JP6420896B2 - Powder enzyme preparation, production method thereof, and use thereof - Google Patents

Description

The present invention relates to a powder enzyme preparation which is excellent in stability over time of aldehyde dehydrogenase activity and can effectively remove an aliphatic aldehyde, a method for producing the same, and a use thereof.

Aliphatic aldehydes are known to damage DNA and cause symptoms of lifestyle-related diseases and gastrointestinal cancers such as the oral cavity and stomach. Many of the aliphatic aldehydes have a specific odor. For example, acetaldehyde which is a causative substance of body odor and tobacco odor, nonenal which is a causative substance of aging odor, and the like are known.
In recent years, there are an increasing number of people who prefer natural things to live a healthy life and those who take care of body odors so as not to be distracted by the people around them. These aliphatic aldehydes are effective with the power of natural enzymes. It is expected to provide products that can be removed easily.

Aldehyde dehydrogenase can convert an aliphatic aldehyde into a carboxylic acid, and thus can be effectively used to decompose aliphatic aldehyde accumulated in and outside the body.
However, aldehyde dehydrogenase is easily affected by external factors such as temperature, humidity, oxygen, etc., and its activity is rapidly reduced during production processing and storage, losing aldehyde resolution.
Therefore, it is extremely difficult to stabilize aldehyde dehydrogenase activity over time and to decompose and remove aldehyde using this activity, particularly at room temperature and in the presence of oxygen.

Conventionally, as a technique for stabilizing enzyme activity over time, a method of adding proteins such as skim milk and albumin, sugars such as sucrose and maltose, polyhydric alcohols and the like is known.
Patent Document 1 describes cosmetics in which enzyme activity is stabilized over time by complexing metal oxide colloidal fine particles having a particle size of 200 μm or less and an enzyme in an ionic interaction manner.

However, when the inventor tried to stabilize the aldehyde dehydrogenase activity over time using the above-described conventional technology, none of the effects of suppressing the decrease in enzyme activity was obtained. In the above prior art, stabilization of enzyme activity over time has been studied for proteolytic enzymes, hydrolases, lipolytic enzymes, etc., and the technology for stabilizing aldehyde dehydrogenase activity over time has not yet been established. I found out.

JP-A-10-251122

An object of the present invention is to provide a powder enzyme preparation which is excellent in aging dehydrogenase activity over time even at room temperature and can effectively remove an aliphatic aldehyde, a method for producing the same, and a use thereof. .

The present inventor has intensively studied to achieve the above object.
as a result,
A powder enzyme preparation comprising aldehyde dehydrogenase and two or more free amino acids or salts thereof,
The two or more free amino acids or salts thereof are adsorbed to aldehyde dehydrogenase,
Surprisingly, the present inventors have found that the influence of the external environment such as temperature, humidity, oxygen, etc. is alleviated, and that the aldehyde dehydrogenase activity is stable over time even at room temperature, and the present invention has been completed.

That is, the present invention
(1) A powder enzyme preparation comprising two or more free amino acids or salts thereof in aldehyde dehydrogenase,
The two or more free amino acids, or salts thereof ,
One or more selected from acidic amino acids glutamic acid and aspartic acid, or salts thereof;
Including at least one selected from basic amino acids lysine and arginine, or salts thereof,
The two or more free amino acids or salts thereof are adsorbed to aldehyde dehydrogenase,
Powder enzyme preparation.
(2) In the powder enzyme preparation of (1),
For the total amount of free amino acids contained in the powder enzyme preparation,
One or more selected from the above-mentioned glutamic acid, aspartic acid, or salts thereof, which are acidic amino acids;
The total content of the basic amino acids and the lysine and arginine, or one or more thereof selected from salts thereof is 25% or more in terms of free amino acids,
Powder enzyme preparation,
(3) In the powder enzyme preparation of (1) or (2),
For the total amount of free amino acids contained in the powder enzyme preparation,
The total content of the glutamic acid and aspartic acid that are acidic amino acids, or salts thereof is 15% or more and 85% or less in terms of free amino acids,
And the total content of the lysine and arginine, or salts thereof, which are basic amino acids, is 15% or more and 85% or less in terms of free amino acids,
Powder enzyme preparation.
(4) To aldehyde dehydrogenase,
One or more selected from acidic amino acids glutamic acid and aspartic acid, or salts thereof;
Formulated with basic amino acids lysine and arginine, or one or more selected from salts thereof,
Powder enzyme preparation,
(5) A method for producing a powder enzyme preparation according to any one of (1) to (4) ,
The aldehyde dehydrogenase;
The glutamic acid and aspartic acid that are acidic amino acids, or salts thereof;
After mixing the basic amino acids lysine and arginine, or salts thereof in solution,
Drying the resulting mixture,
A method for producing a powder enzyme preparation.
(6) In the method for producing a powdered enzyme preparation of (5) ,
The drying of the mixture is lyophilization,
A method for producing a powder enzyme preparation ,
(7) containing the powdered enzyme preparation according to any one of (1) to (4) ,
Food,
(8) The powder enzyme preparation according to any one of (1) to (4), or the powder enzyme preparation obtained by the production method according to (5) or (6),
Food manufacturing methods,
(9) containing the powder enzyme preparation of any one of (1) to ( 4) ,
Cosmetics,
(10) The powder enzyme preparation according to any one of (1) to (4) or the powder enzyme preparation obtained by the production method of (5) or (6) is blended,
Manufacturing method of cosmetics,
It is.

