JPH099986A - Trehalose and its production and use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To readily produce and accumulate trehalose which is a nonreduclng glucide useful for foods, cosmetics, medicines, etc., by making a mlcroorganism having the ability to produce a maltose-trehalose converting enzyme coexist in a nutrient culture medium during the culture. SOLUTION: Maltose in an amount of about 5-10wt./vol.% is contained in a nutrient culture medium during the culture of a mlcroorganism having the ability to produce a maltose-trehalose converting enzyme, e.g. Plmelobacter sp. R48 (FERM BP-4315) or Thermus aquaticus ATCC33923.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to trehalose, a method for producing the same, and uses thereof. More specifically, a trehalose obtained by culturing a microorganism capable of producing maltose / trehalose converting enzyme in a nutrient medium containing maltose is obtained. Or a saccharide containing the same, a method for producing the saccharide, and its use.

[0002]

2. Description of the Related Art Trehalose (α, α-trehalose) has long been known as a non-reducing sugar having glucose as a constituent sugar, and its existence is described in "Advances in Carbohydrate Chemistry (Advanc)."
es in Carbohydrate Chemis
try), Vol. 18, pp. 201-225 (196).
3 years) Academic Press (USA) and “Applied and Environmental Microbiology”
l Microbiology), Vol. 56, No. 32
As described in pages 13 to 3215 (1990) and the like, microorganisms, mushrooms, insects, and the like are spread over a wide range in a small amount. Non-reducing saccharides such as trehalose do not cause aminocarbonyl reaction with substances having amino groups such as amino acids and proteins, and do not damage amino acid-containing substances, so that they can be used and processed without concern about browning and deterioration. Therefore, establishment of an industrial production method is desired.

As a method for producing trehalose, for example, a method using a microbial cell reported in JP-A-50-154485 or maltose phosphorylase and trehalose-proposed in JP-A-58-216695 is proposed. A method of converting maltose in combination with phosphorylase is known. However, in the method using microbial cells, the microbial cells are used as a starting material, and the content of trehalose contained in the microbial cells is usually 1 per solid matter.
It is as low as less than 5 w / w% (hereinafter, w / w% is simply abbreviated as "%" unless otherwise specified in the present specification), and in addition, the steps of extracting and purifying the same are complicated and industrial production. It is not suitable as a method. In addition, the methods using maltose phosphorylase and trehalose phosphorylase both involve glucose-1 phosphate, it is difficult to increase the substrate concentration, and the reaction system of both enzymes is a reversible reaction. The production rate of the product is low, and it is difficult to maintain the reaction system of both enzymes stably to allow the reaction to proceed smoothly, and it has not yet been realized as an industrial production method.

[0004] In connection with this, in "Monthly Food Chemicals", August issue, pp. 67-72 (1992), in the "Oligosaccharides" section of "Current Status and Issues of Starch Utilization Development," Although it is considered to have a remarkably wide range of applications, enzymatic production of this saccharide directly from starch carbohydrates and hydrolysis using a hydrolysis reaction is said to be scientifically impossible at present. " As described, it has heretofore been considered scientifically impossible to produce trehalose by using an enzyme reaction from starch.

On the other hand, starch partially decomposed products produced from starch as a raw material, such as starch liquefaction, various dextrins, various maltooligosaccharides, etc., usually have a reducing group at the end of the molecule and thus show reducibility. Are known. Such a partially degraded starch is referred to as a partially degradable starch in this specification. Generally, the reducing starch partially decomposed product has a degree of reducing power per solid substance that is determined by Dextrose Equivalent (D).
E). The one with the larger value is usually
It has a small molecule, low viscosity, and strong sweetness, but it is highly reactive, and easily undergoes an aminocarbonyl reaction with substances with amino groups such as amino acids and proteins, causing browning, producing a bad odor, and easily degrading quality. Is known to exist.

Various characteristics of such a partially decomposed product of reducing starch depend on the size of DE, and the relationship between the partially decomposed product of reducing starch and DE is extremely important. Traditionally, it has been believed in the industry that it is impossible to break this relationship.

The only method of breaking the relationship between the partially decomposed product of reducing starch and DE is to convert the reducing group of the partially decomposed starch into a sugar alcohol by a high-pressure hydrogenation method or the like to form a non-reducing sugar. Is the way to. However, this method requires a high-pressure autoclave, consumes a large amount of hydrogen and energy, and also requires advanced safety facilities and management from the viewpoint of disaster prevention. In addition, the sugar alcohol of the resulting partially decomposed reducing starch is different in that the raw degradable starch partially decomposed product is composed of glucose and sorbitol, whereas the raw material is composed of glucose and sorbitol. There are concerns about temporary but indigestible and laxative symptoms. Therefore, it is desired to establish a method for reducing or eliminating the reducing power without changing glucose, which is a constituent sugar of the partially decomposed reductive starch.

[0008] In order to solve this, the present inventors previously described in Japanese Patent Application No. 6-144092 a novel maltose-trehalose converting enzyme for converting maltose to trehalose (this enzyme is referred to as Through maltose
It is called trehalose converting enzyme. ) Was disclosed, and a method for producing trehalose and a sugar containing the same from maltose was established using the present maltose-trehalose converting enzyme.

However, in addition to the culture time for culturing the microorganism to produce the enzyme, a time for enzyme recovery and an enzyme reaction time for converting trehalose from maltose are required, which is a considerably long time for industrial implementation. It turned out that there is a drawback that requires. In producing trehalose from maltose, it is desired to establish a method for producing trehalose that can be carried out more easily and easily, including production of the enzyme.

[0010]

DISCLOSURE OF THE INVENTION The present invention establishes a novel method for producing trehalose from maltose easily and in a short time, and at the same time, provides its use.

[0011]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have continued to earnestly study the culture condition of a microorganism capable of producing maltose / trehalose converting enzyme and the production condition of the enzyme. As a result, the microorganism
The present inventors have found that maltose / trehalose converting enzyme is produced from a fairly early stage, and have found that trehalose can be easily produced and accumulated by allowing maltose to coexist in a nutrient medium in culture, and completed the present invention.

That is, the present invention cultivates a microorganism capable of producing maltose / trehalose converting enzyme in a nutrient medium containing maltose, and obtains trehalose or a sugar containing the same, and the production of the sugar. A method and a composition containing the sugar are established. In the present invention, as maltose, among others,
Those obtained by reacting starch liquefaction with β-amylase or a starch debranching enzyme together with β-amylase are preferable, and as microorganisms, among others, maltose / trehalose converting enzyme producing ability is preferable. Utilization of existing microorganisms is extremely advantageous for trehalose production. The trehalose and the saccharide containing the trehalose thus obtained have high stability, are easy to handle, and can be used in a wide variety of applications. For example, they can be advantageously used in various compositions such as food and drink, cosmetics, and pharmaceuticals.

The maltose-trehalose converting enzyme-producing microorganism used in the present invention may be any microorganism that produces an enzyme that converts maltose into trehalose, and is disclosed in, for example, Japanese Patent Application No. 6-144092. Microorganisms belonging to the genus Pimelobacter, the genus Pseudomonas and the genus Thermus are preferred.

The medium used for culturing the microorganism is a nutrient medium in which the microorganism can grow and which can produce maltose / trehalose converting enzyme, and which contains maltose as a substrate for trehalose production. If necessary, other carbon sources that can be assimilated by microorganisms, for example, glucose, fructose, lactose, sucrose, mannitol, sorbitol, sugars such as molasses, further, citric acid, organic acids such as succinic acid, or, It is optional to use the salt together. The concentration of maltose contained in the medium is preferably 20 w / v% or less, preferably 15 w / v% or less, more preferably 5 to 10 w / v%. As the nitrogen source, for example, inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract and meat extract are appropriately used. Also,
As the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate salt, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt and the like are appropriately used. Furthermore, if necessary, amino acids,
Vitamins and the like are also used as appropriate.

The culturing conditions are such that the microorganism grows and produces maltose / trehalose converting enzyme, and the temperature is usually 4 to 80 ° C., preferably 20 to 75 ° C., pH 5 to 9 and preferably 6 to 8.5. It is performed aerobically under the selected conditions. The culturing time may be any time that allows the microorganism to grow, and is preferably 10 to 100 hours.
The dissolved oxygen concentration of the culture solution is not particularly limited, but usually 0.5 to 20 ppm is preferable. For that purpose, means such as adjusting the air flow rate, stirring, adding oxygen to the air flow, and increasing the pressure in the fermenter are adopted. The culture method may be batch culture, continuous culture or semi-continuous culture.

[0016] In this way, a microorganism having the ability to produce maltose / trehalose converting enzyme can be cultured in a nutrient medium containing maltose to produce and accumulate trehalose in the culture. If necessary, a separately prepared maltose / trehalose converting enzyme may be supplemented to the nutrient medium in the culture to increase the production rate of trehalose. It may also be advantageous to add an activator and / or a lytic enzyme such as egg white lysozyme to the nutrient medium in culture to increase the rate of trehalose production. The trehalose thus produced and accumulated is contained in the liquid portion obtained by separating the insoluble matter in the culture.

The culture containing trehalose was prepared as follows.
A known solid-liquid separation method, for example, filtration or centrifugation to obtain a sterilized solution, which is concentrated and decolorized with activated carbon, and then desalted with H-type and OH-type ion exchange resins to be purified, Concentrate to a syrupy product. Further drying to a powdered product is optional. If necessary, the culture is directly subjected to membrane filtration such as flat membrane or hollow fiber membrane to remove soluble proteins such as bacterial cells and high molecular substances such as nucleic acids, or by centrifugation in advance. After removing insoluble matter and then soluble polymer by membrane filtration, concentration, decolorization, desalting and purification are carried out by a conventional method, and trehalose or a sugar product containing the same is advantageously produced. be able to.

Next, the maltose / trehalose converting enzyme contributing to the production of trehalose of the present invention will be described. The enzyme activity is found in both the bacterial cells and the sterilized solution of the culture, and the bacterial cells and the sterilized solution can be collected as a crude enzyme solution, or the entire culture can be used as a crude enzyme solution. A known solid-liquid separation method is adopted to remove the bacterial cells from the culture. For example, a method of centrifuging the culture itself as it is, a method of separating by filtration using a precoat filter, a method of separating by membrane filtration of a flat membrane, a hollow fiber membrane, etc. are appropriately adopted. The sterilized solution can be used as it is as an enzyme solution, but it is generally concentrated before use. As the concentration method, for example,
Ammonium sulfate salting-out method, acetone and alcohol precipitation method, membrane concentration method such as flat membrane and hollow fiber membrane are adopted.

Further, the sterilized liquid and its concentrate can be immobilized by a known method. For example, a binding method to an ion exchanger, a covalent binding / adsorption method with a resin or a membrane, an encapsulation method using a polymer substance, etc. are adopted. Further, the bacterial cells separated from the culture can be directly used as the crude enzyme, but they may be immobilized and used. As an example, this is mixed with sodium alginate and added dropwise into a calcium chloride solution to gel and immobilize into a granular form. This granule may be further treated with polyethyleneimine or glutaraldehyde to be immobilized. It is also possible to extract the enzyme from the cells and use the extract as a crude enzyme solution. For example, the enzyme can be extracted from the bacterial cells by a disruption method by ultrasonic waves, a mechanical disruption method by glass beads and alumina, a disruption method by a French press, etc., and a clear crude enzyme solution can be obtained by centrifugation or membrane filtration. .

