black Tea Milk Void Area Ageless A-ASOD A-ASOD Liquid Part A-ASOD A-ASOD C-ASOD O ₂ DO O ₂ DO O ₂ DO Initial 20.8 1.9 20.6 1.7 20.9 5.2 30 minutes 12.8 0.0 14.6 0.0 15.5 0.0 1 hour 5.6 0.0 10.3 0.0 11.4 0.0 1.5 hours 0.8 0.0 7.3 0.0 8.4 0.0 2 hours 0.0 0.0 5.1 0.0 6.1 0.0 4 hours 0.0 0.0 1.2 0.0 1.1 0.0 6 hours 0.0 0.0 0.2 0.0 0.0 0.0 9 hours 0.0 0.0 0.0 0.0 0.0 0.0 12 hours 0.0 0.0 0.0 0.0 0.0 0.0 ──────────────────────────── O ₂ : Oxygen concentration in air (%) DO: Dissolved oxygen (ppm) C-ASOD: Ascorbate oxidase derived from cucumber A-ASOD: Ascorbate oxidase derived from Acremonium sp .

Example 3. Green tea coloring prevention effect (1) 130 cans of green tea (Itoen) in a 160 ml glass bottle
(1) Mitsubishi Gas Chemical Co., Ltd. Ageless FX-20, so as not to touch the liquid in the voids inside the container.
(2) 3 x 3 with an oxygen-permeable membrane dincome (made by Unitika)
A small bag of square cm was prepared, and 2 ml of an acetic acid buffer solution (pH 4.0) containing 20% sodium ascorbate and Acremonium sp. A container containing 300 units of ascorbate oxidase derived from ( Acremonium sp .) HI-25 was placed and the bottle was sealed. Do this for another 50 days at 35 ° C for 50 days
The color strength was compared by storing at 2 ° C. for 2 days. In addition, the one in which no oxygen absorber was added to the void was used as a control. The color was measured by measuring the absorbance at 420 nm with a spectrophotometer UV-2200 manufactured by Shimadzu Corporation, and the coloration-preventing effect was measured by comparing the absorbance of the control with 100.
It is expressed as a ratio when it is defined as%. The results are shown in Table 5.

[0035] A-ASOD: Ascorbate oxidase derived from Acremonium sp .

Example 4. Effect of preventing green tea from coloring (2 ) 130 ml of green tea (Itoen) in a 160 ml glass bottle
And sodium ascorbate 65mg, Acremonium s
p. ( Acremonium sp .) 260 units of ascorbate oxidase derived from HI-25 are filled so that the voids in the container do not touch the liquid (1) Mitsubishi Gas Chemical Co., Ltd. Ageless FX-20, (2) Make a 3 x 3 cm square pouch with an oxygen-permeable membrane Dincome (made by Unitika), and add 20% sodium ascorbate in the acetate buffer (pH
4.0) 2 ml and Acremonium sp. ( Acremonium s
p .) HI-25-derived ascorbate oxidase 30
The one with 0 units enclosed was placed and the bottle was sealed. This was stored at 35 ° C. for 5 days and further at 50 ° C. for 2 days to compare the color strength. In addition, the one in which no oxygen absorber was added to the void was used as a control. The results are shown in Table 6.

[0037] A-ASOD: Acremonium sp . HI-25
Derived ascorbate oxidase

Example 5. Anti-coloring effect of amino acid solution 100 ml of amino acid solution (2% amino acid W of seasoning powder, 5% xylose dissolved in water) was added to a 160 ml glass bottle.
ml, sodium ascorbate 50 mg, Acremonium sp. ( Acremonium sp .) 200 units of ascorbate oxidase derived from HI-25 was filled so that the void in the container did not come into contact with the liquid (1) Mitsubishi Gas Chemical Co., Inc.
Made Ageless FX-20, (2) Oxygen-permeable membrane Dincome (made by Unitika) is used to make a 3 × 3 cm square pouch, and 2 ml of acetic acid buffer solution (pH 4.0) containing 20% sodium ascorbate and acremonium in it. sp. ( Acremonium
sp .) HI-25-derived ascorbate oxidase 3
A unit enclosing 00 units was placed and the bottle was sealed. After holding this in boiling water for 1 hour, the color strength was compared. In addition, the one in which no oxygen absorber was added to the void was used as a control. The results are shown in Table 7.

[0039] A-ASOD: Acremonium sp . HI-25
Derived ascorbate oxidase

Example 6. Effect of Preventing Coloring of White Wine Using white wine (Suntory) instead of green tea, the color intensity was compared in the same manner as in Example 4. This result is the eighth
Shown in the table. A-ASOD: Ascorbate oxidase derived from Acremonium sp .

