JP2656444B2 - Oxygen absorber and method for preventing deterioration of food and drink using the same - 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 preventing deterioration of food and drink utilizing consumption of oxygen by a reaction between an enzyme oxidizing ascorbic acid and a substrate thereof, and a deoxidizer therefor.

[0002]

2. Description of the Related Art It is known that oxygen is involved in the deterioration of food and drink, and a method of removing oxygen from food and drink with an inorganic iron agent to prevent deterioration is known. However, this method has various limitations and problems due to the fact that the inorganic iron agent is a non-edible material. There is a method in which glucose oxidase or alcohol oxidase, which is an oxygen-consuming enzyme, is used as an oxygen scavenger instead of an inorganic iron agent. However, these enzymes have a drawback that when they react with their substrates, they produce toxic hydrogen peroxide, which is contaminated in foods and drinks.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a method for preventing deterioration of food and drink by using an enzyme oxidizing ascorbic acid as an oxygen-consuming enzyme which does not have the above-mentioned disadvantages, and a deoxygenating agent therefor. To provide.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present invention provides a method of adding an enzyme oxidizing ascorbic acid to a food or drink, or adding an enzyme oxidizing ascorbic acid and a substrate thereof, Accordingly, a method for preventing deterioration of food and drink is provided, wherein deoxidation is performed in food and drink.
The present invention further provides a method for preventing deterioration of food and drink, wherein the food and drink are sealed in the same container so that the enzyme that oxidizes ascorbic acid and its substrate do not come into direct contact with each other.

[0005] The present invention further provides a container having an opening or at least partially composed of a gas-permeable material, wherein an enzyme oxidizing ascorbic acid and a substrate thereof are provided.
Before use, an oxygen scavenger characterized in that these are accommodated so as not to react with each other is provided. The above methods and oxygen scavengers do not have the disadvantages described above because the reaction of the enzyme that oxidizes ascorbic acid with its substrate produces water instead of hydrogen peroxide.

[0006]

DETAILED DESCRIPTION In the present invention, ascorbic acid oxidase, polyphenol oxidase (laccase) and the like can be used as enzymes for oxidizing ascorbic acid.

[0007] It is known that ascorbic acid oxidase, a kind of enzyme that oxidizes ascorbic acid, is distributed in various plants [Enzyme Handbook (Asakura Shoten) 1
58-159], pumpkins (Men Hui Lee and Charle
s R. Dawson, J. Biol. Chem., Vol. 248, No. 19, 6596-6602, 19
73; and Jean Dayan and Charles R. Dawson, Biochemic
al and Biophysical Research Communication, Vol. 73, N
o.2,451-458,1976) and cucumber (T.Nakamura, N.Mak)
ino and Y. Ogura, J. Biol. Chem., Vol. 64, No. 2, 189-195, 1
968), and these can be used in the present invention.

[0008] Ascorbate oxidases derived from microorganisms include Myrothecium verrulia.
caria ) and those derived from Aerobacter aerogenes (Funaki et al., Journal of the Japanese Society of Nutrition and Food Vol. 40, No. 1, 47-
51, 1987) These can also be used. Ascorbate oxidase, in particular, has the following properties: (1) optimal action pH is 3.5-4.5; (2) activated by Fe ⁺⁺ ions and inhibited by Cu ⁺⁺ ; ( 3) Molecular weight: 85,000 ± 5,0 by gel filtration method
And a molecular weight of 23,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis.
(4) Isoelectric point: pI when measured by focal electrophoresis
(5) Temperature stability: pH 8.0, reaction for 30 minutes, 60 ° C.
And (6) pH stability: exhibiting a stable activity at 4 ° C. in the pH range of 4 to 11. Ascorbate oxidase is preferred.

This ascorbate oxidase belongs to Acremonium ( Acremonium ), and a microorganism capable of producing ascorbate oxidase is cultured, and ascorbate oxidase is collected from the culture to produce ascorbate oxidase. It is manufactured by a method.

Novel ascorbate oxidase (producing strain) As a result of searching for a stock strain and several thousand strains isolated from nature to obtain a microorganism producing ascorbate oxidase having a low optimum pH, it was found that the optimum pH was around pH4.
One strain producing ascorbate oxidase having a pH was obtained.

According to the report of the commissioned test for identification of microorganisms by RIKEN, this strain is identified as Deuteromycotin
a), Acremo belonging to the incomplete filamentous fungus network (Hyphomycetes)
It was identified as a genus niumLink. That is, the results of observation of this strain by an optical microscope are as follows. Form phialide and phyarodine conidia. The phialides are gradually tapered, hardly branching, and the color is unclear.