The enzyme solution of the present invention can be used as it is, but can be further purified by a known method before use. As an example, a crude enzyme preparation obtained by salting out a treated product of the culture solution with ammonium sulfate and concentrating it is dialyzed and then manufactured by Tosoh Corporation “DEAE”.
-Anion exchange column chromatography using "Toyopearl", etc., followed by hydrophobic column chromatography using "Butyl Toyopearl" manufactured by the same company, and gel filtration chromatography using "Toyopearl HW-55" manufactured by the same company. By purification using it, an electrophoretically single enzyme can be obtained.

The maltose / trehalose converting enzyme thus obtained generally has, for example, the following physicochemical properties. (1) Action Maltose is converted into trehalose and trehalose is converted into maltose. (2) Molecular weight By SDS-gel electrophoresis, about 57,000 to 12
10,000 daltons. (3) pI of about 3.8 to 5.1 by an isoelectric focusing ampholine-containing electrophoresis method. (4) Activity inhibition 1 mM Cu ⁺⁺ , Hg ^++, or 50 mM Tris-HCl buffer is used for inhibition. (5) Origin It is an enzyme produced by a microorganism.

Specific examples of the difference in the originating microorganism are as follows.

[Pimellobacterium species R48-derived maltose / trehalose converting enzyme] (1) Action Maltose is converted to trehalose and trehalose is converted to maltose. About 1 mol of trehalose or maltose is produced from 1 mol of maltose or trehalose, respectively. (2) Molecular weight By SDS-gel electrophoresis, about 57,000 to 67,
000 Daltons. (3) Isoelectric focusing by ampholine-containing electrophoresis, pI of about 4.1 to 5.1. (4) Activity inhibition 1 mM Cu ⁺⁺ , Hg ^++, or 50 mM Tris-HCl buffer is used for inhibition. (5) Optimum temperature: pH 7.0, reaction for 60 minutes, at around 20 ° C. (6) Optimum pH 25 ° C., reaction for 60 minutes, pH about 7.0 to 8.0. (7) Temperature stability It is stable up to around 30 ° C by holding at pH 7.0 for 60 minutes. (8) pH stability A pH of about 6.0 to 9.0 when kept at 20 ° C. for 60 minutes.

[0023]

[Pseudomonas putida H262-derived maltose / trehalose converting enzyme] (1) Action Maltose is converted to trehalose and trehalose is converted to maltose. About 1 mol of trehalose or maltose is produced from 1 mol of maltose or trehalose, respectively. (2) Molecular weight By SDS-gel electrophoresis, about 110,000 to 12
10,000 daltons. (3) Isoelectric focusing by ampholine-containing electrophoresis, pI of about 4.1 to 5.1. (4) Activity inhibition 1 mM Cu ⁺⁺ , Hg ^++, or 50 mM Tris-HCl buffer is used for inhibition. (5) Optimum temperature: around 37 ° C. after reacting at pH 7.0 for 60 minutes. (6) Optimum pH 35 ° C., reaction for 60 minutes, pH about 7.3 to 8.3. (7) Temperature stability It is stable up to around 40 ° C by keeping at pH 7.0 for 60 minutes. (8) pH stability A pH of about 6.0 to 9.5 when kept at 35 ° C. for 60 minutes.

[0024]

[Thermus aquaticus ATCC 33923-derived maltose / trehalose converting enzyme] (1) Action Maltose is converted to trehalose and trehalose is converted to maltose. About 1 mol of trehalose or maltose is produced from 1 mol of maltose or trehalose, respectively. (2) Molecular weight By SDS-gel electrophoresis, about 100,000 to 11
10,000 daltons. (3) Isoelectric focusing apholine-containing electrophoresis, pI of about 3.8 to 4.8. (4) Activity inhibition 1 mM Cu ⁺⁺ , Hg ^++, or 50 mM Tris-HCl buffer is used for inhibition. (5) Optimum temperature pH of 7.0, reaction for 60 minutes, around 65 ° C. (6) Optimum pH 60 ° C., reaction for 60 minutes, pH about 6.0 to 6.7. (7) Temperature stability It is stable up to around 80 ° C by keeping at pH 7.0 for 60 minutes. (8) pH stability A pH of about 5.5 to 9.5 when kept at 60 ° C. for 60 minutes.

The activity of maltose / trehalose converting enzyme is measured as follows. Maltose 2 as a substrate
0 w / v% (10 mM phosphate buffer, pH 7.0) 1
1 ml of enzyme solution was added to ml, reaction temperature was 25 ℃, 35 ℃
Alternatively, the temperature is set to 60 ° C, and after reacting for 60 minutes, 100 ° C
The reaction is stopped by heating for 10 minutes. This reaction solution was diluted exactly 11-fold with 50 mM phosphate buffer pH 7.5, and 0.4 ml of the diluted solution was added with a trehalase-containing solution (1
Unit / ml) added at 0.1 ml at 45 ° C, 12
After incubating for 0 minutes, the amount of glucose in this reaction solution is quantified by the glucose oxidase method. As a control, the same measurement is performed using an enzyme solution and trehalase that have been inactivated by heating at 100 ° C. for 10 minutes in advance.
Using the above measurement method, the amount of trehalose produced by maltose / trehalose converting enzyme was determined from the increasing amount of glucose, and the activity 1 unit was 1 μmo per minute.
It is defined as the amount of enzyme that produces le trehalose.

The reaction temperature is 25 ° C. when the maltose / trehalose converting enzyme is derived from a microorganism belonging to the genus Pimelobacter, 35 ° C. when derived from a microorganism belonging to the genus Pseudomonas, and is derived from a microorganism belonging to the genus Thermus. To 60 ° C.

Next, the starch used in the present invention may be ground starch such as corn starch, rice starch and wheat starch, or underground starch such as potato starch, sweet potato starch and tapioca starch. To liquefy starch, starch milk is usually suspended in water, and the concentration of starch milk is preferably 10% or more, more preferably about 20 to 50%. Alternatively, it may be liquefied with an enzyme. A relatively low degree of liquefaction is suitable, and DE is preferably
It is preferably less than 15, more preferably less than DE10. In the case of liquefying with an acid, for example, it may be liquefied with hydrochloric acid, phosphoric acid, oxalic acid or the like, and then neutralized to a required pH with calcium carbonate, calcium oxide, sodium carbonate or the like before use. When liquefying with an enzyme, α-amylase,
In particular, the use of thermostable liquefied α-amylase is suitable.

The β-amylase used in the present invention to produce maltose from a liquefied starch solution may be prepared by a known method from plants such as sweet potato, soybean, wheat flour and the like, cultures of microorganisms belonging to the genus Bacillus and the like. Of course, the use of commercially available enzyme preparations is also optional. In addition, the starch debranching enzyme used in the present invention acts on a liquefied starch having a relatively low DE, and desirably acts on a liquefied solution having a DE of less than 15.
As the enzyme that hydrolyzes the branched bond of starch, known pullulanase, isoamylase and the like can be advantageously used, and a commercially available enzyme agent can also be advantageously used.

The amount of the enzyme to be used may be appropriately selected depending on the working conditions and the reaction time, but is usually about 1 to 100 units in the case of β-amylase per gram of solid matter with respect to the starch liquefied solution as the substrate. In the case of starch debranching enzyme, it is selected from about 1 to 2,000 units.

The maltose thus obtained can be advantageously used as trehalose by the culturing method of the present invention or a sugar source for sugar production containing the same. Further, it is also possible to advantageously use commercially available maltose. The maltose may be added to the nutrient medium at a time when trehalose can be produced, and it may be added from the beginning of the culture or from the middle of the culture. Further, in the case of continuous or semi-continuous culture, for example, a part of the culture medium in which trehalose is produced may be extracted, and the nutrient medium in which the same volume of maltose is contained may be additionally cultured. Further, it is optional to carry out the culture in a nutrient medium in which β-amylase or β-amylase together with starch debranching enzyme coexist in a liquefied starch solution.

The trehalose-containing culture thus obtained is filtered, centrifuged and the like to remove insolubles such as bacterial cells, and then concentrated, decolorized with activated carbon,
-Type, OH-type ion-exchange resin for desalination, purification, concentration,
Syrup-shaped product. Further, it is optionally dried to give a powder product. If necessary, further purification, for example, fractionation by column chromatography such as ion exchange column chromatography, activated carbon column chromatography, silica gel column chromatography, fractionation by organic solvents such as alcohol and acetone, a membrane having appropriate separation performance. The trehalose product of the highest purity can be obtained by purifying by one or a combination of two or more methods such as separation by yeast, further fermentation treatment with yeast, decomposition and removal of remaining reducing sugars by alkali treatment, etc. Is also easy to obtain.

In particular, as an industrial mass production method,
Ion exchange column chromatography is suitable for use, and examples thereof include JP-A-58-23799 and JP-A-SHO-5.
Contaminant saccharides can be removed by column chromatography using a strongly acidic cation exchange resin disclosed in Japanese Unexamined Patent Publication No. 8-72598, and a target low-reducing saccharide having an improved trehalose content can be advantageously produced. At this time, any one of a fixed bed system, a moving bed system, and a simulated moving bed system may be adopted.

The trehalose-containing saccharide of the present invention thus obtained is, if necessary, decomposed with glucoamylase or α-glucosidase to adjust sweetness, reducing power and the like, or to reduce viscosity. In addition, it is optional to perform further processing such as converting the reducing sugar remaining after hydrogenation into a sugar alcohol to eliminate the reducing power.

In particular, glucoamylase or α-glucosidase is allowed to act on the trehalose-containing carbohydrate of the present invention to give a mixed solution of trehalose and glucose, which is then purified by the above-mentioned purification method, for example, ion exchange column chromatography. If glucose is removed by chromatography or the like, a trehalose-rich fraction can be collected. It can be advantageously carried out by purifying and concentrating it to obtain a syrupy product, and further concentrating it to supersaturate it and crystallizing it to obtain trehalose hydrous crystals or anhydrous crystalline trehalose.

In order to produce trehalose hydrous crystals, for example, a trehalose-rich liquid having a purity of about 60% or more and a concentration of about 65 to 90% is placed in an auxiliary crystal can and, if necessary, added to the auxiliary crystal can.
In the coexistence of 1 to 20% of seed crystals, the mixture is gradually cooled with stirring at a temperature of 95 ° C or lower, preferably 10 to 90 ° C to produce a mass kit containing trehalose hydrous crystals. Further, it is also advantageous to employ a continuous crystallization method in which crystallization is performed while concentrating under reduced pressure. As a method for producing the trehalose hydrous crystal or the syrup-containing crystal containing the trehalose from the musket, a known method such as a mashing method, a block pulverizing method, a fluidized granulation method, and a spray drying method may be adopted.

In the case of the syrup method, a masquette is usually placed in a basket centrifuge to separate the trehalose-containing crystals and the syrup (mother liquor), and the crystals are washed with a small amount of cold water if necessary. Is also an easy way
It is suitable for producing trehalose hydrous crystals of higher purity. In the case of the spray drying method, a mass kit having a concentration of 70 to 85% and a crystallization rate of 20 to 60% is usually sprayed from a nozzle by a high pressure pump, and hot air at a temperature at which the crystal powder is not dissolved, for example, 60 to 100 ° C is used. A non-hygroscopic or hardly hygroscopic syrup-containing crystal can be easily produced by drying at 37 ° C. and then aging with warm air at 30 to 60 ° C. for about 1 to 20 hours. In the case of the block pulverization method, usually, a mass kit having a water content of 10 to 20% and a crystallization rate of 10 to 60% is allowed to stand for about 0.1 to 3 days to crystallize and solidify the whole in a block form. If this is powdered by a method such as crushing or cutting and dried, a non-hygroscopic or hardly hygroscopic honey-containing crystal can be easily produced.