Example 7. Removal of dissolved oxygen by oxygen absorber
Thermostatically, filled with water and sealed in a container (capacity 47ml) to maintain the,
Put an oxygen absorber in it. The oxygen scavenger used was an oxygen-permeable membrane, Dincome (made by Unitika), and a 3 x 3 cm square pouch was made into which 2 ml of acetic acid buffer (pH 4.0) containing 20% sodium ascorbate and acremmonium s were added.
p. ( Acremonium sp .) HI-25-derived ascorbate oxidase 300 units and iron powder type oxygen scavenger (ageless contents) are used as a small bag (3 x 3 cm square) of an oxygen permeable membrane dincome (made by Unitika). ) Enclosed in two types. This was covered with a lid to prevent air from entering, and dissolved oxygen was measured. The temperature of the container is 25
The temperature was kept at 0 ° C., and the liquid was stirred. The results are shown in Table 9. Dissolved oxygen can be removed by putting an oxygen absorber in the liquid.

[0042] A-ASOD: Ascorbate oxidase derived from Acremonium sp .

Example 8. Polyphenol oxidase
Dissolved oxygen was removed using a polyphenol oxidase in place by the deoxygenation ascorbate oxidase. That is, in the No. 2 of Example 1 (3), 10 mg (30 units) of polyphenol oxidase (Takara Shuzo Co., Ltd.) was added to 27 ml of a 1% catechin (Nacalai Tesque, Inc.) solution instead of A-ASOD to remove dissolved oxygen. I went. The voids were deoxygenated with Ageless FX-20 (Mitsubishi Gas Chemical Co., Ltd.) and the container temperature was kept at 25 ° C. The results are shown in Table 10.

[0044] O ₂ : Oxygen concentration in the void (%) DO: Dissolved oxygen in the buffer (ppm)

As in this example, food or drink is added with a substance that becomes a substrate of an enzyme that oxidizes tyrosine or phenols, or food or drink contains a substance that becomes a substrate of an enzyme that oxidizes tyrosine or phenols. If so, it is possible to remove dissolved oxygen of food and drink by adding an enzyme that oxidizes tyrosine or phenols to these, and it is possible to prevent deterioration of food by combining with an agent that can remove oxygen in voids. Become.

[0046]

As described above, according to the present invention, deterioration of various foods and drinks can be prevented. Reference example 1. Cultivation of Acremonium SP HI-25
Nutrient glycerol 1% (W / V), polypeptone 1% (W /
V), meat extract 1% (W / V), dipotassium phosphate 0.
1% (W / V) and magnesium sulfate heptahydrate 0.000
A liquid medium (pH 6.0) having a composition of 1% (W / V) was dispensed into 180 500-ml shaking culture flasks for each 100 ml, sterilized by a conventional method, and then acremonium sp. After inoculation with HI-25, shake culture was performed at 30 ° C. for 10 days with an amplitude of 7 cm and 120 R.PM to obtain a specific activity of ascorbate oxidase (unit per protein, uni
ts / A280) 0.0589 culture was obtained.

This culture was filtered off using Toyo Filter Paper No. 2 to obtain 11.5 l of culture filtrate. Use this as enzyme solution, 1
Ammonium sulphate was added to 1.5 liter so that it was saturated to 80% 6.45.
Then, kg was added, and static sedimentation was performed in the refrigerator. The supernatant portion was discarded and the precipitated portion was centrifuged to obtain a sedimentation section. 20 mM sodium acetate-hydrochloric acid buffer solution (pH 5.5) (hereinafter,
After suspending in 700 ml of a sodium acetate buffer), it was dialyzed overnight with water as an external solution. After dialysis, 1 liter of DEAE cellulose equilibrated with sodium acetate buffer was added and filtered. The active fraction was eluted with 2 liters of a sodium acetate buffer solution containing 0.1 M sodium chloride in the obtained precipitate fraction, and 80% saturated ammonium sulfate salting out was performed on the eluate to obtain a precipitate fraction in the same manner as described above. After suspending this in 270 ml of sodium acetate buffer, the solid portion was removed by centrifugation to obtain 265 ml of ascorbate oxidase active fraction.