According to the present invention, a powder enzyme preparation excellent in aging dehydrogenase activity over time can be produced at low cost and in large quantities. Furthermore, aldehyde dehydrogenase activity can be maintained even in foods and cosmetics stored for several years at a room temperature of about 25 ° C.
Amino acids are conventionally used in foods and cosmetics and can be used safely.
Therefore, the powdered enzyme preparation of the present invention is harmful to living organisms and has an unpleasant odor when used as a food, supplement, food additive, oral preparation, oral cleanser, cosmetic, external preparation, deodorant, etc. It becomes possible to remove the causative aliphatic aldehyde.
Therefore, it can contribute to improvement of people's health and living environment.

The present invention will be described in detail below.
In the present invention, “%” means “mass%” and “part” means “part by mass”.

<Features of the present invention>
The powder enzyme preparation of the present invention is a powder enzyme preparation comprising aldehyde dehydrogenase and two or more free amino acids or salts thereof, wherein the two or more free amino acids or salts thereof are aldehyde dehydrogenation. By being adsorbed to the enzyme, the influence of the external environment such as temperature, humidity, oxygen, etc. is alleviated, and the aging dehydrogenase activity is stable over time even at room temperature.

<Average particle size of powder enzyme preparation>
The powdered enzyme preparation of the present invention includes two or more free amino acids or salts thereof mixed with aldehyde dehydrogenase, and the two or more free amino acids or salts thereof are adsorbed on aldehyde dehydrogenase. It is a powder.
The average particle diameter of the powder enzyme preparation is not particularly limited as long as the effect of the present invention is exerted, and may be, for example, about 1 to 1000 μm.

<Aldehyde dehydrogenase>
The aldehyde dehydrogenase contained in the powdered enzyme preparation of the present invention may be plant-derived or animal-derived, but is derived from microorganisms such as yeast and acetic acid bacteria that have been widely used in the field of food industry and the like. It is preferable to use those derived from acetic acid bacteria with particularly high specific activity.
Further, since the aldehyde dehydrogenase derived from microorganisms exists as a complex penetrating the cell membrane, the raw material of the aldehyde dehydrogenase of the present invention includes the isolated aldehyde dehydrogenase alone. Alternatively, the complex integral with the cell membrane may be used.
When using a single enzyme, for example, a surfactant is added to isolate and purify the enzyme. When the complex integrated with the cell membrane is used, for example, the cells are crushed and the cytoplasm is removed by a treatment such as a French press. Alternatively, the cells are used as they are without being disrupted.
The aldehyde dehydrogenase thus obtained may be subjected to a treatment such as drying within a range not impairing the effects of the present invention.
In the powdered enzyme preparation of the present invention, when the complex integrated with a cell membrane is used as an aldehyde dehydrogenase, the temporal stability of the aldehyde dehydrogenase activity increases.
Furthermore, when a cell itself that is not disrupted, for example, a microbial cell itself, is used as the complex integral with the cell membrane, the stability over time is excellent. In particular, when a microbial cell itself is used as a dead cell, alteration due to the metabolic action of live cells is suppressed, and stability over time is excellent.
Unless otherwise specified, hereinafter, the complex integrated with the cell membrane or a crushed product thereof is abbreviated as aldehyde dehydrogenase.

<Free amino acids or salts thereof>
The free amino acids blended in the powdered enzyme preparation of the present invention are 20 types constituting the protein.
Specifically, alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, aspartic acid, glutamic acid, arginine, histidine, lysine.
Examples of the free amino acid salts include the 20 types of sodium salts, potassium salts, hydrochlorides, and other salts.
The free amino acid used in the present invention may be any optical isomer of L-form, D-form, or DL-form, and may be a hydrate.
In addition, the compounding quantity of a free amino acid or those salts should just be the quantity which has the effect of this invention, and should just be 1 to 99% in conversion of a free amino acid with respect to powder enzyme preparation whole quantity specifically ,. The increase or decrease in the amount of free amino acid during storage is negligible relative to the amount of the free amino acid of the present invention or a salt thereof, and does not affect the amount.

<Glutamic acid, aspartic acid, lysine, arginine>
When the powdered enzyme preparation of the present invention contains two or more free amino acids selected from glutamic acid, aspartic acid, lysine, and arginine among the 20 types of free amino acids, or aldehyde dehydrogenase activity. Excellent in stability over time.

<Total content of glutamic acid, aspartic acid, lysine and arginine>
When the powdered enzyme preparation of the present invention contains 4 kinds of free amino acids of glutamic acid, aspartic acid, lysine and arginine, or salts thereof in terms of free amino acids, 25% or more, and further 35% or more of the total amount of free amino acids, aldehyde dehydrogenation Excellent stability over time of enzyme activity.
The upper limit of the total content of the above four kinds of free amino acids or salts thereof is not particularly limited as long as the effect of the present invention is exhibited, but specifically, it is 100% or less.
In the present invention, the amount of free amino acid means an amino acid that contributes to the stabilization of aldehyde dehydrogenase activity over time, that is, a free amino acid present in a powder enzyme preparation, or a salt thereof in terms of free amino acid. It means the value measured, and does not include amino acids that exist as proteins or peptides.
The amount of free amino acid may be determined by a commonly used measurement method. For example, as in Test Example 2 described later, ion-exchanged water is added to a powdered enzyme preparation, shaken, centrifuged, and the supernatant is obtained. To separate. Sulfosalicylic acid is added to the separated supernatant for protein removal treatment, followed by shaking and centrifugation. The obtained supernatant is dried under reduced pressure, dissolved by adding a citrate buffer, and measured using an amino acid automatic analyzer.