Further, in order to produce anhydrous crystalline trehalose, trehalose hydrous crystals can be dried and converted, but generally, a high-concentration trehalose-rich solution having a water content of less than 10% is taken in a supporting crystal can. A mass kit containing anhydrous crystalline trehalose was produced while stirring in the range of 50 to 160 ° C., preferably 80 to 140 ° C. in the presence of seed crystals. It is produced by crystallizing by a method such as granules or spray drying and pulverizing.

The trehalose of the present invention thus produced, or the saccharide containing the same, has low reducibility and is stable, and is mixed with other materials, particularly substances having amino acids such as amino acids, oligopeptides and proteins. Even if it is processed, it does not turn brown, does not generate an offensive odor, and hardly damages other mixed materials. Further, it has a low viscosity even though it has a low reducing power, and has a good quality and a sweet taste.

Furthermore, since the trehalose of the present invention is easily decomposed into glucose by trehalase, it is digested and absorbed by ingestion and used as a calorie source. It can also be used as a sweetener that is unlikely to be fermented by caries-inducing bacteria and is unlikely to cause caries. Further, it has properties such as osmotic pressure controllability, shaping property, luster imparting property, moisturizing property, viscosity, crystallization preventing property of other sugars, fermentability, and aging preventing property of gelatinized starch.

Further, the trehalose of the present invention is used parenterally as a tube feeding agent, infusion solution, etc., is well metabolized and utilized without fear of toxicity and side effects, and is advantageously used for energy supply to the living body. can do. Further, since it is a stable sweetener, in the case of trehalose hydrous crystals, it can be advantageously used in combination with a binder such as pullulan, hydroxyethyl starch, polyvinylpyrrolidone and the like as a sugar coating agent for tablets.

In the case of anhydrous crystalline trehalose,
It can be advantageously used as a dehydrating agent for water-containing substances such as foods, pharmaceuticals, cosmetics, raw materials thereof, or processing intermediates, and stable and high-quality solid materials such as powders, granules and tablets can be easily produced.

Therefore, the trehalose of the present invention or the sugar containing the trehalose is used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, a dehydrating agent, etc., foods, drinks, feeds, feeds, It can be advantageously used for various compositions such as foodstuffs, cosmetics and pharmaceuticals.

The trehalose of the present invention or the sugar containing the trehalose can be used as it is as a seasoning for sweetening. If necessary, for example, starch syrup, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, isomalt oligosaccharide, galactooligosaccharide, fructooligosaccharide, lactosucrose, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α -Glycosyl stevioside, rebaudioside,
It may be used in admixture with one or more suitable amounts of other sweeteners such as glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, etc., and if necessary, dextrin. It can also be used as a mixture with a bulking agent such as starch, lactose or the like.

The trehalose of the present invention, or a sugar powder or crystalline product containing the trehalose, may be used as it is or, if necessary, mixed with a bulking agent, an excipient, a binder or the like to form granules. It is also possible to mold it into various shapes such as a spherical shape, a short rod shape, a plate shape, a cube, and a tablet for use.

The trehalose of the present invention, or the sweetness of sugar containing the trehalose, is in good harmony with various substances having other tastes such as sourness, salt to taste, astringency, umami and bitterness, and has acid resistance. Since it has high heat resistance, it can be advantageously used for sweetening and improving the taste of general food and drink, and for improving the quality.

For example, amino acids, peptides, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hishio, sprinkle,
Mayonnaise, dressing, vinegar, three tablespoons vinegar, powdered sushi vinegar, Chinese ingredients, tentsuyu, noodle soup, sauce, ketchup,
Grilled meat sauce, curry roux, stew ingredients, soup ingredients,
It can be advantageously used as various seasonings such as dashi stock, nucleic acid-based seasonings, complex seasonings, mirin, new mirin, table sugar, and coffee sugar.

Further, for example, various Japanese sweets such as rice crackers, hail, rice cakes, rice cakes, steamed buns, uiro, bean paste, yokan, water yoyo, nishikidama, jelly, castella, candy, etc.,
Bread, biscuits, crackers, cookies, pies, pudding, butter cream, custard cream, puff cream, waffles, sponge cakes, donuts, chocolate, chewing gum, caramel, candy and other confectionery, ice cream, sherbet and other ice confections,
Fruit syrup pickles, syrups such as ice honey, flower paste, peanut paste, fruit paste, pastes such as spreads, jam, marmalade, syrup pickles, fruits such as sugar fruit, processed foods of vegetables, Fukugami pickles,
Pickles such as betta pickles, senmai pickles, lucky pickles, pickles such as takuan pickles, Chinese cabbage pickles, meat products such as ham and sausage, fish ham, fish sausage,
Fish products such as kamaboko, chikuwa, tempura, seafood, various delicacies such as salted squid, vinegar konbu, sakisume, fugumirin dried, seaweed, wild vegetables, sardines, small fish, boiled tsukudani, boiled beans , Potato salad, konbu-maki and other prepared foods, yogurt, cheese and other dairy products, fish meat,
Meat, fruit, vegetable bottled, canned, sake, synthetic liquor, liqueur, liquor such as Western liquor, coffee, tea, cocoa,
Soft drinks such as juices, carbonated drinks, lactic acid drinks, and lactic acid bacteria drinks, pudding mixes, hot cake mixes, instant foods, instant foods such as instant soups, and even baby foods
It can be advantageously used for sweetening various foods and drinks such as therapeutic foods, drinks, peptide foods, frozen foods and dried foods, for improving taste and for improving quality.

It can also be used for the purpose of improving palatability of feed, feed, etc. for domestic animals, poultry and other domestic animals such as bees, silkworms and fish. In addition, tobacco, toothpaste, lipstick, lip balm, oral solution, tablets, troches, liver oil drops, mouthwash, mouthwash, mouthwash, various solid materials such as paste, liquid, etc., preference, cosmetics, pharmaceuticals, etc. Can be advantageously used as a sweetening agent for various compositions, or as a taste improving agent, a corrigent, a quality improving agent, a stabilizer, and the like.

As the quality improver and stabilizer, active ingredients,
It can be advantageously applied to various physiologically active substances which easily lose their activity or the like, health foods and pharmaceuticals containing the same. For example, interferon-α, interferon-β, interferon-γ, Tumor necrosis factor-α, Tumor necrosis factor-β, macrophage migration inhibitory factor, colony-stimulating factor, transfer factor, lymphokines such as interleukin II, and insulin. , Hormones such as growth hormone, prolactin, erythropoietin, egg cell stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, pox seedling, tetanus toxoid, hub antitoxin, biologics such as human immunoglobulin, penicillin, erythromycin, Antibiotics such as chloramphenicol, tetracycline, sprepomycin, kanamycin sulfate,
Thiamine, riboflavin, L-ascorbic acid, liver oil,
Carotenoids, vitamins such as ergosterol, tocopherol, lipase, elastase, urokinase,
Enzymes such as protease, β-amylase, isoamylase, glucanase, lactase, ginseng extract, soft-shelled turtle extract, chlorella extract, aloe extract, propolis extract and other extracts, viruses, lactic acid bacteria, yeasts and other viable bacteria, royal jelly, etc. Physiologically active substances
The stable and high-quality liquid, paste or solid health food or pharmaceutical product can be easily produced without losing the active ingredient and activity.

The method of incorporating trehalose or a sugar containing the same into various compositions as described above may be included in the steps until the product is completed. For example, mixing, dissolving, melting, Known methods such as dipping, infiltration, spraying, coating, coating, spraying, pouring, crystallization and solidification are appropriately selected. The amount is usually 0.1% or more, preferably 1% or more. Next, the present invention will be described more specifically by experiments.

First, the maltose trehalose converting enzyme from the novel microorganisms Pimelobacters species R48, Pseudomonas putida H262 and Thermus aquaticus ATCC 33923 will be described.
Next, maltose / trehalose converting enzyme from other known microorganisms will be described.

[0052]

[Experiment 1 Enzyme production] Glucose 2.0 w / v%, polypeptone 0.5 w / v%, yeast extract 0.1 w / v
%, Dipotassium phosphate 0.1 w / v%, monosodium phosphate 0.06 w / v%, magnesium sulfate heptahydrate 0.7.
A liquid medium consisting of 05 w / v%, calcium carbonate 0.5 w / v%, and water was placed in a 500 ml Erlenmeyer flask at a concentration of 10%.
Add 0 ml each, sterilize in an autoclave at 115 ° C for 30 minutes, cool, and then pimerobacters species R
48 (FERM BP-4315), 27 ° C,
What was cultivated by rotating and shaking at 200 rpm for 24 hours was used as a seed culture.

About 20 liters of a medium having the same composition as in the case of seed culture was placed in a fermenter having a volume of 30 liters, sterilized by heating and cooled to a temperature of 27 ° C., and 1 v / v% of seed culture was inoculated,
While maintaining the temperature at 27 ° C and pH 6.0 to 8.0, about 40
The cells were cultured with aeration and stirring for hours.

The maltose / trehalose converting enzyme activity of the culture broth was 0.55 unit / ml. A part of the culture solution is collected and centrifuged to separate the cells and the culture solution supernatant, and the cells are suspended in 50 mM phosphate buffer solution (pH 7.0) to obtain the same solution as the original culture solution. After determining the amount, the maltose / trehalose converting enzyme activity of the bacterial cell suspension and the culture supernatant was measured. As a result, 0.5 unit / ml of activity was found in the bacterial supernatant in the bacterial suspension. Was observed to have an activity of 0.05 unit / ml. The activity of this enzyme was measured at a reaction temperature of 25 ° C.

[0055]

[Experiment 2 Purification of Enzyme] The culture solution obtained in Experiment 1 was centrifuged to recover a bacterial cell having a wet weight of about 0.5 kg.
It was suspended in M phosphate buffer (pH 7.0). About 5 liters of this cell suspension was applied to Edmund Buehler's "Vibrogen Cell Mill" to crush the cells, and the disrupted solution was centrifuged (15,000 G, 30 minutes) to give about 4.5 liters. A supernatant was obtained. The degree of saturation was 0.
Ammonium sulphate was added to the solution to give a solution of 3, and the mixture was allowed to stand at 4 ° C. for 4 hours and then centrifuged to collect the supernatant.

Further, ammonium sulfate was added to the solution so as to have a saturation level of 0.8, dissolved, allowed to stand at 4 ° C. overnight, and then centrifuged to recover a salted-out product of ammonium sulfate.

The obtained ammonium sulfate salt-out product was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against the same buffer for 24 hours, and centrifuged to remove insoluble materials. The dialysate (400 ml) was divided into two portions and subjected to ion exchange column chromatography (gel amount 300 ml) using "DEAE-Toyopearl".

The maltose / trehalose converting enzyme of the present invention was adsorbed on "DEAE-Toyopearl" and eluted from the column with the same buffer containing sodium chloride. After the eluted enzyme active fraction was collected, it was dialyzed against the same buffer containing 1M ammonium sulfate,
The dialysate was centrifuged to remove insoluble matter, and then subjected to hydrophobic column chromatography (gel amount 300 ml) using "Butyl Toyopearl 650" manufactured by Tosoh Corporation. The adsorbed maltose / trehalose converting enzyme was eluted from the column with a linear gradient of ammonium sulfate 1M to 0M to collect an enzyme active fraction.

Subsequently, ion exchange chromatography (gel amount 10 ml) using "Mono Q HR5 / 5" manufactured by Pharmacia LCAB was performed, and the eluted enzyme active fraction was collected. Table 1 shows the amount of enzyme activity, the specific activity, and the yield in each step of purification.