Phyaroconidium is colorless, smooth, one cell,
Spherical-subspherical (rarely elliptical-oval), 1.5-2 diameter
(〜4) μm, viscous. The amygdala shell that forms slightly later is an incomplete filamentous fungal network, Agyriella Sac
c. Similar to the conidial locus formed by the genus Bacteria. This fruiting body (Conidiomata) is white, sub-spherical-elliptical, diameter 1
Although it is less than mm, it can be visually confirmed and is a naked conidium that is not wrapped by a hypha shell wall or the like, and has a phialide formed on a branched hypha inside.

The phialide is often in the form of a flask, the base of which is subspherical, and the tip of which is extremely tapered and curved.
Phialo- type conidia are morphologically indistinguishable from those of Acremonium synanamorph , but are clumpy, highly prolific, and accumulate between branched hyphae. Therefore, this strain was transformed into Acremonium sp HI-25 ( Acremonium sp HI-25).
-25).

Next, the mycological properties of this strain will be described. (Growing state in each medium) The medium was cultured at 30 ° C. for 14 days on four types of medium (potato, dextrose agar medium, Sabouraud agar medium, malt extract agar medium, YpSs agar medium), and the growing state in each medium was as follows. .

[Potato dextrose agar medium] Growth; late, 6.0 cm in diameter b. Surface: White, short villiform aerial hyphae form dense colonies, exhibit radial grooves at the center, and form a somewhat delayed amygdala-like fruiting body (Conidiomata). Often light brown drops form on the surface. C. Back side: wrinkles, cream color. D. Pigment; no formation.

[Sabouraud agar medium] Growth; late, 5.6 cm in diameter b. Surface; white, short villi-like, with radial grooves with small central ridges. C. Back side: wrinkles, lemon color. D. Pigment; no formation.

[Malt extract agar medium] Growth; slow, 5.1cm in diameter b. Surface: white, short villi, radial grooves near the center. C. Back: wrinkles, yellow-brown. D. Pigment; no formation.

[YpSs agar medium] Growth; late, 6.0 cm in diameter b. Surface: White, short fluffy aerial hyphae are formed and flat colonies are formed. Some time later, the oocyst-like fruiting body (Co
nidiomata). Often light brown drops form on the surface. C. Back side: no wrinkles, cream color. D. Pigment; no formation. (Growing conditions on potato dextrose medium)

[Optimal growth conditions] Optimum temperature 28-31 ° C Optimum growth pH 6.0-7.0 [Range of growth] Temperature range of growth 15-35 ° C pH range of growth 5.0-7.8

The Acremonium spp. Was deposited at the Patented Microorganisms Depositary Center of the Institute of Microbial Industry and Technology, Ministry of International Trade and Industry, as Microbial Laboratories Deposit No. 11236, and was microfabricated on January 29, 1990. Kenjo Yori No. 3124 (FER
MBP-3124) and transferred to an international deposit under the Budapest Treaty. The strain used for producing the ascorbate oxidase of the present invention is not limited to the above-mentioned strain, but it is needless to say that mutants thereof, and other strains that produce the enzyme of the present invention can also be used. .

(Culture) Next, any culture method may be used as a culture method for producing ascorbate oxidase from the genus Acremonium. For example, a liquid culture method will be described below. The medium used in the present invention may be any medium as long as it is a medium belonging to the genus Acremonium and capable of growing ascorbate oxidase-producing microorganisms, for example, used for culturing fungi or actinomycetes. Used medium is used. For example, carbon sources such as glucose, sucrose, glycerin, dextrin, molasses, and organic acids, and one or more selected from, for example, amino acids, vitamins, yeast extracts, meat extracts, koji juice, polypeptone, protein hydrolysates, and the like. Are used, and potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts, sub-salts, inorganic salts such as copper salts or the like, or those in which one or more are appropriately added are preferably used. Can be

The initial pH of the medium is, for example, about 3.5 to 3.5.
9.0, preferably adjusted to about 5.5 to 6.5, and the culture temperature is usually 15 to 42 ° C, preferably about 25 to 35 ° C.
Culture for about 1 to 20 days, preferably about 3 to 12 days. It is preferable to culture under aerobic conditions, for example, by a shaking culture method or an aerobic submerged culture method using a jar fermenter.

(Purification method) Next, the cells are removed from the culture thus obtained to obtain a culture filtrate. Any method may be used to remove the cells from the culture, such as a conventional centrifugation method or a filtration method. Then, ascorbate oxidase is isolated from the culture filtrate thus obtained to obtain the ascorbate oxidase of the present invention.