<Optimal combination of glutamic acid, aspartic acid, lysine and arginine>
Furthermore, the powder enzyme preparation of the present invention is one or more selected from acidic amino acids glutamic acid and aspartic acid, or salts thereof, and basic amino acids lysine and arginine, or salts thereof. In combination, the aldehyde dehydrogenase activity is excellent in stability over time.

<Contents of acidic amino acids and basic amino acids>
The aldehyde dehydrogenase activity of the powder enzyme preparation of the present invention is such that the total content of glutamic acid and aspartic acid, which are acidic amino acids, or salts thereof is 15 in terms of free amino acid, relative to the total amount of free amino acids contained in the powder enzyme preparation. When the total content of lysine and arginine, which are basic amino acids, or salts thereof is 15% or more and 85% or less in terms of free amino acids, the stability over time is excellent.
Further, the total content of the acidic amino acids or salts thereof is 25% or more and 75% or less in terms of free amino acids, and the total content of the basic amino acids or salts thereof is 25% or more and 75 in terms of free amino acids. When it is at most%, the stability over time is particularly excellent.

<Action of free amino acids or salts thereof>
Although the mechanism of action of the two or more free amino acids or their salts on the stabilization of the aldehyde dehydrogenase activity over time is not clear, the presence of different types of free amino acids makes it possible to control temperature, humidity, oxygen, etc. It is thought that the influence of external factors is alleviated and contributes to stabilization of aldehyde dehydrogenase activity over time at room temperature.
In particular, the four free amino acids, glutamic acid, aspartic acid, lysine, and arginine, or their salts are all polar and exhibit aldehyde dehydration by developing an electrostatic interaction with aldehyde dehydrogenase. It is considered to be advantageous for stabilizing the enzyme activity over time.
Further, under neutral conditions, acidic amino acids glutamic acid and aspartic acid are negatively charged, and basic amino acids lysine and arginine are positively charged. In addition to strengthening the electrostatic interaction expressed with aldehyde dehydrogenase by adding free amino acids having different positive and negative charges, or salts thereof, free amino acids or salts thereof It is considered that this interaction also strengthens and contributes greatly to the stabilization of aldehyde dehydrogenase activity over time.

<Content ratio of aldehyde dehydrogenase and free amino acids or salts thereof>
The content ratio of free amino acids or salts thereof in the powdered enzyme preparation of the present invention is 0.1 parts or more and 20 parts or less in terms of free amino acids with respect to 1 part of the aldehyde dehydrogenase converted in terms of microorganism, When it is 2 parts or more and 15 parts or less, the stability over time of the aldehyde dehydrogenase activity is excellent.

<Other raw materials>
In the powdered enzyme preparation of the present invention, raw materials other than the essential aldehyde dehydrogenase and two or more free amino acids or salts thereof are appropriately selected and blended within a range not impairing the effects of the present invention. Can do.
Specifically, glucose, galactose, mannose, lactose, maltose, sucrose, mannitol, trehalose, starch hydrolyzate, starch syrup, reduced starch syrup, starch, pectin, cellulose and other sugars, whole milk powder, skim milk powder, casein, albumin Such as protein, ascorbic acid, tocopherol, tannin, catechin, curcumin, hesperidin, antioxidants such as polyphenols such as isoflavone, various surfactants, jojoba oil, palm oil, shea fat, diatomaceous earth, silica, activated carbon, etc. Porous materials and the like.

<Method for producing powdered enzyme preparation>
As described above, the powder enzyme preparation of the present invention is a plant-derived or animal-derived aldehyde dehydrogenase, preferably one derived from bacteria such as yeast and acetic acid bacteria, which have been widely used in the field of food industry, etc. The one derived from acetic acid bacteria with high specific activity is used. And it can manufacture as follows, for example by adding 2 or more types of free amino acids, or those salts to an aldehyde dehydrogenase.

<Preparation of aldehyde dehydrogenase derived from acetic acid bacteria>
Acetic acid bacteria are cultured in a medium containing at least a carbohydrate derived from glucose, vitamins and minerals derived from yeast extract, and ethanol.
In this case, the ethanol concentration in the medium is 3% or more, the product temperature is 25 to 35 ° C., and the aeration rate is preferably cultured under culture conditions of 0.02 to 5 L / min per 1 L of the medium.
This culture is performed at a degree of acetic acid of less than 4% in the culture medium. That is, with the growth of acetic acid bacteria in the medium, ethanol in the medium is oxidized to acetic acid through aldehyde, and the acetic acid degree in the medium rises, but in the present invention, before the acetic acid degree reaches 4%, Preferably, the culture is stopped when the acetic acidity is 0.5% or more and 3.9% or less, particularly 1% or more and 3.5% or less.
The acetic acidity is calculated by adding sodium hydroxide dropwise until neutralization using the color reaction of phenolphthalein as an index, and in terms of the amount of sodium hydroxide dropped.