[0060]

[Table 1]

The purified enzyme preparation was subjected to gel electrophoresis containing 7.5 w / v% concentration polyacrylamide to examine the purity of the enzyme preparation. As a result, the protein band was single and had a high purity.

[0062]

[Experiment 3 Properties of enzyme] The purified maltose / trehalose-converting enzyme preparation obtained by the method of Experiment 2 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration 10 w / v%), and molecular weight markers (Nippon Bio・ The molecular weight of this enzyme was measured in comparison with Rad Laboratories Co., Ltd.), and the molecular weight was about 57,000 to 6
It was 7,000 daltons.

The purified maltose / trehalose converting enzyme preparation was subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2 w / v% ampholine (manufactured by Pharmacia LCAB), and after electrophoresis, the pH of the protein band and gel was measured. When the isoelectric point of this enzyme was determined by the method, the isoelectric point was pI of about 4.1 to 5.1.

The effects of temperature and pH on the enzyme activity were examined according to the activity measuring method. The results are shown in FIG. 1 (effect of temperature) and FIG. 2 (effect of pH). The optimum temperature of the enzyme is pH 7.0, around 20 ℃ for 60 minutes reaction, the optimum pH
Was about 7.0 to 8.0 after reacting at 25 ° C. for 60 minutes. The temperature stability of this enzyme was determined by holding the enzyme solution (50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water, and measuring the remaining enzyme activity. The pH stability was determined by maintaining the enzyme in 50 mM buffer of each pH at 20 ° C. for 60 minutes, adjusting the pH to 7.0, and measuring the remaining enzyme activity. The results are shown in Fig. 3 (temperature stability) and Fig. 4 (pH).
Stability). The temperature stability of this enzyme was up to around 30 ° C., and the pH stability was about 6.0 to 9.0.
The enzyme activity is 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 m
Inhibited with M Tris-HCl buffer.

[0065]

[Experiment 4 Action on Various Carbohydrates] Using various sugars, it was tested whether they could be substrates. Glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, soluble starch, amylose (average degree of polymerization 18), trehalose, neotrehalose, gentiobiose, kojibiose, isomaltose, cellobiose, multi Tol, sucrose, maltulose, turanose, palatinose, trehalulose, or a solution of lactose, and glucose equivalent to α-glucose / 1-phosphate, or β-glucose / 1-phosphate and equal glucose. Was prepared.

To each of these solutions, 2 units of the purified maltose-trehalose-converting enzyme obtained by the method of Experiment 2 were added per gram of substrate solid, and the substrate concentration was 5 w / v%.
Was adjusted so that it was allowed to act for 24 hours at 20 ° C. and pH 7.0. "Kieselgel 60" manufactured by Merck & Co., Inc. before and after the enzymatic reaction (aluminum plate, 20 x 20 cm)
Was subjected to thin-layer chromatography (hereinafter, abbreviated as TLC) using, to confirm the presence or absence of enzyme action on each sugar. TLC was developed once at room temperature using 1-butanol: pyridine: water = 6: 4: 1 (volume ratio) as a developing solvent. Color development was performed by spraying a 20% sulfuric acid-methanol solution and heating at 110 ° C. for 10 minutes. Table 2 shows the results.

[0067]

[Table 2]

As is clear from the results shown in Table 2, the enzyme of the present invention acts only on maltose and trehalose among the various kinds of sugars tested, and other sugars, especially α-glucose monophosphate. Since it does not act on a system containing glucose and glucose, or a system containing β-glucose / 1-phosphate and glucose, it is a novel enzyme, unlike phosphorylases such as conventionally known maltose phosphorylase and trehalose phosphorylase. It turned out to be

[0069]

[Experiment 5 Products from maltose or trehalose]
To the maltose aqueous solution having a final concentration of 5%, 2 units of the purified maltose-trehalose converting enzyme obtained by the method of Experiment 2 was added per gram of the solid substrate, and the mixture was allowed to act at 20 ° C. and pH 7.0 for 24 hours. The sugar composition of the enzyme reaction solution was analyzed by gas chromatography (hereinafter abbreviated as GLC). A part of the enzyme reaction solution was dried, dissolved in pyridine and then trimethylsilylated to obtain an analytical sample. The gas chromatograph is “GC-16A” manufactured by Shimadzu Corporation, the column is a stainless steel column (3 mmφ × 2 m) filled with “2% silicon OV-17 / chromosolve W” manufactured by GL Science Co., and the carrier gas is Nitrogen gas flow rate 40ml / min, column oven temperature 160 ℃
To 320 ° C at a heating rate of 7.5 ° C / min.
A hydrogen flame ion detector was used for detection. Table 3 shows the results.

[0070]

[Table 3]

As is clear from the results in Table 3, it was found that the reaction product X was produced in a large amount and the retention time thereof was in agreement with that of the commercially available trehalose. The following confirmation test was performed to identify the reaction product X. That is, the above-mentioned enzyme reaction solution using maltose as a substrate was diluted with 20 mM acetate buffer, pH 4.5 to a sugar concentration of 2%, and 0.5 ml of glucoamylase (Seikagaku Corporation) 0.1 A unit was added and reacted at 40 ° C. for 20 hours.

Similarly, the enzyme reaction solution was diluted with 20 mM phosphate buffer, pH 7.0 so that the sugar concentration was 2%, 0.5 unit of trehalase was added to 0.5 ml, and the mixture was reacted at 40 ° C. for 20 hours. It was When the enzyme reaction solution using maltose as a substrate, its glucoamylase treatment solution and trehalase treatment solution were analyzed and compared by GLC, maltose was completely decomposed into glucose by the glucoamylase treatment,
The reaction product X remained without being decomposed.

On the other hand, although the maltose remained due to the trehalase treatment, the reaction product X was completely decomposed into glucose. Considering the reaction characteristics of glucoamylase and trehalase, the oligosaccharide from maltose produced by the novel enzyme of the present invention is judged to be trehalose.

Furthermore, using trehalose as a substrate, a purified enzyme was allowed to act under the same conditions as in the case of maltose, and the reaction solution was also subjected to GLC analysis. It was found that maltose was produced from trehalose by the enzyme of the present invention. did. The above GLC analysis results are summarized in Table 4.

[0075]

[Table 4]

As is clear from the results shown in Table 4, the enzyme of the present invention converts maltose into trehalose and trehalose into maltose. It was found that the equilibrium point was offset to the trehalose side, and the conversion rate from maltose to trehalose was high, which was about 70% or more.

[0077]

[Experiment 6 Effect of maltose concentration on trehalose production] Maltose concentration was 2.5%, 5%, 10%, 20
% Or 40%, at a temperature of 20 ° C. and a pH of 7.0, 2 units of the purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 was added to the maltose gram, and the reaction was carried out. The enzyme was inactivated by heating at 0 ° C for 10 minutes.

The total sugar amount of this reaction solution was quantified by the Anthuron-sulfuric acid method and the reducing sugar amount was quantified by glucose conversion by the Somogy-Nelson method, and the ratio of the reducing sugar amount to the total sugar amount was calculated as the reducing power.

The reaction solution was diluted to a sugar concentration of about 1%, deproteinized with a small amount ultrafilter, "Morcut II LGC" manufactured by Japan Millipo Limited, and subjected to high performance liquid chromatography (hereinafter, referred to as HPLC). Will be abbreviated.)
The sugar composition was analyzed at. The HPLC apparatus is "CCPD system" manufactured by Tosoh Corporation, and the analytical column is "YMC-pack PA-03" manufactured by YMC Corporation (4.
6 mmφ × 250 mm), the eluent is acetonitrile: water = 78: 22 (volume ratio) at a flow rate of 1.2 ml / min,
Detection was performed with a differential refractometer. The results are shown in Table 5.

[0080]

[Table 5]

As is clear from the results in Table 5, the conversion reaction from maltose to trehalose proceeded well regardless of the maltose concentration of the substrate, and about 80% conversion to trehalose was achieved.

[0082]

[Experiment 7 Effect of temperature on trehalose production] At a maltose concentration of 20%, pH 7.0, 2 units of the purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 were added per maltose gram, and the temperature was kept at 5 ° C and 10 ° C. ℃, 1
The reaction was carried out at 5 ° C., 20 ° C. or 25 ° C., the reaction solution was collected over time, and heated at 100 ° C. for 10 minutes to inactivate the enzyme. This enzyme reaction solution was subjected to HPLC as in Experiment 6.
The sugar composition was analyzed at. Table 6 shows the trehalose content at each temperature and each time.

[0083]

[Table 6]

As is clear from the results in Table 6, the higher the reaction temperature, the higher the trehalose production rate, but the conversion reaction from maltose to trehalose proceeded well even at a temperature of 5 ° C. 82% conversion.

[0085]

[Experiment 8 Preparation of Trehalose from Maltose] 10 parts by weight of maltose (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was dissolved in 40 parts by weight of water, and the temperature was 15 ° C. and the pH was 7.0.
The purified maltose-trehalose converting enzyme obtained by the method of Experiment 2 was added in an amount of 2 units per maltose gram and reacted for 48 hours, and then heated at 100 ° C. for 10 minutes to inactivate the enzyme. This solution contains about 7 trehalose per solid.
It contained 4%. This solution was decolorized with activated carbon, desalted with an ion exchange resin (H-type and OH-type), purified, and concentrated to a concentration of about 78%, and trehalose hydrous crystals were used as seed crystals to give 0.1% per solid matter. When added, and allowed to stand at room temperature overnight, crystals were precipitated. The obtained musket is dehusked, the crystals are sprayed with a small amount of water to wash the crystals, and the purity is 99.
About 3.0 parts by weight of an extremely high-purity trehalose hydrous crystal of 8% was obtained.

[0086]

[Experiment 9 Enzyme production] Glucose 2.0 w / v%, ammonium sulfate 1.0 w / v%, dipotassium phosphate 0.
1 w / v%, monosodium phosphate 0.06 w / v%, magnesium sulfate 0.05 w / v%, calcium carbonate 0.
Liquid medium consisting of 3 w / v% and water was put in a 500 ml Erlenmeyer flask by 100 ml, sterilized in an autoclave at 115 ° C. for 30 minutes, cooled, and inoculated with Pseudomonas putida H262 (FERMBP-4579), What was subjected to rotary shaking culture at 27 ° C. and 200 rpm for 24 hours was used as a seed culture.

About 20 liters of a medium having the same composition as in the case of seed culture was put in a fermenter having a volume of 30 liters, sterilized by heating, cooled to a temperature of 27 ° C., and then inoculated with 1 v / v% of the seed culture,
Approximately 20 while keeping the temperature at 27 ° C and pH 6.5 to 8.0.
The cells were cultured with aeration and stirring for hours.

The maltose / trehalose converting enzyme activity of the culture broth was 0.12 unit / ml. A part of the culture solution is collected and centrifuged to separate the cells and the culture solution supernatant, and the cells are suspended in 50 mM phosphate buffer solution (pH 7.0) to obtain the same solution as the original culture solution. After determining the amount, the maltose / trehalose converting enzyme activity of the bacterial cell suspension and the culture supernatant was measured, and it was found that 0.11 unit / ml of activity was found in the culture supernatant in the bacterial suspension. Was observed to have an activity of 0.01 unit / ml. The activity of this enzyme was measured at a reaction temperature of 35 ° C.