First, ascorbate oxidase can be isolated and purified from the culture filtrate by any of ammonium sulfate fractionation treatment, alcohol fractionation treatment, DEAE-cellulose treatment, sephacryl treatment, Sephadex treatment, TSK gel DEAE column treatment and the like. These are combined as necessary, and then subjected to dehydration or drying, if necessary, to produce the desired enzyme.

(Method for Measuring Enzyme Activity) The method for measuring the novel ascorbate oxidase activity obtained by the present invention is as follows. 1 ml of a 0.1 M sodium acetate buffer (pH 4.0) containing 0.5 mM ascorbic acid and 0.5 mM disodium ethylenediaminetetraacetate was added to 30 ml.
After preliminarily heating at 5 ° C. for 5 minutes, 0.1 ml of the enzyme solution of the specimen is added and reacted at 30 ° C. for 5 minutes. Then, 3 ml of 0.2M hydrochloric acid is added as a reaction stopping solution, and the absorbance at 245 nm is measured. At this time, the activity is determined from the decrease in the absorbance at 245 nm of the unreacted mixture. One unit is the amount of the enzyme that oxidizes 1 μmol of ascorbic acid per minute under the above-mentioned enzyme activity measurement conditions.

(Properties of the enzyme) The properties of ascorbate oxidase produced by Acremonium sp. HI-25 were as follows. (1) Optimum action pH Using various buffers in the range of pH 2.6 to 7.0, 30
When the reaction was performed at 5 ° C. for 5 minutes, the results shown in FIG. 1 were obtained. The enzyme of the present invention has an optimal action pH between pH 3.5 and 4.5.

(2) pH Stability The residual relative activity after incubating the enzyme at 30 ° C. or 4 ° C. for 17 hours at various pHs was as shown in FIG. The enzyme of the present invention is stable at 4 ° C. in the range of pH 4-11. (3) Optimum temperature of action This enzyme was added to a sodium acetate buffer (0.5 mM
The relative activities when reacted at various temperatures for 5 minutes in DTA) were as shown in FIG. This enzyme works well at 40 ° C to 55 ° C at pH 4.0.

(4) Temperature stability The relative activity remaining after the enzyme was incubated at various temperatures for 30 minutes in a 50 mM phosphate buffer (pH 8.0) was as shown in FIG. The enzyme is 6
Stable below 0 ° C.

(5) Molecular weight and subunit structure Using a TSKgel G2000 SW Glass column, bovine serum albumin (molecular weight 6) was used as a molecular weight standard.
6,000) and ribonuclease A (from bovine pancreas;
When measured by the gel filtration method using a molecular weight of 13,700), it shows about 85,000 ± 5,000 molecules. By sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis, phosphorylase b (derived from rabbit muscle; molecular weight 94,000), bovine serum albumin (molecule 67,000), ovalbumin (derived from egg white; molecular weight 43,000) were used as molecular weight standards. ), Carbonic anhydrase (derived from bovine erythrocytes; molecular weight 30,000),
Trypsin inhibitor (from soybean; molecular weight 20,10)
0) and α-lactalbumin (derived from milk; molecular weight 1)
4,400), the molecular weight is 23,00.
A single band corresponding to 0 ± 2,000 is observed. Therefore, this enzyme is presumed to be a tetramer of the subunit.

(6) Influence of enzyme inhibitor The relative activity remaining after incubation at 30 ° C for 10 minutes with the following 1 mM inhibitor was as follows.

[0031]

[Table 1]

As described above, the present enzyme uses sodium sulfide (N
a ₂ S) and sodium azide.
Sodium N-diethyldithiocarbamate and EDT
A does not inactivate. (7) Effects of metal ions The effects of various metal ions at a concentration of 4.5 mM were as follows.

[0033]

[Table 2] As described above, this enzyme was inactivated by Cu ⁺⁺ ions, and Fe
⁺⁺ Activated by ions. (8) Isoelectric point When measured by a focal electrophoresis method, it shows an isoelectric point of 4.0. Comparison of the above results with known enzymes is as follows.