The culture solution after the culture is stopped contains aldehyde dehydrogenase as the complex integrated with the cell membrane of acetic acid bacteria. Further, the culture solution contains fermentation components such as acetic acid produced by the culture in addition to the medium components.
In the present invention, from the viewpoint of stabilizing the activity of aldehyde dehydrogenation with time, it is preferable to separate an acetic acid bacterium containing aldehyde dehydrogenase and the fermentation component.
As a method for separation, for example, centrifugation (gravity conversion, 1000 to 20000 g), membrane concentration treatment, drying treatment, and the like are performed, and a fraction of acetic acid cells containing aldehyde dehydrogenase is fractionated.
Next, washing may be performed to remove medium components and fermentation components from the fraction of acetic acid bacteria.
The washing method uses, for example, the feature that the concentrated fraction of acetic acid bacterial cells is insoluble in water, and after diluting and mixing in a buffered aqueous solution of citric acid or phosphoric acid, it is centrifuged again to remove the supernatant. To remove medium components and fermentation components.

The acetic acid bacterium containing the aldehyde dehydrogenase thus obtained can be used as it is as an aldehyde dehydrogenase, and can be used as a dead bacterium by subjecting it to treatment such as heating and oxygen blocking.
In addition, since aldehyde dehydrogenase is present in the bacterial membrane, the cell body may be subjected to treatment with ultrasonic waves, French press, etc. to use a cell membrane crushed material, and a surfactant or enzyme is allowed to act on the aldehyde dehydration. You may use as an enzyme simple substance which fractionated the elementary enzyme.

<Free amino acid mixing step>
Next, two or more free amino acids or their salts are mixed with the aldehyde dehydrogenase described above.
The mixing method is roughly divided into a method of mixing powders and a method of mixing a part or all of them in a solvent and drying.

<Mixing process>
The aldehyde dehydrogenase produced by the above method and two or more kinds of free amino acids or salts thereof are mixed, and the free amino acids or salts thereof are adsorbed on the aldehyde dehydrogenase.
The mixing method includes, for example, a method of mixing an aldehyde dehydrogenase powder and a free amino acid or a salt thereof, a method of mixing an aldehyde dehydrogenase powder and a free amino acid or a salt solution thereof, an aldehyde dehydrogenase And free amino acids or their salts are mixed in a solution.
When the aldehyde dehydrogenase powder and the free amino acid or their salt powder are mixed, for example, the powder may be pulverized by a conventional method such as adding both into a rotating cylinder and granulating by rolling.
When mixing a solution of aldehyde dehydrogenase powder and free amino acid or salt thereof, spray the solution of free amino acid or salt thereof to aldehyde dehydrogenase powder by a usual method such as spraying, etc. And mix.
When mixing an aldehyde dehydrogenase and a free amino acid or a salt thereof in a solution, the aldehyde dehydrogenase and the free amino acid or a salt thereof may be added to the solvent and mixed. You may mix a liquid and the solution of free amino acids, or those salts.
The solvent used in the solution may be appropriately selected within a range that does not impair the effects of the present invention, such as fresh water and pH buffer solution.
In this step, other raw materials such as commonly used excipients such as sugars can be blended within a range not impairing the effects of the present invention.

Among the above mixing methods, in particular, when a part or all of a free amino acid or a salt thereof is used after mixing as a solution, since the free amino acid or a salt thereof exists in a molecular state, it is compared with the case of mixing powders. Thus, free amino acids or their salts are easily adsorbed to the aldehyde dehydrogenase, and the stability over time of the aldehyde dehydrogenase activity is excellent.

<Drying process>
In the production method in which part or all of the raw material is used for mixing as a solution, it is pulverized by a conventional method such as spray drying or freeze drying.
When spray drying is performed, for example, the solution is sprayed and dried at an inlet temperature of 100 to 130 ° C and an outlet temperature of 50 to 70 ° C.
When lyophilization is performed, for example, the solution is poured into a metal vat and frozen at -20 to -50 ° C, and then the shelf is heated to 5 to 50 ° C under reduced pressure to remove the solvent and dry.
In the present invention, the aldehyde dehydrogenase activity of the obtained powder enzyme preparation is excellent in stability over time when pulverized by freeze-drying in which the influence of heat on the aldehyde dehydrogenase is suppressed.
In addition, when mixing the solution of aldehyde dehydrogenase powder and a free amino acid or those salts, you may perform the said mixing process and the said drying process simultaneously like the method of granulation normally implemented.

<Application of powder enzyme preparation>
The powder enzyme preparation of the present invention has an effect of removing aliphatic aldehydes.
In particular, it has a special effect in removing aliphatic aldehydes having 1 to 10 carbon atoms and not containing nitrogen atoms.
Specific examples include formaldehyde, acetaldehyde, butanal, glyceraldehyde, malondialdehyde, pentanal, hexanal, heptanal, octanal, nonenal, decanal, isooctanal, isononenal, isodecanal and the like.