[0089]

[Experiment 10 Purification of Enzyme] The culture solution obtained in Experiment 9 was centrifuged to recover a bacterial cell having a wet weight of about 0.45 kg.
The cells were suspended in 0 mM phosphate buffer (pH 7.0). Approximately 2 liters of this microbial cell suspension was treated with an ultra-high pressure microbial cell disruption device (“Minilab” sold by Dainippon Pharmaceutical Co., Ltd.) to disrupt the bacterial cells, and the disrupted treatment liquid was centrifuged (15,000 G, 30 minutes). ), About 1.7 l of supernatant was obtained. Ammonium sulfate was added to the supernatant to dissolve it at a saturation level of 0.7,
After allowing to stand overnight, the salted out product of ammonium sulfate was recovered by centrifugation.

The obtained ammonium sulfate salt precipitate was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against the same buffer for 24 hours, and centrifuged to remove insolubles. The dialysate (400 ml) was divided into two portions and subjected to ion exchange column chromatography (gel amount 300 ml) using "DEAE-Toyopearl".

The maltose-trehalose converting enzyme of the present invention was adsorbed on "DEAE-Toyopearl" and eluted from the column with the same buffer containing salt. After the eluted enzyme activity fraction was collected, it was dialyzed against the same buffer solution, and again "DEA
Ion exchange column chromatography (gel amount 80 ml) using "E-Toyopearl" was performed. The adsorbed maltose / trehalose converting enzyme was converted to 0.1M to 0.3M of sodium chloride.
A linear gradient of M was used to elute from the column, and the enzyme active fraction was collected.

Then, gel filtration chromatography (400 ml of gel amount) using "Toyopearl HW-55S" manufactured by Tosoh Corporation was carried out to collect the eluted enzyme active fraction. Table 7 shows the amount of enzyme activity, the specific activity, and the yield at each purification step.

[0093]

[Table 7]

The purified enzyme preparation was subjected to gel electrophoresis containing 7.5 w / v% concentration polyacrylamide to examine the purity of the enzyme preparation. As a result, the protein band was single and had a high purity.

[0095]

[Experiment 11 Properties of enzyme] The purified maltose-trehalose-converting enzyme preparation obtained by the method of Experiment 10 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration: 7.5 w / v).
%), And the molecular weight of this enzyme was measured by comparison with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.) that was electrophoresed at the same time.
It was 0 to 120,000 daltons.

The purified maltose / trehalose converting enzyme preparation was subjected to an isoelectric point polyacrylamide gel electrophoresis method containing 2 w / v% ampholine (Pharmacia LCA), and the pH of the protein band and gel was measured after the electrophoresis. When the isoelectric point of this enzyme was determined by the method, the isoelectric point was pI of about 4.1 to 5.1.

The effects of temperature and pH on the activity of this enzyme were examined according to the activity measuring method. The results are shown in FIG. 5 (effect of temperature) and FIG. 6 (effect of pH). The optimum temperature of the enzyme is pH 7.0, around 37 ℃ for 60 minutes reaction, the optimum pH
Was about 7.3 to 8.3 after reacting at 35 ° C. for 60 minutes. The temperature stability of this enzyme was determined by holding the enzyme solution (50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water, and measuring the remaining enzyme activity. The pH stability was determined by holding the enzyme in a 50 mM buffer of each pH at 35 ° C. for 60 minutes, adjusting the pH to 7.0, and measuring the remaining enzyme activity. The results are shown in Fig. 7 (temperature stability) and Fig. 8 (pH).
Stability). The temperature stability of this enzyme was up to around 40 ° C., and the pH stability was about 6.0 to 9.5.
The enzyme activity is 1 mM Cu ⁺⁺ , Hg ⁺⁺ or 50 m
Inhibited with M Tris-HCl buffer.

[0098]

[Experiment 12 Action on various carbohydrates] The purified enzyme of Pseudomonas putida H262 obtained in Experiment 10 was allowed to act on various carbohydrates according to the method of Experiment 4 except that the reaction temperature was 35 ° C. It was tested whether it could become. As a result, the enzyme of Pseudomonas putida H262 was
Like the enzyme of Pimelobacter species R48,
It acts only on maltose and trehalose to convert maltose to trehalose and trehalose to maltose. The equilibrium point is offset to the trehalose side,
High conversion rate from maltose to trehalose, about 7
It turned out to be 0%.

[0099]

[Experiment 13 Effect of maltose concentration on trehalose production] Purified maltose-trehalose conversion obtained by the method of Experiment 10 at a maltose concentration of 5%, 10%, 20% or 30% at a temperature of 35 ° C and pH 7.0. The enzyme was added to the maltose gram in an amount of 2 units for reaction, the reaction solution was sampled with time, and heated at 100 ° C. for 10 minutes to inactivate the enzyme.

Using this reaction solution, the reducing power and the sugar composition were measured in the same manner as in Experiment 6. Table 8 shows the results.

[0101]

[Table 8]

As is clear from the results in Table 8, about 70% of trehalose was produced regardless of the maltose concentration of the substrate.

[0103]

[Experiment 14 Preparation of Trehalose from Maltose] 10 parts by weight of maltose (available from Hayashibara Biochemical Laboratories Co., Ltd.) was dissolved in 40 parts by weight of water, and the temperature was 35 ° C. and the pH was 7.0. The purified maltose / trehalose converting enzyme of the present invention was added in an amount of 2 units per maltose gram, reacted for 48 hours, and then heated at 100 ° C. for 10 minutes to inactivate the enzyme. This solution contained about 69% trehalose per solid. This solution was decolorized with activated carbon, desalted with an ion exchange resin (H-type and OH-type), purified, and concentrated to a concentration of about 78%, and trehalose hydrous crystals were used as seed crystals to give 0.1% per solid matter. When added, and allowed to stand at room temperature overnight, crystals were precipitated. The obtained mass kit was mashed and the crystals were washed by spraying a small amount of water to obtain about 2.3 parts by weight of extremely high-purity trehalose crystals having a purity of 99.7%.

[0104]

[Experiment 15 Enzyme production] Polypeptone 0.5 w / v
%, Yeast extract 0.1 w / v%, sodium nitrate 0.0
7 w / v%, disodium phosphate 0.01 w / v%, magnesium sulfate 0.02 w / v%, calcium chloride 0.
A liquid medium consisting of 01 w / v% and water was adjusted to pH 7.5, 100 ml each was put in a 500 ml Erlenmeyer flask, sterilized in an autoclave at 120 ° C. for 20 minutes, cooled, and cooled by Thermus aquaticus ATCC3.
The seed culture was obtained by inoculating 3923 and culturing with shaking at 60 ° C. and 200 rpm for 24 hours.

Approximately 20 liters of a medium having the same composition as in the case of seed culture were placed in two fermenters each having a volume of 30 liters, sterilized by heating and cooled to 60 ° C., and then 1 v / v of the seed culture solution was added.
% Of the solution was inoculated, and the culture was carried out with aeration and stirring for about 20 hours while maintaining the temperature at 60 ° C. and pH 6.5 to 8.0.

The maltose / trehalose converting enzyme activity of the culture was 0.35 unit / ml. Taking a part of the culture solution, centrifuging to separate the cells into a culture supernatant liquid, and further suspending the cells in 50 mM phosphate buffer (pH 7.0),
After making the same volume as the original culture solution, the maltose / trehalose converting enzyme activity of the bacterial cell suspension and the culture supernatant solution was measured, and it was found that the bacterial cell suspension had an enzyme activity of 0.33 unit / ml. However, 0.02 units / ml of enzyme activity was observed in the culture supernatant. The activity of this enzyme is 6
It was measured at 0 ° C.

[0107]

[Experiment 16 Purification of Enzyme] The culture solution obtained in Experiment 15 was centrifuged to recover a microbial cell having a wet weight of about 0.28 kg, which was suspended in a 10 mM phosphate buffer solution (pH 7.0). About 1.9 l of the cell suspension was treated with an ultrasonic crusher (“Model US300” manufactured by Nippon Seiki Seisakusho Co., Ltd.) to crush the cells. Centrifuge (15,000) the crushing solution.
G, 30 minutes) to obtain about 1.8 l of supernatant. Ammonium sulfate was added to the supernatant so as to give a saturation degree of 0.7, dissolved, allowed to stand at 4 ° C. overnight, and then centrifuged to recover a salt of ammonium sulfate.

The obtained ammonium sulfate salt-out product was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against the same buffer for 24 hours, and centrifuged to remove insoluble materials. The dialysate (1560 ml) was used as "DEAE" manufactured by Tosoh Corporation.
-Ion exchange column chromatography (gel amount 530 ml) using "Toyopearl 650" was performed in three times.

The maltose / trehalose converting enzyme of the present invention was adsorbed on "DEAE-Toyopearl" and eluted from the column with the same buffer containing sodium chloride. After the eluted enzyme active fraction was collected, it was dialyzed against the same buffer containing 1M ammonium sulfate,
Next, hydrophobic column chromatography (gel amount 380 ml) using "Butyl Toyopearl 650" was performed. The adsorbed maltose / trehalose converting enzyme was eluted from the column with a linear gradient of ammonium sulfate 1M to 0M to collect an enzyme active fraction.

Next, gel filtration chromatography using "Toyopearl HW-55S" (gel amount 380 ml).
Then, the eluted enzyme active fraction was collected.

Subsequently, ion exchange chromatography (gel amount 1.0 ml) using "Mono Q HR5 / 5" manufactured by Pharmacia LCAB was performed, and the enzyme was eluted with a linear gradient of 0.1 M to 0.35 M sodium chloride. The active fraction was collected. Table 9 shows the amount of enzyme activity, specific activity and yield in each step of purification.

[0112]

[Table 9]

The purified enzyme preparation was assayed for the purity of the enzyme preparation by gel electrophoresis containing 5 w / v% concentration polyacrylamide. As a result, the protein band was single and had a high purity.

[0114]

[Experiment 17 Properties of enzyme] The purified maltose / trehalose-converting enzyme preparation obtained by the method of Experiment 16 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration 7.5 w / v).
%), And the molecular weight of this enzyme was measured by comparison with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.) that was electrophoresed at the same time.
It was 0 to 110,000 daltons.

The purified maltose / trehalose converting enzyme preparation was subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2% w / v ampholine (manufactured by Pharmacia LCA), and the pH of the protein band and gel was measured after the electrophoresis. Then, the isoelectric point of this enzyme was determined, and the isoelectric point was pI of about 3.8 to 4.8.

The effects of temperature and pH on the enzyme activity were examined according to the activity measuring method. The results are shown in FIG. 9 (effect of temperature) and FIG. 10 (effect of pH). The optimum temperature of the enzyme is pH 7.0, around 65 ° C for 60 minutes reaction, and the optimum pH.
Was about 6.0 to 6.7 after reacting at 60 ° C. for 60 minutes. The temperature stability of the present enzyme was determined by holding the enzyme solution (containing 50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water, and measuring the remaining enzyme activity. In addition, the pH stability of this enzyme is 50
After maintaining in mM buffer at 60 ° C. for 60 minutes, the pH was adjusted to 7.0 and the residual enzyme activity was measured to obtain the value. The respective results are shown in FIG. 11 (temperature stability) and FIG. 12 (pH stability). The temperature stability of this enzyme is up to about 80 ° C., and the pH stability is about 5.5 to 9.
It was 5. The enzyme activity is 1 mM Cu ⁺⁺ , Hg
^{+ +} Or 50 mM Tris-HCl buffer.