Further, this enzyme solution was equilibrated with sodium acetate buffer containing 0.2 M sodium chloride to obtain Sephacryl S.
Gel filtration was performed on a -300 column (5 x 40 cm) to collect active fractions. Next, this active fraction was dialyzed overnight against 20 mM sodium acetate buffer (pH 5.0). Further, after dialysis, a salt gradient elution was performed using a DEAE-cellulose column (5 × 9 cm) equilibrated with a sodium acetate buffer (pH 5.0) to obtain an active fraction around 0.02 to 0.08M salt concentration. It was

This active fraction was concentrated to 12 ml by vacuum concentration and subjected to gel filtration on a Sephadex G-100 column equilibrated with sodium acetate buffer (pH 5.0) containing 0.2 molar sodium chloride to obtain the active fraction. collected. Next, this activity segment was eluted with a sodium chloride gradient on a TSKgel DEAE-5PW column (5 mm x 5 cm) equilibrated with a sodium acetate buffer (pH 5.0), and activity around 0.04 to 0.06 M sodium chloride concentration was obtained. Purified ascorbate oxidase was obtained. The ascorbate oxidase thus obtained has the following properties.

(1) Optimum action pH Various buffer solutions are used in the pH range of 2.6 to 7.0, and 30
When the reaction was carried out at 5 ° C. for 5 minutes, the results shown in FIG. 1 were obtained. The enzyme of the present invention has an optimum working pH of pH 3.5 to 4.5. (2) pH stability The residual relative activity after incubating this enzyme for 17 hours at 30 ° C. or 4 ° C. at various pH was as shown in FIG. The enzyme of the present invention is stable in the pH range of 4 to 11 at 4 ° C.

(3) Optimum temperature of action This enzyme was treated with sodium acetate buffer (0.5 mM) having a pH of 4.0.
The relative activity when reacted in DTA) at various temperatures for 5 minutes was as shown in FIG. The enzyme acts well at 40 ° C to 55 ° C at pH 4.0. (4) Temperature stability The residual relative activity after incubating this enzyme for 30 minutes at various temperatures in 50 mM phosphate buffer (pH 8.0) was as shown in FIG. The enzyme is stable at 60 ° C or lower under the above conditions.

(5) Molecular weight and subunit structure Using a TSKgel G2000 SW Glass column, bovine serum albumin (molecular weight 66,000) and ribonuclease A (derived from bovine pancreas; molecular weight 13,700) were used as molecular weight standards.
85,000 when measured by gel filtration method using
± 5,000 molecules are shown. By sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis, phosphorylase b (derived from rabbit muscle; molecular weight 94,000), bovine serum albumin (molecular weight 67,000), ovalbumin (derived from egg white; molecular weight 43,000) as a molecular weight standard. ), Carbonic anhydrase (from bovine erythrocyte; molecular weight 30,000), trypsin inhibitor (from soybean; molecular weight 20,100), and α-lactalbumin (from milk; molecular weight 14,400) , A single band corresponding to a molecular weight of 23,000 ± 2,000 is observed. Therefore, this enzyme is presumed to be a tetramer of subunits.

(6) Effect of enzyme inhibitor The residual relative activity after incubation with the following 1 mM concentration of the inhibitor for 10 minutes at 30 ° C. was as follows. ───────────────────────────────── Inhibitor Residual relative activity (%) Control (no addition) 100 N, Sodium N-diethyldithiocarbamate 101 Na ₂ S 3 EDTA 106 Sodium azide 0 ───────────────────────────────── As described above, the enzyme is sodium sulfide ( It is inactivated by Na ₂ S) and sodium azide, but not by sodium N, N-diethyldithiocarbamate and EDTA.

(7) Effects of Metal Ions The effects of various metal ion concentrations of 4.5 mM were as follows. ──────────────────────────── Metal ion (4.5mM) Metal salt used Relative residual activity (%) Reference (no addition) 100 Mg ⁺⁺ MgCl ₂・ 6H ₂ O 79.2 Co ⁺⁺ CoSO ₄・ 7H ₂ O 108 Ca ⁺⁺ CaCl ₂・ 2H ₂ O 105 Zn ⁺⁺ ZnSO ₄・ 7H ₂ O 100 Mn ⁺⁺ MnSO ₄・ 5H ₂ O _{^{113 Fe ++ FeSO 4 · 7H 2}} O 254 Cu ++ CuSO 4 · 5H 2 O 29.8 ──────────────────────────── more As shown in the figure, this enzyme is inactivated by Cu ⁺⁺ ion,
⁺⁺ Activated by ions.

(8) Isoelectric point An isoelectric point of 4.0 is shown when measured by the focus electrophoresis method.

Claims (1)

[Claims] 1. An agent which comprises adding an enzyme that oxidizes ascorbic acid to food or drink, or an enzyme that oxidizes ascorbic acid and its substrate, or is edible and capable of removing dissolved oxygen from food or drink. In addition to removing the dissolved oxygen in the food and drink, it is possible to remove the oxygen in the space by adding an agent capable of removing oxygen to the space in the container containing the food and drink. A characteristic method for preventing deterioration of food and drink.