The powdered enzyme preparation of the present invention can be used in any scene where an aliphatic aldehyde is desired to be removed.
For example, in order to remove an in vivo or in vitro aliphatic aldehyde, it may be taken orally or applied as an external preparation.
Aldehydes have a concentration of only 0.1 to several tens of ppm at the time of volatilization and cause oxidative stress and unpleasant odors on living bodies.
When the powdered enzyme preparation of the present invention is used by oral ingestion or application, the amount used may be such that aldehyde can be decomposed and removed, and can be, for example, 1 to 100 mg per oral ingestion or 100 square centimeters of application. .
In addition, the powder enzyme preparation of the present invention is for removing aldehydes in the environment such as odors of buildings and public facilities where people gather.
You may apply | coat or spray on building materials, such as a wall material.

<Food containing powdered enzyme preparation>
When the powder enzyme preparation of the present invention is used for food, the following raw materials can be used in combination as long as the effects of the powder enzyme preparation of the present invention are not impaired.
Specifically, sweeteners such as trehalose, aspartame, phenylalanine, acidulants such as citric acid, acetic acid, lactic acid, phosphoric acid, citrus juice, glucose, sucrose, lactose, salt, soy sauce, other seasonings, pectin, Gelatin, agar, alginic acid, soybean polysaccharide, thickeners such as cellulose, xanthan gum, guar gum, carrageenan, cosmetic raw materials such as collagen, hyaluronic acid, placenta, coenzyme Q10, chondroitin, royal jelly, turmeric, liver extract, caihi, fennel , Herbal medicines such as peony, licorice, fragrances, pigments, preservatives and the like.

<Cosmetics containing powdered enzyme preparation>
When the powder enzyme preparation of the present invention is used in cosmetics, the following raw materials can be used in combination as long as the effects of the powder enzyme preparation of the present invention are not impaired.
Specifically, porous fine particles such as diatomaceous earth, activated carbon and silica, clay minerals such as talc, mica, bentonite, kaolin and sericite, polysaccharides such as cellulose, carboxymethylcellulose, dextrin, cyclodextrin, corn starch and potato starch , Plant extracts such as polyphenol, eucalyptus extract, catechin, evening primrose extract, silicone oil such as polysiloxane, cyclic siloxane, isopropyl myristate, paraffin, squalane, beeswax, palm oil, jojoba oil, shea fat, etc., ethanol , Isopropylmethylphenol, benzalkonium chloride, triclosan, chlorhexidine, trichlorocarbanilide, methylparaben, ethylparaben, propylparaben, phenoxyethanol, lysozyme chloride, silver compound Bactericides or preservatives such as aluminum chloride, chlorohydroxyaluminum, alum, paraphenol sulfonic acid, zinc oxide, hydroxyapatite, magnesium oxide, calcium carbonate, tartaric acid, etc., astringent or deodorant, glycyrrhizic acid, urea, ougon extract, Anti-inflammatory agents such as buckwheat extract, collagen, hyaluronic acid, coenzyme Q10, chondroitin, placenta, ceramide, elastin, astaxanthin, resveratrol, lycopene, royal jelly, aloe extract, platinum nanocolloid, retinoic acid, retinol, hydrolyzed wheat, Beauty ingredients such as winged bean extract, lactoferrin, fermented soy milk, EGF, whitening ingredients such as vitamin C derivatives, arbutin, fullerene, tranexamic acid, hydroquinone, and yokuinin extract UV absorbers such as titanium oxide, benzophenone derivatives, paraaminobenzoic acid derivatives, methoxycinnamic acid derivatives, humectants such as pentylene glycol, hexanediol, caprylyl glycol, glycol, propylene glycol, butylene glycol, polyethylene glycol, adenosine, alkylbenzene Anionic surfactants such as sulfonate, polyoxyalkylene alkyl ether sulfate, alkyl sulfate ester, olefin sulfonate, fatty acid salt, dialkylsulfosuccinate, polyethylene glycol hydrogenated castor oil, polyoxyethylene fatty acid ester, Nonionic surfactant such as polyoxyethylene hydrogenated castor oil derivative, alkyltrimethylammonium salt, dialkyldimethylammonium salt, alkylpyridinium Cationic surfactants such as salt, stearyltrimethylammonium chloride, amphoteric surfactants such as alkylbetaine, alkylamidopropylbetaine, imidazolinium betaine, egg yolk lecithin, soybean lecithin, various amino acids, various vitamins, various peptides, Examples include higher fatty acids, polyhydric alcohols, thickeners, carbonates, emulsifiers, and fragrances.

Hereinafter, the present invention will be specifically described based on examples, comparative examples, and test examples. The present invention is not limited to these.

[Preparation Example 1]
Acetic acid bacteria (Gluconacetobacter konbuchae NBRC 14816 strain) 0.1% was added to ethanol 4%, yeast extract 0.2%, and fresh water 95.7%, the product temperature was 30 ° C, and the aeration rate per culture solution was 0.4L. The culture was performed for 24 hours under the conditions of / min.
The obtained acetic acid bacterium solution was subjected to centrifugal concentration treatment (10000 g) and washed several times with fresh water to prepare a 10% acetic acid bacterium solution.
The obtained solution was subjected to a French press treatment to crush and kill acetic acid bacteria, thereby obtaining a 10% aqueous solution of aldehyde dehydrogenase (the complex in which the enzyme is integrated with the cell membrane) in terms of microorganisms.