[0117]

[Experiment 18 Action on Various Carbohydrates] The purified enzyme of Thermus aquaticus ATCC33923 obtained in Experiment 16 was allowed to act on various sugars according to the method of Experiment 4 except that the reaction temperature was 50 ° C. It was tested whether it could be a substrate. As a result, Thermus Aquaticus ATC
The enzyme of C33923 is Pimelobacter species
R48 enzyme or Pseudomonas putida H
Similar to the enzyme of No. 262, it acted only on maltose and trehalose to convert maltose to trehalose and trehalose to maltose. It was found that the equilibrium point was offset to the trehalose side, and the conversion rate from maltose to trehalose was high, reaching 70% or more.

[0118]

[Experiment 19 Effect of maltose concentration on trehalose production] Maltose concentration was 2.5%, 5%, 10%, 2
0% or 40%, temperature 60 ℃, pH 6.5,
Thermus aquaticus AT obtained by the method of Experiment 16
2.5 units of purified maltose / trehalose converting enzyme of CC33923 was added per maltose gram for reaction, a reaction solution was collected at 72 hours, and heated at 100 ° C. for 30 minutes to inactivate the enzyme. Using this reaction solution, the reducing power and the sugar composition were measured in the same manner as in Experiment 6. Table 10 shows the results.

[0119]

[Table 10]

As is clear from the results in Table 10, about 70% of trehalose was produced regardless of the maltose concentration of the substrate.

[0121]

[Experiment 20 Effect of temperature on trehalose production] Thermus aquaticus ATCC339 obtained by the method of Experiment 16 at a maltose concentration of 20% and a pH of 6.5
Add 23 units of purified maltose-trehalose converting enzyme of 23 units per maltose gram, and heat at 40 ° C, 50 ° C.
The reaction was carried out at 60 ° C., 60 ° C. or 70 ° C., the reaction solution was sampled with time, and heated at 100 ° C. for 30 minutes to inactivate the enzyme. This reaction solution was analyzed for sugar composition by HPLC in the same manner as in Experiment 6. Table 11 shows the trehalose content at each temperature and each time.

[0122]

[Table 11]

As is clear from the results shown in Table 11, the conversion rate from maltose to trehalose was higher as the reaction temperature was lower, and about 80% was converted to trehalose at 40 ° C.

[0124]

[Experiment 21: Production of maltose / trehalose converting enzyme from other microorganisms and its properties] Among known microorganisms, a specific microorganism confirmed to have the ability to produce maltose / trehalose converting enzyme of the present invention was used in the same manner as in Experiment 15. It culture | cultivated in the Erlenmeyer flask for 48 hours. After examining the enzyme activity of the culture broth, according to the method of Experiment 16, the culture broth was applied to a disruption device, and the supernatant was dialyzed to obtain a partially purified enzyme.
The property was investigated according to the method of. The results are shown in Table 12.

[0125]

[Table 12]

Using the partially purified enzymes derived from these known microorganisms belonging to the genus Thermus shown in Table 12, according to the method of Experiment 18, the action on various sugars was examined. In the case of the enzyme derived from Thermus aquaticus ATCC 33923, Similar to, only acts on maltose and trehalose,
It was found to produce trehalose from maltose.

Also, Thermus Louver ATCC35
The maltose-trehalose converting enzyme of 948 had lower optimum temperature and stable temperature than the enzyme of Thermus aquaticus ATCC 33923, but other enzymes of the genus Thermus belong to Thermus aquaticus ATCC3.
It was found that it has almost the same properties as the enzyme of 3923 and has high thermostability.

[0128]

[Experiment 22 Physicochemical properties of the prepared trehalose] The physicochemical properties of the trehalose prepared by the method of Experiment 8 were examined using a highly pure preparation. Melting point is 97.0 ° C., specific rotation is [α] ²⁰ _D + 199 ° (c = 5), and heat of fusion is 57.8.
KJ / mole, the solubility was 77.0 g as an anhydride in water at 25 ° C. These physical property values were in good agreement with those of a commercially available trehalose hydrous crystal (manufactured by Wako Pure Chemical Industries, Ltd.) that was measured at the same time.

[0129]

[Experiment 23: In-vivo utilization test] Kouji et al., "Clinical Nutrition", Vol. 41, No. 2, pp. 200-208 (19
High purity trehalose preparation (purity 99.8%) 3 prepared in Experiment 8 according to the method reported in (1972) 3
0 g was made a 20 w / v% aqueous solution, and this was volunteer 3
Orally administered to healthy individuals (healthy 26-year-old, 27-year-old, and 30-year-old male), and blood samples were collected over time to measure blood glucose level and insulin level. Glucose was used as a control. As a result, trehalose behaved similarly to the case of glucose, and both the blood glucose level and the insulin level showed maximum values about 0.5 to 1 hour after administration. Trehalose is
It was found to be easily digested, absorbed, and metabolized and used as an energy source. Therefore, the trehalose obtained by the method of the present invention and the sugar containing the same are suitable as a sugar source for energy supply.

[0130]

[Experiment 24 Acute toxicity test] Using a mouse, the high-purity trehalose hydrous crystal prepared in Example A-6 was orally administered to perform an acute toxicity test. As a result, trehalose was a low-toxic substance, and no death was observed even at the maximum dose that could be administered. Therefore, although not an accurate value, the LD ₅₀ value was 50 g / kg or more.

[0131]

[Experiment 25 Effect of maltose concentration on trehalose production by culture method] Maltose was added to maltose in an amount of 2 to 20 w
It was cultured in a nutrient medium containing / v%, and the effect of maltose concentration on the trehalose yield in the culture was examined.
The culturing method is Pimelobacter species R48.
In the case of (FERM BP-4315), the same as in Experiment 1 except that the nutrient medium was a medium containing separately sterilized maltose in an amount of 2 to 20 w / v% instead of 2.0 w / v% glucose. Incubate for 72 hours at 27 ° C in a fermenter, then add 0.1v / v% of surfactant (polyoxyethylene sorbitan monopalmitate, "Tween 40" sold by Wako Pure Chemical Industries, Ltd.) for 24 hours. Continued. Also, Thermus Aquaticus A
In the case of TCC33923, the nutrient medium was separately sterilized, and a medium containing 2 to 20 w / v% of maltose was used.
After culturing for 24 hours, the surfactant "Tween 40" was further added at 0.1 v / v% and the culturing was continued for 24 hours. The culture was centrifuged and the trehalose content (mg / ml) contained in the supernatant was measured by HPLC. The results are shown in Table 13.
Shown in

[0132]

[Table 13]

As is clear from the results shown in Table 13, the concentration of maltose in the nutrient medium is 20 w / v% or less, preferably 15 w / v% or less, more preferably 5 to 10 w / v%.
It was found that the trehalose yield was high in the vicinity, which was suitable for trehalose production.

Hereinafter, a method for producing trehalose or a sugar containing the same by using a microorganism having the ability to produce maltose / trehalose converting enzyme of the present invention will be described in Example A in which trehalose or a sugar containing the same is added. Such a composition is shown in Example B.

[0135]

Example A-1 10% Concentration Potato Starch Milk (pH 5.
To 5), 2 units of α-amylase (“Spitase HS” manufactured by Nagase Seikagaku Co., Ltd.) was added per gram of starch to heat-gelatinize and liquefy with stirring, and immediately after autoclaving (120 ° C.) for 20 minutes, Temperature 50 ℃, pH
Adjusted to 5.0. To this, β-amylase (manufactured by Nagase Seikagaku Co., Ltd.) was added in an amount of 20 units per gram of starch, and isoamylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) in a ratio of 500 units per gram of starch.
The mixture was reacted for a time, then heated to 95 ° C. to inactivate the enzyme, filtered and decolorized to obtain a sugar solution having a maltose content of about 92%. As a sugar source, instead of 2.0 w / v% glucose, the above sugar solution was separately sterilized to obtain 10 w / v% per solid matter.
Except that the nutrient medium prepared according to the method of Experiment 1 was used as a fermenter, and the seed culture of Pimelobacter species R48 (FERM BP-4315) capable of producing maltose / trehalose converting enzyme was added at 1 v / v% inoculation and 72 at 27 ° C as in Experiment 1.
Culturing with aeration and stirring for 0.2 hours, and 0.2v of surfactant (alkylphenol polyoxyethylene ether, "Triton X-100" sold by Wako Pure Chemical Industries, Ltd.)
/ V% was added and the culture was continued for 24 hours. This culture was filtered to remove insoluble matter, and the resulting filtrate was heated to 95 ° C. to deactivate the enzyme, then concentrated, decolorized and filtered with activated carbon according to a conventional method, and H-type and OH-type. It was desalted with an ion exchange resin for purification, and further concentrated to obtain a syrup having a concentration of about 70% in a yield of about 65% based on a solid substance. This product contains about 44% trehalose per solid matter, has a mild sweetness, moderate viscosity and moisturizing property, and is a sweetener, a taste improver,
It can be advantageously used as a stabilizer, an excipient, etc. in various compositions such as various foods, drinks, cosmetics and pharmaceuticals.

[0136]

[Example A-2] To the filtrate of the culture obtained by the method of Example A-1, 10 units of glucoamylase per gram of solid matter, "glucozyme" manufactured by Nagase Seikagaku Co., Ltd. was added.
A sugar solution obtained by reacting at H5.0 and 50 ° C. for 24 hours, followed by heat inactivation, decolorization, and desalting purification is used as a raw sugar solution. In order to increase the content of trehalose, a sodium-type strongly acidic cation exchange resin ( "XT-" manufactured by Tokyo Organic Chemical Industry Co., Ltd.
1016 ″, cross-linking degree 4%) was used for ion exchange column chromatography. The resin was packed in four jacketed stainless steel columns having an inner diameter of 5.4 cm and connected in series to give a resin layer total length of 20 m. Column temperature 60 ℃
While maintaining at 5%, add 5% v / v of sugar solution to the resin,
Warm water at 60 ° C. was flown through this with SV 0.15 to fractionate, glucose was removed, and a trehalose-rich fraction was collected. Further purification, concentration, vacuum drying and crushing gave a trehalose-rich powder in a yield of about 35% based on solids. This product contains about 97% trehalose,
It has extremely low reducibility, mellow and elegant sweetness, and is advantageously used as a sweetener, taste improver, quality improver, stabilizer, excipient, etc. in various compositions such as food and drink, cosmetics, and pharmaceuticals. it can.

[0137]

Example A-3 The trehalose-rich fraction obtained by the method of Example A-2 was decolorized with activated carbon and desalted with an ion exchange resin according to a conventional method to obtain a purified solution, which had a concentration of about 70%.
After concentrating to 2, the mixture was taken in an auxiliary crystallizer, and about 2% of trehalose hydrous crystals was added as a seed crystal and gradually cooled to obtain a maskit with a crystallization rate of about 45%. From the nozzle on the drying tower 1
It was sprayed at a high pressure of 50 kg / cm ² . At the same time 8
Hot air of 5 ° C is blown from the top of the drying tower to collect it on the transfer wire mesh conveyor provided at the bottom, and while blowing warm air of 45 ° C from under the conveyor, the crystal powder collected on the wire mesh conveyor is dried. It was gradually moved out of the tower and taken out. The crystal powder thus taken out is filled in an aging tower and aged for 10 hours while sending warm air, crystallization and drying are completed, and trehalose hydrous crystal powder is added to a raw material trehalose-rich sugar solution to a solid content of about Obtained in a yield of 90%. This product has virtually no hygroscopicity, is easy to handle, has a sweetener, a taste improver,
It can be advantageously used as a stabilizer or the like in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

[0138]

Example A-4 The trehalose-rich fraction obtained by the method of Example A-2 was purified in the same manner as in Example A-3, then placed in an evaporator and boiled under reduced pressure to a water content of about 3. It was 0% syrup. Then, it was transferred to an auxiliary crystallizer, to which anhydrous crystalline trehalose was seeded as 1% per syrup solid.
In addition, a block was prepared by stirring and assisting crystallization at 120 ° C., then taking out to an aluminum vat and aging at 100 ° C. for 6 hours for crystallization. Then, crush this block with a cutting machine,
By fluidizing and drying, anhydrous crystalline trehalose powder having a water content of about 0.3% was obtained in a yield of about 85% based on the solid content, based on the raw trehalose-rich sugar solution. This product can be used not only as a dehydrating agent for water-containing products such as foods, cosmetics, pharmaceuticals, raw materials thereof, or processed intermediates, but also as a white powder sweetener having an elegant sweetness for various foods, beverages, cosmetics, pharmaceuticals, etc. It can be advantageously used in a composition.