[0034]

[Table 3]

[0035]

[Table 4]

In the present invention, the enzymes that oxidize ascorbic acid include various ascorbate oxidases and polyphenol oxidases (laccases) including the above-mentioned known enzymes. The present invention provides a method for preventing deterioration of food and drink based on consumption of oxygen by an enzyme that oxidizes ascorbic acid. In a first embodiment of this method, an enzyme that oxidizes ascorbic acid is added to a food or drink that contains enough ascorbic acid, or ascorbic acid is added to a food or drink that does not or does not contain ascorbic acid sufficiently. An oxidizing enzyme and its substrate, such as ascorbic acid or a salt thereof or isoascorbic acid or a salt thereof, are added. Thereby, when a substrate such as ascorbic acid in the food and drink is oxidized by the enzyme that oxidizes ascorbic acid, oxygen in the food and drink is consumed, 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 enzymes already known as described above can be used. However, in the case of acidic foods and drinks such as fruit juices, the known enzymes do not function sufficiently. Therefore, it is preferable to use the enzyme of the present invention having an optimum pH in the acidic region. The amount of enzyme used depends on the type of food and drink, etc., but is generally 0.00 g / g of food and drink.
1 unit or more, and preferably food or drink g or
0.02 units or more per ml. Even if the amount of the enzyme is large, there is no disadvantage such as deterioration of the food, but even if the amount of the enzyme is increased beyond a certain level, the deterioration preventing effect does not always increase correspondingly.

The amount of ascorbic acid or a salt thereof to be 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. To 1.0% by weight, preferably 0.01 to
0.5% by weight. In addition, as a 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.

According to the second aspect of the method for preventing deterioration of food and drink according to the present invention, an enzyme oxidizing ascorbic acid and a substrate thereof such as ascorbic acid or a salt thereof or isoascorbic acid or a salt thereof are added to an aqueous medium such as a buffer. An enzyme consuming system is prepared by dissolving in a liquid, and this and the food and drink are sealed in the same container without bringing them into contact with each other. According to this method, the oxygen in the container is consumed by the oxygen consuming system, and as a result, oxygen in the food and drink is deoxygenated.

In this embodiment, the optimal action of the enzyme used as an aqueous medium constituting the oxygen consuming system, for example, a buffer solution
Use a material having a pH value equal to or close to the pH value. For example, since cucumber-derived ascorbate oxidase has an optimum action pH around 5.6, it is preferable to use a phosphate buffer having a pH of 5 to 7, and the enzyme produced by the Acremonium strain of the present invention is acidic. It is convenient to use, for example, an acetate buffer having a pH of 4 to 5 because it has an optimum action pH in the region.

The quantitative relationship between the enzyme that oxidizes ascorbic acid and its substrate in the second embodiment and the food and drink is similar to that of the first embodiment, but in the second embodiment, The amount of the enzyme and its substrate is preferably slightly larger than that in the first embodiment, because it is necessary to consume oxygen in the environment (that is, in the container) in addition to the above oxygen.

In the implementation of the second embodiment, a solution of the enzyme and a solution of the substrate can be separately prepared, mixed, put into a food and drink storage 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, put the enzyme and its substrate in a food and drink storage container without reacting,
It is preferable to start the enzymatic reaction after sealing the container. For this purpose, for example, the enzyme solution and the substrate solution can be separately housed and put in a storage container, and after sealing the storage container, one liquid can be injected into the other liquid.

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

The present invention further provides an oxygen scavenger for use in a method for preventing deterioration of food and drink. The oxygen scavenger contains ascorbate oxidase and its substrate in a container having an opening or at least partially composed of a gas permeable material such that they do not react with each other before use. It is characterized by the following. The oxygen scavenger container consumes air in the environment outside the oxygen scavenger container, e.g., inside a food storage container, when the enzyme oxidizing ascorbic acid and its substrate react therein. , Openings or gas permeable portions. Various means can be used as means for preventing the enzyme and its substrate from reacting before using the oxygen scavenger.

For example, the enzyme solution and the substrate solution may be placed in separate containers, and these containers may be accommodated in the oxygen absorber container so that these solutions can be mixed at the time of use. For example, the oxygen absorber container is reversed. Alternatively, the enzyme solution and the substrate solution are stored in a deoxygenator container with a partition therebetween, and the partition can be broken at the time of use. As a specific example, a bag made of a film or sheet that can be easily broken is used as the partition wall, and when used, the bag can be broken by, for example, a needle-shaped member.

Alternatively, one of the enzyme and its substrate is made into a solution, and the other is put into a deoxidizer container as a dry powder,
This can be mixed during use. As another method, the enzyme and its substrate are stored in a dry powder state in a deoxidizer container containing a reaction buffer, and the buffer and the dry powder can 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 during use to allow the powder to contact the buffer. Further, in the case of liquid foods and drinks, the object can be achieved by coating a mixture of the enzyme powder and the substrate powder with a film or the like which is permeable to oxygen and water so that the mixture is brought into contact with the food and drink at the time of use.