[Preparation Example 2]
In the same manner as in Preparation Example 1, except that acetic acid bacteria were replaced with Acetobacter Aceti NBRC 14818 strain, an aqueous solution of 10% aldehyde dehydrogenase (the above-mentioned complex in which the enzyme was integrated with the cell membrane) in terms of microorganism was obtained.

[Example 1]
10% aldehyde dehydrogenase of Preparation Example 1 (the complex in which the enzyme is integrated with the cell membrane), 3% sodium L-glutamate, 3% sodium L-aspartate, 3% L-arginine, 10% dextrin, fresh water 71% was stirred and mixed. This is rapidly frozen at a product temperature of −30 ° C., and then subjected to a vacuum freeze-drying treatment at a shelf temperature of 15 ° C. under reduced pressure, and free amino acids or salts thereof are adsorbed on the aldehyde dehydrogenase. A formulation was obtained.

[Example 2]
Except that L-arginine was replaced with lysine, all were prepared according to Example 1 to obtain a powdered enzyme preparation of the present invention in which a free amino acid or a salt thereof was adsorbed on an aldehyde dehydrogenase.

[Example 3]
L-sodium glutamate 3%, L-sodium aspartate 3%, L-arginine 3%, dextrin 10%, L-glutamate 6.5%, L-arginine 2.5%, mannitol 2.5%, fresh water The powder enzyme preparation of the present invention was prepared in the same manner as in Example 1 except that 13.7% was replaced, and free amino acids or their salts were adsorbed to aldehyde dehydrogenase.

[Example 4]
L-sodium glutamate 3%, L-sodium aspartate 3%, L-arginine 3%, sodium L-aspartate 3%, lysine 6%, glycine 0.2%, dextrin 2.5%, fresh water 4.5 The powder enzyme preparation of the present invention in which free amino acids or their salts were adsorbed to aldehyde dehydrogenase was prepared in the same manner as in Example 1 except that% was replaced.

[Example 5]
Other than replacing 3% sodium L-glutamate, 3% sodium L-aspartate, 3% L-arginine with 1% sodium L-glutamate, 1% sodium L-aspartate, 1% L-arginine, 6% fresh water Were prepared according to Example 1 to obtain a powdered enzyme preparation of the present invention in which a free amino acid or a salt thereof was adsorbed on an aldehyde dehydrogenase.

[Example 6]
L-sodium glutamate 3%, L-sodium aspartate 3%, L-arginine 3%, sodium L-glutamate 0.1%, sodium L-aspartate 0.1%, L-arginine 0.2%, glycine Except for the replacement with 0.6% and fresh water 8%, all were prepared according to Example 1 to obtain a powdered enzyme preparation of the present invention in which free amino acids or their salts were adsorbed to aldehyde dehydrogenase. .

[Comparative Example 1]
Prepared according to Example 1 except that 3% of sodium L-glutamate, 3% of sodium L-aspartate, and 3% of L-arginine were replaced with 9% of glycine. A powdered enzyme preparation adsorbed with these salts was obtained.
The residual activity with respect to the initial activity of aldehyde dehydrogenase determined from the measurement of aldehyde dehydrogenase activity was 0%.

[Comparative Example 2]
Prepared according to Example 1 except that 3% sodium L-glutamate, 3% sodium L-aspartate and 3% L-arginine were replaced with 9% sodium L-glutamate, and released to aldehyde dehydrogenase. A powdered enzyme preparation adsorbed with amino acids or salts thereof was obtained.
The residual activity with respect to the initial activity of aldehyde dehydrogenase determined from the measurement of aldehyde dehydrogenase activity was 4%.

[Comparative Example 3]
Prepared according to Example 1 except that 3% sodium L-glutamate, 3% sodium L-aspartate, and 3% L-arginine were replaced with 9% L-arginine. Or a powdered enzyme preparation adsorbed with salts thereof.
The residual activity with respect to the initial activity of aldehyde dehydrogenase determined from the measurement of aldehyde dehydrogenase activity was 10%.

[Test Example 1] Effect of aldehyde dehydrogenase on aging stability due to blending of free amino acids 0.05 g of the powdered enzyme preparations obtained in Examples 1 to 6 and Comparative Examples 1 to 3 were placed on a 5 cm square aluminum pouch. Encapsulated and subjected to accelerated storage test at 37 ° C. for 2 months.
The temporal stability of the aldehyde dehydrogenase was determined using the following free amino acid content measurement method, aldehyde dehydrogenase activity measurement method, and residual activity evaluation criteria.
<Method for measuring free amino acid content>
Add 10 mL of ion-exchanged water to 2 g of the powdered enzyme preparation, shake for 15 minutes, heat at 50 ° C. for 30 minutes, centrifuge, separate the supernatant, transfer to a volumetric flask, and transfer to 50 mL. Up.
20 mL of 3% sulfosalicylic acid was added to 20 mL of the measured supernatant, shaken for 15 minutes (protein removal treatment), centrifuged, and the supernatant was separated and dried under reduced pressure. A citrate buffer solution was added thereto, and after filtration using a membrane filter having a pore size of 0.45 μm, free amino acids present in the powdered enzyme preparation were measured using an amino acid automatic analyzer.
<Method for measuring aldehyde dehydrogenase activity>
The aldehyde dehydrogenase activity immediately after production and after the accelerated storage test was measured as follows.
That is, 0.125 mL of pH 5 McKilvein buffer, 0.2 mL of 0.1 M acetaldehyde, and 0.1 mg / mL powdered enzyme preparation suspension were mixed, and distilled water was added to adjust the total amount to 0.8 mL.
To this was added 0.1 mL of 0.1 M potassium ferricyanide, a heat treatment was performed at 37 ° C. for 20 minutes, and 0.5 mL of Dupanol solution was added to stop the enzyme reaction.
Next, 2.5 mL of distilled water was mixed and allowed to stand at room temperature for 20 minutes, and then the coloration of the produced Prussian blue was measured at an absorbance of 660 nm.
In this case, the sample which did not mix | blend a powder enzyme formulation suspension was measured as a blank test.
The residual activity of the aldehyde dehydrogenase is a value calculated with the value of the aldehyde dehydrogenase activity immediately after production of the powder enzyme preparation as 100%.
<Evaluation criteria>
A: Residual activity is 50% or more B: Residual activity is 30% or more C: Residual activity is less than 30%