[0139]

Example A-5 0.1% calcium carbonate was added to corn starch milk with a concentration of 33%, and the pH was adjusted to 6.5.
In addition to this, α-amylase ("Tamamir 60" manufactured by Novo Co.
L ″) is added at 0.2% per gram of starch and the mixture is added at 95 ° C. for 15
Allowed to react for minutes. The reaction solution was autoclaved (120
C.) for 30 minutes and then cooled to 55.degree. C., to which 500 units of isoamylase (manufactured by Hayashibara Biochemical Research Co., Ltd.) per gram of starch and .beta.-amylase (manufactured by Nagase Seikagaku Corp.) are added. Add 30 units per gram,
The mixture was reacted for 48 hours, then heated to 95 ° C. to inactivate the enzyme, filtered and decolorized to obtain a sugar solution having a maltose content of about 84%. As a sugar source, a nutrient medium prepared according to the method of Experiment 9 was placed in a fermenter except that the above-mentioned sugar solution was separately sterilized and 10 w / v% was added per solid matter, and maltose / trehalose converting enzyme production was performed on the nutrient medium. Pseudomonas putida H262 (FERM BP-4 with ability)
The seed culture of (579) was inoculated with 1 v / v% and cultured under aeration and stirring at 27 ° C. for 48 hours in the same manner as in Experiment 9, and then 0.2 v / v% of the surfactant “Tween 40” was added for 24 hours. The culture was continued. The culture is filtered to remove insolubles,
The obtained filtrate is heated to 95 ° C. to deactivate the enzyme, then concentrated, decolorized and filtered with activated carbon according to a conventional method, purified by desalting with H-type and OH-type ion exchange resins, and further concentrated. And syrup with a concentration of about 70% per solid
% Yield. This product contains about 64% of trehalose per solid, has low reducing property, mild sweetness, moderate viscosity and moisturizing property, and is advantageous for various compositions such as various foods, drinks, cosmetics and pharmaceuticals. Available for

[0140]

[Example A-6] The syrup obtained by the method of Example A-5 was concentrated to a concentration of about 80% and placed in an auxiliary crystallizer, to which about 1% of trehalose hydrous crystalline powder was added as a seed crystal and gradually cooled with stirring. ,
Crystallized. Then, the mixture was mashed with a basket type centrifuge, and the crystals were sprayed with a small amount of water and washed to obtain high-purity trehalose-containing crystals in a yield of about 20% based on solids. This product exhibits the same physicochemical properties as in Experiment 22, and can be advantageously used as various sweeteners, taste improvers, quality improvers, stabilizers and the like in various compositions such as various foods and drinks, cosmetics and pharmaceuticals. Further, it can be advantageously used as an industrial reagent or a chemical raw material.

[0141]

[Example A-7] Corn starch milk (pH 10%)
To 5.5), add 2 units of α-amylase "Spitase HS" per gram of starch and heat-gelatinize / liquefy under stirring.
Immediately after autoclaving (120 ° C.) for 20 minutes, the temperature was adjusted to 55 ° C. and pH 5.0. Isoamylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was added to this at 300 units per gram of starch and β-amylase (Nagase Seikagaku Co., Ltd.) at a ratio of 20 units per gram of starch, and reacted for 24 hours. Then, it was heated to 95 ° C. to inactivate the enzyme, filtered and decolorized to obtain a sugar solution having a maltose content of about 92%. As a sugar source, a nutrient medium prepared according to the method of Experiment 15 was placed in a fermenter except that the above-mentioned sugar solution was separately sterilized and 10 w / v% was added per solid matter, and maltose / trehalose-converting enzyme production was performed on the nutrient medium. Thermus Aquaticus ATCC with ability
The seed culture of 33923 was inoculated with 1 v / v% and experiment 15
Similarly, culture with aeration and stirring at 60 ° C. for 40 hours, and then add 0.1% v / v% of the surfactant “Triton X-100”.
And 50 mg of egg white lysozyme was added per 1 liter of the culture solution, and the culture was continued for 16 hours. This culture was filtered to remove insoluble matter, and the resulting filtrate was heated to 95 ° C. to deactivate the enzyme, then concentrated, decolorized and filtered with activated carbon according to a conventional method, and H-type and OH-type. It was desalted with an ion exchange resin, purified, and further concentrated to obtain a syrup having a concentration of about 70% in a yield of about 55% based on a solid matter. This product contains about 68% trehalose per solid, has mild sweetness, moderate viscosity and moisturizing property, and is used as a sweetener, taste improver, stabilizer, excipient, etc. for various foods and drinks. , It can be advantageously used for various compositions such as cosmetics and pharmaceuticals.

[0142]

[Example A-8] The syrup obtained by the method of Example A-7 was concentrated to a concentration of about 85% and placed in an auxiliary crystallizer. After mixing about 1% of seed crystals, the syrup was placed in a vat and kept at 20 ° C for 4 days. The mixture was left to stand to be crystallized and solidified, then pulverized with a cutting machine and dried to obtain a trehalose hydrous crystal powder containing honey in a yield of about 95% based on solids. This product has virtually no hygroscopicity, is easy to handle, and is useful as a sweetener, taste improver, quality improver, stabilizer, etc. for various compositions such as food and drink, cosmetics, and pharmaceuticals. Available for

[0143]

Example A-9 The syrup obtained by the method of Example A-7 is concentrated to a concentration of about 80% and taken in an auxiliary crystallizer. Crystallization is performed in the same manner as in Example A-6. Water-containing trehalose crystals were obtained in a yield of about 20% based on solids. Experiment 22
Showing the same physicochemical properties as in Example A-6,
It can be advantageously used for various compositions such as various foods and drinks, cosmetics and pharmaceuticals, as well as industrial reagents, industrial raw materials and chemical raw materials.

[0144]

[Example B-1 Sweetener] 1 part by weight of trehalose hydrous crystal powder obtained by the method of Example A-3 was mixed with α-glycosyl stevioside (“αG sweet” sold by Toyo Seika Co., Ltd.).
0.01 parts by weight and 0.01 parts by weight of L-aspartyl-L-phenylalanine methyl ester (“Aspartame” sold by Ajinomoto Co., Inc.) were uniformly mixed and subjected to a granule molding machine to obtain a granular sweetener. This product has an excellent sweetness and has about twice the sweetness of sucrose, and the calorie per sweetness is reduced to about 1/2 that of sucrose. This sweetener has excellent stability without decomposition of the high-intensity sweeteners mixed in it, and as a low-calorie sweetener, it is a low-calorie food and drink for obese people, diabetics, etc. whose calorie intake is restricted. It is suitable for sweetening things. Further, the present sweetener is suitable for sweetening foods and beverages that suppress tooth decay because it produces less acid due to cavities-inducing bacteria and less insoluble glucan.

[0145]

[Example B-2 Hard candy] 100 parts by weight of a 55% sucrose solution were mixed with 30 parts by weight of the trehalose-containing syrup obtained by the method of Example A-1 by heating, and then the water content was reduced to less than 2% under reduced pressure. Concentrate by heating, mix with 1 part by weight of citric acid and an appropriate amount of lemon flavor and color,
A product was obtained by molding according to a conventional method. This product is a high-quality hard candy that is crisp, has a good taste, and does not crystallize or deform sucrose.

[0146]

[Example B-3 Chocolate] 40 parts by weight of cocoa paste, 10 parts by weight of cocoa butter, 30 parts by weight of sucrose, high-purity trehalose hydrous crystal 20 obtained by the method of Example A-6
After mixing parts by weight and passing through a refiner to reduce the particle size, the mixture is put in a conche and kneaded at 50 ° C. for 2 days and nights. During this period, 0.5 part by weight of lecithin was added and thoroughly mixed and dispersed. Next, adjust the temperature to 31 ° C with a temperature controller, pour into the mold immediately before the butter hardens, and remove the foam with a vibrator.
It was solidified by passing through a 10 ° C. cooling tunnel for 20 minutes. This was die-cut and packaged to obtain a product. This product has no hygroscopicity, has good color and gloss, has a good internal structure, melts smoothly in the mouth, and has an elegant sweetness and mellow flavor.

[0147]

[Example B-4 Chewing gum] 3 parts by weight of a gum base was heated and melted to a softness, and 4 parts by weight of sucrose and 3 parts by weight of the trehalose hydrous crystal powder obtained by the method of Example A-3 were added to the chewing gum. A proper amount of flavor and color was mixed, and kneaded by a roll, molded and packaged according to a conventional method to obtain a product. This product is a chewing gum with good texture and flavor.

[0148]

Example B-5 Sweetened condensed milk: In 100 parts by weight of raw milk, 3 parts by weight of the trehalose-containing syrup obtained by the method of Example A-5 and 1 part by weight of sucrose were dissolved, sterilized by heating with a plate heater, and then the concentration was 70. %, And aseptically canned to obtain the product. The product has a mild sweetness and a good flavor, and can be advantageously used for seasoning baby food, fruit, coffee, cocoa, tea and the like.

[0149]

[Example B-6 Lactic acid bacterium beverage] 175 parts by weight of skim milk powder,
Powder containing a high amount of trehalose obtained by the method of Example A-2 80
1 part by weight and 50 parts by weight of the powder containing a high amount of lactosucrose disclosed in JP-A-4-281795 are added to water 1,2.
Dissolve in 00 parts by weight, sterilize at 65 ℃ for 30 minutes, 40 ℃
After cooling to 30 ° C., in accordance with a conventional method, 30 parts by weight of a starter of lactic acid bacteria was inoculated and cultured at 37 ° C. for 8 hours to obtain a lactic acid bacteria drink. This product is a lactic acid bacteria beverage with good flavor. In addition, the product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacterium growth promoting effect.

[0150]

[Example B-7 Powdered juice] Example A-6 was prepared based on 33 parts by weight of orange juice powder prepared by spray drying.
50 parts by weight of high-purity trehalose hydrous crystal obtained by the method of
Sucrose 10 parts by weight, anhydrous citric acid 0.65 parts by weight, malic acid 0.1 parts by weight, L-ascorbic acid 0.1 parts by weight, sodium citrate 0.1 parts by weight, pullulan 0.5 parts by weight, powdered flavor An appropriate amount was thoroughly mixed and stirred, pulverized into a fine powder, charged into a fluidized bed granulator, and the exhaust air temperature was set to 40 ° C., and the trehalose-rich syrup obtained by the method of Example A-5 was used as a binder. Spray, granulate for 30 minutes,
The product was obtained by weighing and packaging. This product has a fruit juice content of about 30.
% Powdered juice. In addition, this product had no offensive taste or odor and was stable for a long period of time.