[0047]

According to the method of the present invention, oxygen is consumed by reacting with an enzyme that oxidizes ascorbic acid and its substrate, that is, ascorbic acid or a salt thereof or isoascorbic acid or a salt thereof, whereby food and drink are consumed. It is possible to prevent the deterioration by deoxygenation. In this case, hydrogen peroxide is not generated as a result of the enzymatic reaction,
Unlike using glucose oxidase or the like, food and drink are not contaminated with harmful hydrogen peroxide.

Further, since the enzyme oxidizing ascorbic acid and its substrate are dissolved in an aqueous solution, the appearance thereof is not impaired even when added to a clear food or beverage. Furthermore, since the enzyme of the present invention produced by the microorganism of the genus Acremonium has an optimum pH in the acidic region, it can be used to prevent deterioration of acidic foods and drinks such as fruit juice.

Thus, according to the present invention, brewed foods and drinks such as miso, soy sauce, alcoholic beverages, juices such as orange juice and tomato juice, favorite drinks such as tea and coffee, retort foods, dairy products, oils and fats, etc. A wide variety of foods and drinks can be prevented from deteriorating. Next, the present invention will be described more specifically with reference to examples.

Example 1 (Reference example) Acremonium S
Cultured glycerol 1% (W / V), polypeptone 1% (W / V)
V), meat extract 1% (W / V), dipotassium phosphate 0.
1% (W / V) and magnesium sulfate heptahydrate 0.000
A 100% liquid medium (pH 6.0) having a composition of 1% (W / V) was dispensed into 180 500 ml shaking culture flasks, sterilized by a conventional method, and then added to Acremonium sp. After inoculation with HI-25, the cells were cultured with shaking at 30 ° C. for 10 days at an amplitude of 7 cm and 120 RPM, and the specific activity of ascorbate oxidase (units per protein, un
(its / A280) 0.0589 culture was obtained.

Example 2 (Reference example) Acremonium S
Purification of ascorbate oxidase from P.HI-25
The culture obtained in manufacturing example 1 was filtered with Toyo filter paper No. 2, to obtain a culture filtrate 11.5 l. This is used as the enzyme solution.
6.45 so that the ammonium sulfate becomes 80% saturated with respect to 1.5 l.
kg, and sedimentation was carried out in a refrigerator. The supernatant was discarded and the sediment was centrifuged to obtain a sedimented fraction. This was suspended in 700 ml of 20 mM sodium acetate-hydrochloric acid buffer (pH 5.5) (hereinafter referred to as sodium acetate buffer),
Dialysis was performed overnight using water as an external solution.

After further dialysis, 1 liter of DEAE cellulose equilibrated with a sodium acetate buffer was added, followed by filtration. The active fraction was eluted with 2 L of a sodium acetate buffer solution containing 0.1 M sodium chloride added to the obtained precipitate fraction, and the eluate was subjected to 80% saturated ammonium sulfate salting out to obtain a precipitate fraction in the same manner as described above. this,
After suspending in 270 ml of sodium acetate buffer, the solid portion was removed by centrifugation to obtain 265 ml of ascorbate oxidase activity section.

Further, Sephacryl S was prepared by equilibrating the enzyme solution with a sodium acetate buffer solution containing 0.2 M salt.
Gel filtration was performed on a -300 column (5 x 40 cm) to collect the active fractions. This active fraction was then dialyzed overnight against a 20 mM sodium acetate buffer (pH 5.0). Further, after dialysis, salt gradient elution was carried out with a DEAE-cellulose column (5 × 9 cm) equilibrated with a sodium acetate buffer (pH 5.0) to obtain an activity class around 0.02 to 0.08 M salt concentration. Was.

This active fraction was concentrated to 12 ml by vacuum concentration, gel-filtered on a Sephadex G-100 column equilibrated with a sodium acetate buffer (pH 5.0) to which 0.2 molar salt was added, and the active fraction was separated. collected. Next, a TSKgel DEAE-5PW column (5 mm × 5 mm) in which this active fraction was equilibrated with a sodium acetate buffer (pH 5.0).
cm), elution with a salt gradient was carried out at 0.04 to 0.
Purified ascorbate oxidase was obtained in the active category near the 06M salt concentration. Total protein for each purification step,
The total activity, specific activity and yield were as shown in the table below.