※（ ）内は遊離アミノ酸換算の値

* Values in parentheses are in terms of free amino acids.

From Table 1, the powder enzyme preparation which mix | blended 2 or more types of free amino acids, or those salts, especially the powder enzyme containing 2 or more types of free amino acids chosen from glutamic acid, aspartic acid, arginine, and lysine, or those salts The preparations (Examples 1 to 6) had a residual activity of 30% or more, and the aging dehydrogenase activity was highly stable over time. Further, the content ratio of the two or more kinds of free amino acids or salts thereof relative to 1 part of aldehyde dehydrogenase (microorganism equivalent) was in the range of 0.1 part or more and 20 parts or less.
On the other hand, the powder enzyme preparations (Comparative Examples 1 to 3) containing only one kind of free amino acids or salts thereof have a significantly reduced residual activity of less than 30%, and the aging dehydrogenase activity is stable over time. It was lacking.
Therefore, it can be seen that by adding two or more free amino acids or salts thereof to the aldehyde dehydrogenase, the temporal stability of the aldehyde dehydrogenase activity is increased even at room temperature.

In addition, when one or more selected from the acidic amino acids glutamic acid and aspartic acid, or salts thereof, and one or more selected from the basic amino acids lysine and arginine, or salts thereof are combined, It can also be seen that the stability over time of the dehydrogenase activity is increased (Examples 1-6).
Further, at least one selected from glutamic acid and aspartic acid, which are acidic amino acids, or salts thereof is 25% or more and 75% or less, and at least one selected from lysine and arginine, which are basic amino acids, or salts thereof. In the powder enzyme preparations (Examples 1 to 5) containing 25% or more and 75% or less, the residual activity was higher and kept at 50% or more, and the stability over time of the aldehyde dehydrogenase activity was excellent.
Therefore, it is prepared so as to contain a specific amount of at least one selected from glutamic acid and aspartic acid that are acidic amino acids, or salts thereof, and at least one selected from lysine and arginine that are basic amino acids, or salts thereof. It can be seen that the powdered enzyme preparation is excellent in aging dehydrogenase activity over time even at room temperature.

[Test Example 2] Deodorization test (acetaldehyde, nonenal)
First, prepare a 300 mL Erlenmeyer flask with filter paper and pour 1 μL of acetaldehyde and 1 mL of an aqueous solution of the powder enzyme preparation of Example 1 on this filter paper. Immediately place a butyl rubber lid on the Erlenmeyer flask. And sealed. After sealing, a heat treatment for 10 minutes was performed at a product temperature of 37 ° C.
Similarly, a heat treatment was performed by replacing acetaldehyde with nonenal.
For Examples 2 to 6 and Comparative Examples 1 to 3, the same test as in Example 1 was performed.
About the obtained two conical flasks, the deodorizing test was implemented on the following evaluation criteria.
<Evaluation criteria>
A: Almost acetaldehyde and nonenal odor disappeared.
B: Acetaldehyde and nonenal odor were reduced.
C: Acetaldehyde or nonenal odor was not reduced.

From Table 2, the powder enzyme preparation (Examples 1-6) in which the residual activity with respect to the initial activity of the aldehyde dehydrogenase was 30% or more in Test Example 1 reduced acetaldehyde and Nonenal odor. In particular, the powder enzyme preparations (Examples 1 to 5) in which the residual activity with respect to the initial activity of acetaldehyde dehydrogenase in Test Example 1 was 50% or more almost lost the acetaldehyde and nonenal odor.
On the other hand, the powder enzyme preparations (Comparative Examples 1 to 3) in which the residual activity relative to the initial activity of aldehyde dehydrogenase in Test Example 1 was less than 30% could not reduce acetaldehyde and nonenal odor.

[Example 7]
A powder enzyme preparation of the present invention was prepared in accordance with Example 1 except that the vacuum freeze-drying treatment was replaced with a spray-drying treatment. The spray drying treatment was performed at an inlet product temperature of 170 ° C. and an outlet product temperature of 65 ° C. of the spray dryer to obtain the powder enzyme preparation of the present invention.
When compared with the powder enzyme preparation of Example 1 based on the evaluation criteria of Test Example 2, a deodorizing effect was obtained, but Example 1 was superior to Example 7 in deodorizing effect.