[0055]

[Table 5]

Example 3 (Example) Tangerines and Valenciao
As an example of a food for preventing deterioration of a range juice, an enzyme prepared by dissolving ascorbic acid oxidase prepared in accordance with Example 2 and freeze-dried ascorbic acid oxidase in a 0.1 mol sodium acetate buffer solution in 100% fruit juice mixed with concentrated and reduced mandarin orange and Valencia orange Liquid (7.0u
nits / ml), and 10 μl of the solution was injected into a dissolved oxygen measurement cell filled with pre-sorted mandarin orange / Valencia orange juice at 25 ° C. to perform deoxygenation. The results were as follows. In the table, cucumber-derived ascorbate oxidase (manufactured by Toyobo Co., Ltd.) 23 units /
The results obtained by injecting 10 μl of the active substance of ml (pH 6.0) into the cell in the same manner as described above and performing deoxygenation are also shown. The dissolved oxygen is measured by DO-METER MODE manufactured by Toko Chemical Laboratory Co., Ltd.
LT-100 was used.

[0057]

[Table 6]

The pH of mandarin orange / Valencia orange juice (manufactured by Glico) was 3.52, and deoxygenation by cucumber-derived ascorbate oxidase at such a low pH was hardly performed. However, when ascorbate oxidase derived from Acremonium sp. HI-25 was used, an oxygen concentration of 0.01 ppm could be achieved in 20 minutes.

Example 4 (Example) As another example of a food for preventing deterioration of milk, a solution obtained by adding and dissolving 0.1% of ascorbic acid to milk was poured into a dissolved oxygen measurement cell, and the milk was heated at 25 ° C.
Was kept at a constant temperature. An enzyme solution prepared by dissolving ascorbate oxidase prepared in accordance with Example 2 and freeze-dried in a 0.1 M phosphate buffer (pH 6.0, 7.0 units / m 2) was used.
Deoxygenation was performed by injecting 50 μl of l). The results are as follows. In the table, 23.0 units / ml of ascorbate oxidase derived from cucumber (pH 6.
The results obtained by injecting 50 μl of the active substance of 0) into the cell in the same manner as described above and performing deoxygenation are also shown.

[0060]

[Table 7]

Since milk (made by Snow Brand) has a high pH of 6.74, even ascorbate oxidase derived from cucumber was able to remove dissolved oxygen close to ascorbate oxidase derived from Acremonium sp. HI-25.

Example 5 (Example) pH 4.5 sodium acetate
Acid by ascorbate oxidase in serum buffer
Enzyme solution prepared by dissolving ascorbic acid oxidase was lyophilized prepared according removal Example 2 containing the pH4.5,0.1 molar sodium acetate buffer (7.0units / ml) 10μl,
The following table shows the results of measuring the dissolved oxygen at 25 ° C. by adding to 0.1 M sodium acetate buffer (pH 4.5) containing 0.1% ascorbic acid. The table also shows the removal of dissolved oxygen by cucumber-derived ascorbate oxidase. Cucumber-derived ascorbate oxidase has an activity of 23 units / ml at pH 6.0.
μl was added and measured as described above.

[0063]

[Table 8]

The removal of dissolved oxygen by ascorbate oxidase at pH 4.5 was carried out by reaction at 25 ° C. for 20 minutes to remove as much as 0.02 ppm of ascorbate oxidase derived from Acremonium sp. HI-25. And that from cucumber could only be removed up to 4.52 ppm oxygen concentration.

Example 6 (Example) pH 6.0 sodium phosphate
By ascorbate oxidase in calcium buffer
Removal of Oxygen 10 μl of an enzyme solution (7.0 units / ml) prepared by dissolving lyophilized ascorbate oxidase prepared according to Example 2 in 0.1 mol phosphate buffer at pH 6.0 was added to 0.1%
It was added to a 0.1 molar sodium phosphate buffer (pH 6.0) containing ascorbic acid, and the dissolved oxygen at 25 ° C. was measured. The table also shows the removal of dissolved oxygen by cucumber-derived ascorbate oxidase. 10 μl of cucumber-derived ascorbate oxidase having an activity of 23 units / ml and pH 6.0 was added in the same manner as described above, and the dissolved oxygen was measured.

[0066]

[Table 9]

When the pH increases, even ascorbate oxidase derived from cucumber, Acremonium sp. H
Dissolved oxygen removal was not significantly different from that of I-25-derived ascorbate oxidase.

Example 7 (Example) Ascorbic acid oxy
Elimination of oxygen in the container with dase In a 20 ml glass container, Beckman OXYGEN ANALYZER 39
550 OXYGEN ELECTRODE was inserted, and 133 mg of ascorbic acid and 8.0 units of ascorbic acid oxidase prepared in Example 2 were dissolved in a 0.1 mol sodium phosphate buffer at pH 6.0 to a volume of 5 ml in a container.
While constantly stirring with a small stirrer, the oxygen content (O ₂ %) in the sealed glass container was measured. The glass container is 2
It was kept at a constant temperature controlled at 5 ± 0.1 ° C. The results are shown in the table below.

The table also shows the removal of oxygen in the container by cucumber-derived ascorbate oxidase. 3. In the case of cucumber-derived ascorbate oxidase.
7 units were used. Other conditions were the same as for Acremonium SP HI-25.

[0070]

[Table 10] When a buffer solution of pH 6.0 is used, it is clear that the amount of oxygen in the container, whether from cucumber or from acremonium, is deoxygenated.

Example 8 (Reference Example) In this reference example, a dry stabilizer of enzyme BSA, ED
When ascorbic acid is oxidized by ascorbate oxidase in the presence of TA or the like, sodium azide (Na
This shows that inactivation of ascorbate oxidase by N ₃ ) is suppressed, and that the ascorbate oxidase of the present invention can be used in a measurement system containing NaN ₃ .

0.1% BSA was added to each 50 mM buffer. Then, each of the ascorbate oxidase addition, even in NaN ₃ silage on the addition of NaN ₃ to be 0.2%, the residual activity was measured after storage for two days 5 ° C. and 28 ° C.. The results are as shown in the table below.

[0073]

[Table 11]

As can be seen from the relative activity when the buffer alone was taken as 100, the decrease in the activity due to the addition of NaN ₃ was more in the case of the acremonium-derived enzyme.
As can be seen from the residual activity at 28 ° C. when the activity was set to be 28 ° C. and the residual activity at 28 ° C. when the activity immediately after the adjustment was set to 100, the decrease in activity due to the addition of NaN ₃ could not be said to be greater for the acremonium-derived enzyme.

In this case, ascorbate oxidase activity was measured as follows. Ascorbic acid activity measurement method for reducing elimination (NTB
Method) After preliminarily heating 0.1 ml of 50 mM potassium dihydrogen phosphate-sodium hydroxide buffer (pH 5.6) containing 1 mM ascorbic acid and 1 mM disodium ethylenediaminetetraacetate at 37 ° C for 5 minutes, Add 0.02 ml of enzyme solution,
After exactly 5 minutes of reaction at 37 ° C., 2 mM nitro blue tetrazolium (NTB) and 4% polyoxyethylene (10) octyl phenyl ether (Triton X)
1.0 ml of 50 mM potassium dihydrogen phosphate-sodium hydroxide buffer (pH 7.6) containing -100) was added to stop the enzyme reaction, and the reaction between NTB and ascorbic acid was carried out at 37 ° C. for 15 minutes. . 0.1% Trit
2.5 ml of 0.1 M hydrochloric acid containing on X-100 was added, and the absorbance at 530 nm was measured. At this time, the activity was determined from the difference in absorbance from the reaction without addition of the enzyme.

Example 9 (Reference example) oxyascorbate
Comparative stability between purified and purified ascorbate oxidase (A. ASOD) derived from Acremonium sp. HI-25 and conventional cucumber derived ascorbate oxidase (C. ASOD) from Amano Pharmaceutical Co., Ltd. The test was performed. A. ASOD and C.I. Both ASOD were sterilized with a 0.2 μm membrane filter, added to a predetermined amount of sterilized 50 mM phosphate buffer (pH 6.0 and 6.85), and stored at 4 ° C. and 30 ° C. for 3 days. did. The results are as shown in the table below.

[0077]

[Table 12]

In this case, the ascorbate oxidase activity was determined by the above-mentioned enzyme activity measurement method. From these results, Acremonium-derived ascorbate oxidase (A. ASOD) has a higher residual activity in any of the first to fourth sections as compared to cucumber-derived ascorbate oxidase (C. ASOD), and is stable. there were.

Example 10 (Reference example) of phospholipids in serum
For the measurement of phospholipids in serum, phospholipase D
(PLD) acts to produce choline, and oxidizes the choline with choline oxidase to produce hydrogen peroxide. This hydrogen peroxide is oxidized by peroxidase, and the chromogen is oxidized and colored by oxygen generated at the same time, and the phospholipid is measured. In this measurement, ascorbic acid in the sample inhibits the peroxidase reaction involved in color development and interferes with the correct quantification of phospholipids. To avoid this, ascorbate oxidase has been used.

Conventionally, cucumber-derived ascorbate oxidase has been used. But,
As shown in the stability test in the previous section, cucumber-derived ascorbate oxidase is inferior in stability, and cannot be stored for a long time after preparing the reagent, which is inconvenient and lossy. , Resulting in higher costs. The use of ascorbate oxidase from acremonium improves these disadvantages.