[Example 8]
10% aldehyde dehydrogenase powder separated from 10% aldehyde dehydrogenase (complex of enzyme and cell membrane bound) of Preparation Example 1, L-sodium glutamate powder 3%, L-sodium aspartate powder 3%, L -3% arginine powder and 10% dextrin powder were charged into a powder mixer at room temperature (20 ° C), and then stirred and mixed at 100 rpm for 5 hours, and free amino acids or their salts were adsorbed on aldehyde dehydrogenase. A powdered enzyme preparation of the present invention was obtained.
When the deodorization test was performed based on the evaluation criteria of Test Example 2, a deodorization effect was obtained. Further, Example 1 was superior to Example 8 in the deodorizing effect.

[Example 9] After dry-mixing 0.1 parts of the powder enzyme preparation of Food Example 1, 0.5 parts of dextrin, 0.3 parts of crystalline cellulose, 0.05 parts of corn starch and 0.05 parts of sucrose fatty acid ester A tablet tablet having a diameter of 9 mm, a thickness of 4 mm, a weight of 290 mg, and a circular plan view was manufactured by applying a load of 1.6 t per square centimeter.
When the obtained tablet tablet was ingested, bad breath due to acetaldehyde was improved.

[Example 10] Powder enzyme preparation 0.1 parts obtained in the same manner as in Example 1 except that the aldehyde dehydrogenase of Food Preparation Example 2 was used, 4 parts sucrose, 0.05 parts turmeric powder, vitamin C0 .05 parts, vitamin B 20.05 parts, indigestible dextrin 0.05 parts, citric acid 0.005 parts, acetic acid 0.005 parts, fresh water 95.69 parts are mixed, and a 100 g container is packed by a conventional method. A beverage was produced.
After drinking 800 mL of beer, the resulting container-packed beverage was ingested, which reduced the intoxication caused by drinking acetaldehyde.

[Example 11] 0.1 parts of powder enzyme preparation of cosmetic example 2, 90 parts of liquefied petroleum gas, 7.7 parts of cetyl 2-ethylhexanoate, 1 part of silicic anhydride, 1 part of polysiloxane copolymer After mixing, a spray of 180 mL of can was produced by a conventional method.
When the obtained spray was sprayed and applied to cotton underwear worn by men in their fifties for 24 hours, the aging odor caused by Nonenal was reduced.

[Example 12] 0.1 parts of powder enzyme preparation, 10 parts of ethanol, 2 parts of isopropylmethylphenol, 1 part of talc obtained in the same manner as in Example 2 except that the aldehyde dehydrogenase of Cosmetic Preparation Example 2 was used. Then, 0.05 part of menthol and 86.85 parts of fresh water were mixed and immersed in a 10 cm square polyester sheet according to a conventional method.
When a man in his 50s wiped his neck using the obtained sheet-shaped cosmetic, the aging odor due to Nonenal was reduced.

Claims (10)

A powder enzyme preparation comprising aldehyde dehydrogenase and two or more free amino acids or salts thereof,
The two or more free amino acids, or salts thereof ,
One or more selected from acidic amino acids glutamic acid and aspartic acid, or salts thereof;
Including at least one selected from basic amino acids lysine and arginine, or salts thereof,
The two or more free amino acids or salts thereof are adsorbed to aldehyde dehydrogenase,
Powder enzyme preparation. The powdered enzyme preparation according to claim 1,
For the total amount of free amino acids contained in the powder enzyme preparation,
One or more selected from the above-mentioned glutamic acid, aspartic acid, or salts thereof, which are acidic amino acids;
The total content of the basic amino acids and the lysine and arginine, or one or more thereof selected from salts thereof is 25% or more in terms of free amino acids,
Powder enzyme preparation. In the powder enzyme preparation according to claim 1 or 2,
For the total amount of free amino acids contained in the powder enzyme preparation,
The total content of the glutamic acid and aspartic acid that are acidic amino acids, or salts thereof is 15% or more and 85% or less in terms of free amino acids,
And the total content of the lysine and arginine, or salts thereof, which are basic amino acids, is 15% or more and 85% or less in terms of free amino acids,
Powder enzyme preparation. To aldehyde dehydrogenase,
One or more selected from acidic amino acids glutamic acid and aspartic acid, or salts thereof;
Formulated with basic amino acids lysine and arginine, or one or more selected from salts thereof,
Powder enzyme preparation. A method for producing a powdered enzyme preparation according to any one of claims 1 to 4 ,
The aldehyde dehydrogenase;
The glutamic acid and aspartic acid that are acidic amino acids, or salts thereof;
After mixing the basic amino acids lysine and arginine, or salts thereof in solution,
Drying the resulting mixture,
A method for producing a powder enzyme preparation. In the manufacturing method of the powdered enzyme formulation of Claim 5 ,
The drying of the mixture is lyophilization,
A method for producing a powder enzyme preparation . Containing the powdered enzyme preparation according to any one of claims 1 to 4 ,
Food. The powdered enzyme preparation according to any one of claims 1 to 4 , or the powdered enzyme preparation obtained by the production method according to claim 5 or 6,
A method for producing food. Containing the powdered enzyme preparation according to any one of claims 1 to 4 ,
Cosmetics. The powdered enzyme preparation according to any one of claims 1 to 4 , or the powdered enzyme preparation obtained by the production method according to claim 5 or 6,
A method for producing cosmetics.