Preparation of “Merck Type Reagent Solution-1” Phospholipase D (PLD) (Merck enzyme solution-1 (7
7037) 1) and ascorbic acid oxidase (Merck 77049 1) in 100 ml of a 0.5 mmol solution of 3,5-dimethoxy-N-ethyl-N- (2-hydroxy-3-sulfopropyl) -aniline (Merck buffer- 1 (7
7038) Dissolve in 1).

Preparation of "Acremo Type Reagent Solution-1" Phospholipase D (PLD) (Merck enzyme solution-1 (7
7037) 1) and 150 units of ascorbate oxidase derived from acremonium in 100 ml of 0.5 mmol solution of 3,5-dimethoxy-N-ethyl-N- (2-hydroxy-3-sulfopropyl) -aniline (Merck buffer solution- 1 (77038) one).

Using the "Merck type reagent solution-1" and "Accremo type reagent solution-1", the measurement of phospholipids using human serum as a sample is shown in the following table. Although it was not converted to the actual amount of phospholipid in serum, it was shown from the absorbance at 600 nm that a value that was the same even when using ascorbate oxidase from cucumber or using ascorbate oxidase derived from acremonium was obtained. .

In addition to the measurement of phospholipids in Reference Example 3, the present invention can also be used for the measurement of a test drug using ascorbate oxidase, for example, cholesterol, triglyceride, uric acid, free fatty acid and the like.

[0085]

[Table 13]

Example 11 (Reference example) Using an enzyme sensor
Measurement of Ascorbic Acid Ten aminated polyacrylonitrile (PAN) membranes (1.5 cm 2) were added to 5 ml of a 12.5% glutaraldehyde solution and reacted at 0 ° C. for 20 minutes. After washing 500 ml of 0.1 M borate buffer (pH 8.5) membrane, cucumber-derived and the present ascorbate oxidase (1200 units) dissolved in 10 ml of 0.1 M phosphate buffer (pH 7.5) were added, The reaction was carried out at 30 ° C. for 1 hour to immobilize the enzyme on the membrane. 0.1 M phosphate buffer (pH
After washing in 7.5), the immobilized enzyme membrane was attached to the surface of the oxygen electrode and used for measurement of ascorbic acid. Ascorbic acid was measured by adding 30 microliters of a solution containing ascorbic acid to 1.5 ml of a 0.1 M phosphate buffer (pH 7.5) and determining the amount of dissolved oxygen at 37 ° C. The results are shown in FIG. 5 (cucumber-derived enzyme) and FIG. 6 (acremonium-derived enzyme).

Example 12 (Reference Example) The two kinds of immobilized enzyme membranes obtained in Example 11 were stored at 37 ° C. in a 0.1 M phosphate buffer (pH 7.5), taken out with time, and subjected to measurement of ascorbic acid. As shown in FIG. 7, it was found that the use of the acremonium-derived enzyme was more excellent in stability than the cucumber-derived enzyme.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of Acremonium sp. It is a graph which shows the optimal action pH of ascorbate oxidase which HI-25 produces.

FIG. 2 is a graph showing the pH stability of the enzyme.

FIG. 3 is a graph showing the optimal action temperature of the enzyme.

FIG. 4 is a graph showing the temperature stability of the enzyme.

FIG. 5 shows the relationship between L-ascorbic acid concentration in a sample and signal when L-ascorbic acid is measured using a membrane on which cucumber-derived ascorbate oxidase is immobilized (Example 11).

FIG. 6 shows a relationship between L-ascorbic acid concentration in a sample and a signal when L-ascorbic acid is measured using a membrane on which ascorbate oxidase derived from acremonium of the present invention is immobilized ( Example 12).

FIG. 7 is a graph showing the change over time of the performance of the membrane immobilized with acremonium-derived ascorbate oxidase of the present invention and the membrane immobilized with cucumber-derived ascorbate oxidase during the storage period.

Continuation of the front page (72) Inventor Shigeki Fuyasu 3-4 Gotan, Goyucho, Toyokawa-shi, Aichi

Claims (3)

(57) [Claims] (1) The present invention does not contain ascorbate oxidase.
Or the addition of ascorbic acid oxidase to have food and drink,
Alternatively, a method for preventing food and drink deterioration, comprising adding ascorbate oxidase and a substrate thereof to thereby perform deoxidation in the food and drink. 2. A method for preventing deterioration of food and drink, wherein the food and drink, and ascorbate oxidase and its substrate are sealed in the same container so as not to come into direct contact with each other. 3. A container having an opening or at least partially composed of a gas-permeable material, wherein ascorbate oxidase and its substrate are accommodated before use so that they do not react with each other. An oxygen scavenging device characterized by being.