[0151]

Example B-8 Custard Cream 100 parts by weight of corn starch, 100 parts by weight of syrup containing trehalose obtained by the method of Example A-1, 80 parts by weight of maltose, 20 parts by weight of sucrose and 1 part by weight of salt were thoroughly mixed. , Chicken eggs 2
Add 80 parts by weight and stir, then boil the milk 1,0
00 parts by weight was gradually added, and the mixture was further heated and stirred, and when the cornstarch was completely gelatinized and the whole became translucent, turn off the heat, cool it, add an appropriate amount of vanilla flavor, and measure. The product was filled, packed, and obtained. This product is
It has a smooth luster and is mildly sweet and delicious.

[0152]

[Example B-9 Uironomoto] 90 parts by weight of rice flour, 20 parts by weight of corn starch, 40 parts by weight of sucrose, Example A-
80 parts by weight of trehalose hydrous crystal powder obtained by the method of 3 and 4 parts by weight of pullulan were uniformly mixed to produce iuirosin. Uironin, an appropriate amount of matcha and water were kneaded, put in a container and steamed for 60 minutes to produce matcha uiro. This product has good shine, mouthfeel, and flavor. In addition, the aging of starch is suppressed and the shelf life is good.

[0153]

[Example B-10 Apricot] 10 parts by weight of raw azuki bean was added with water according to a conventional method, and boiled, astringently cut, and drained to remove water-soluble impurities, and about 21 parts by weight of azuki bean paste. Obtained. This raw bean paste, 14 parts by weight of sucrose, Example A
Add 5 parts by weight of trehalose-containing syrup obtained by the method of -7 and 4 parts by weight of water, boil, and add a small amount of salad oil to the mixture to knead it so as not to break the mash and obtain about 35 parts by weight of bean paste. It was This product has no color burn, has a good texture, and has a good flavor, and is suitable as an ingredient for red bean bread, steamed bun, dumpling, monaka, frozen dessert and the like.

[0154]

[Example B-11 Bread] 100 parts by weight of wheat flour, 2 parts by weight of yeast, 5 parts by weight of sugar, 1 part by weight of trehalose-rich powder obtained by the method of Example A-2, and 0.1 parts of inorganic food.
According to a conventional method, knead parts by weight with water, and add 2 seeds at 26 ° C.
Fermented for a period of time, then aged for 30 minutes and baked. This product is a high-quality bread with good hue and color, moderate elasticity and mild sweetness.

[0155]

[Example B-12 Ham] 1,000 parts by weight of pork thigh meat are uniformly rubbed with 15 parts by weight of salt and 3 parts by weight of potassium nitrate, and are deposited in a cold room for one day. 500 parts by weight of water, 100 parts by weight of salt, 3 parts by weight of potassium nitrate, 40 parts by weight of trehalose-rich powder obtained by the method of Example A-2 and spices are soaked in a salting solution for 7 days in a cold room, and then, According to a conventional method, the product was washed with cold water, wrapped with a string, smoked, cooked, and cold-packed to obtain a product. This product is a high quality ham with good color and good flavor.

[0156]

Example B-13 Powder Peptide 1 part by weight of a soybean peptide solution for foods (“High New S” manufactured by Fuji Oil Co., Ltd.) at a concentration of 40% was added to a high-purity trehalose hydrous crystal obtained by the method of Example A-6. 2 parts by weight were mixed, put in a plastic vat, dried under reduced pressure at 50 ° C., and pulverized to obtain a powdered peptide. The product has a good flavor and can be advantageously used not only as a material for confectionery such as premixes and frozen desserts, but also as a baby food such as oral liquid food and tube liquid food, and a therapeutic nutrient.

[0157]

Example B-14 Powdered Miso 3 parts by weight of the anhydrous crystalline trehalose powder obtained by the method of Example A-4 were mixed with 1 part by weight of red miso and poured into a metal plate having a large number of hemispherical recesses. The mixture was allowed to stand overnight at room temperature to solidify, and the mold was released to obtain about 4 g of solid miso, and the solid miso was crushed to obtain a powdered miso. This product can be advantageously used as a seasoning for instant ramen, instant soup, etc. The solid miso can be used not only as a solid seasoning but also as a miso confectionery or the like.

[0158]

Example B-15 Powdered Egg Yolk Egg yolk prepared from raw eggs is sterilized by a plate-type heat sterilizer at 60 to 64 ° C., and 1 part by weight of the obtained liquid yolk is obtained by the method of Example A-4. The anhydrous crystalline trehalose powder was mixed at a ratio of 4 parts by weight, transferred to a vat and left overnight to be converted into trehalose hydrous crystals to prepare a block. This block was pulverized with a cutting machine to obtain powder egg yolk. The product can be advantageously used not only as a material for confectionery such as premixes, frozen desserts and emulsifiers, but also as baby foods such as oral liquid foods and tube liquid foods, nutritional supplements for therapy and the like. Further, it can be advantageously used as a skin beautifying agent, a hair restorer and the like.

[0159]

Example B-16 Cosmetic Cream 2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glyceryl monostearate, 2 parts by weight of trehalose-rich powder obtained by the method of Example A-2, 1 part by weight of α-glycosyl rutin, 1 part by weight of liquid paraffin, 10 parts by weight of glyceryl trioctanoate, and an appropriate amount of a preservative,
It is heated and dissolved according to a conventional method, and 2 parts by weight of L-lactic acid is added thereto.
5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water were added, emulsified by applying a homogenizer, and an appropriate amount of flavor was further added and mixed with stirring to prepare a cream. This product is
Has high anti-oxidant, high stability, high quality sunscreen,
It can be advantageously used as a skin beautifying agent and a whitening agent.

[0160]

[Example B-17 Powdered ginseng extract] 0.5 part by weight of ginseng extract was mixed with 1.5 parts by weight of the anhydrous crystalline trehalose powder obtained by the method of Example A-4, and the mixture was transferred to a vat for 2 days. The mixture was allowed to stand and converted into trehalose hydrous crystals to prepare blocks. This block was pulverized with a cutting machine and classified to obtain a powdered ginseng extract. This product was granulated with a proper amount of Vitamin B1 and Vitamin B2 powder in a granulating machine to obtain a vitamin-containing granular ginseng extract. This product can be advantageously used as a fatigue recovery agent, tonic, tonic agent, etc. It can also be used as a hair restorer.

[0161]

[Example B-18 Solid formulation] A human natural interferon-α preparation (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was applied to an immobilized anti-human interferon-α antibody column according to a conventional method, and contained in the preparation. Human natural interferon-α was adsorbed and the stabilizer, bovine serum albumin, was passed through to remove it, and then the pH was changed to obtain human natural interferon-α obtained by the method of Example A-6. Elution was performed using physiological saline containing 5% pure trehalose hydrous crystals. This solution is subjected to microfiltration and dehydrated and pulverized by adding about 20 times the amount of anhydrous crystalline maltose powder and "Fine Tose" sold by Hayashibara Shoji Co., Ltd., and tabletted with a tableting machine to give 1 tablet (about 200 mg. ), A tablet containing about 150 units of human natural interferon-α was obtained. As a sublingual tablet, about 1 to 10 tablets for adults are orally administered per day, and this product can be advantageously used for treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., for which the number of patients is increasing rapidly in recent years. In this product, the non-reducing sugar of the present invention and anhydrous crystalline maltose both act as stabilizers, and their activity is maintained well for a long period of time even when left at room temperature.

[0162]

Example B-19 Sugar-coated tablet A plain tablet having a weight of 150 mg was used as a core, to which 40 parts by weight of high-purity trehalose hydrous crystals obtained by the method of Example A-9 and pullulan (average molecular weight 20
10,000 parts), 2 parts by weight, 30 parts by weight of water, 25 parts by weight of talc and 3 parts by weight of titanium oxide were used to sugar-coat until the tablet weight became about 230 mg, and then 65 parts by weight of the same trehalose hydrous crystalline powder. , A coating solution consisting of 1 part by weight of pullulan and 34 parts by weight of water, sugar coating, and
It was polished with a wax solution to obtain a sugar-coated tablet having an excellent glossy appearance. The product has excellent impact resistance and maintains high quality for a long time.

[0163]

[Example B-20 Toothpaste] Compounded dicalcium phosphate 45.0 parts by weight Pullulan 2.95 parts by weight Sodium lauryl sulfate 1.5 parts by weight Glycerin 20.0 parts by weight Polyoxyethylene sorbitan laurate 0.5 parts by weight Antiseptic Agent 0.05 part by weight Trehalose hydrous crystal powder obtained by the method of Example A-8 12.0 parts Maltitol 5.0 parts water 13.0 parts by weight The above materials are mixed according to a conventional method, and toothpaste is prepared. Got This product has a moderate sweetness and is particularly suitable as a toothpaste for children.

[0164]

[Example B-21 Solid formulation for liquid foods] 500 parts by weight of high-purity trehalose hydrous crystals produced by the method of Example A-6, 270 parts by weight of powdered egg yolk, 209 parts by weight of skim milk powder, 4.4 parts by weight of sodium chloride, 1.8 parts by weight of potassium chloride, 4 parts by weight of magnesium sulfate, 0.01 part by weight of thiamine, 0.1 part by weight of sodium ascorbate, 0.6 part by weight of vitamin E acetate and 0.0 part of nicotinic acid amide.
A compound consisting of 4 parts by weight was prepared, and 25 g of this compound was filled in a moisture-proof laminated pouch and heat-sealed to obtain a product. This product is about 150 to 300m per bag
Orally, or nasal cavity, stomach,
It is used in the intestine and the like by a tube-based method, and can be advantageously used for supplying energy to the living body.

[0165]

[Example B-22 Infusion] A highly pure trehalose-containing crystal produced by the method of Example A-6 was added to water to a concentration of about 10 w /.
It was dissolved in v% and then microfiltered by a conventional method to make it pyrogen-free, and aseptically filled in a plastic bottle and stoppered to obtain a product. This product is a stable infusion solution that does not change over time and is suitable for intravenous, intraperitoneal, etc. administration. This product is isotonic with blood at a concentration of 10 w / v% and can be supplemented with energy at twice the concentration of glucose.

[0166]

[Example B-23 Infusion] A high-purity trehalose hydrous crystal produced by the method of Example A-9 and an amino acid mixture having the following composition were dissolved in water to give 5 w / v% and 30 w / v%, respectively. It was mixed and dissolved, and then purified in the same manner as in Example B-22 to be pyrogen-free, further, filled in a plastic bag and stoppered to obtain a product. Composition of amino acid formulation (mg / 100 ml) L-isoleucine 180 L-leucine 410 L-lysine hydrochloride 620 L-methionine 240 L-phenylalanine 290 L-threonine 180 L-tryptophan 60 L-valine 200 L-arginine hydrochloride 270 L-histidine hydrochloride 130 glycine 340 This product is a stable infusion solution that does not change over time because trehalose does not show reducibility despite the complex infusion solution of sugar and amino acid. It is suitable for administration to the inside. This product can be advantageously used not only for supplying energy to the living body but also for supplying amino acids.

[0167]

Example B-24 Ointment for Treatment of Wounds To 200 parts by weight of trehalose hydrous crystal powder and 300 parts by weight of maltose produced by the method of Example A-8, 50 parts by weight of methanol in which 3 parts by weight of iodine were dissolved was added and mixed. , 10 w / v
% 200 parts by weight pullulan aqueous solution was added and mixed to obtain a plaster for treating wounds, which has a proper extension and shows adhesiveness. This product not only acts as a bactericidal action by iodine, but also acts as an energy supplement to cells by trehalose, so the healing period is shortened and the wound surface is healed cleanly.

[0168]

As is apparent from the above, by culturing a microorganism capable of producing maltose / trehalose converting enzyme in a nutrient medium containing maltose, a non-reducing sugar can be extremely easily and shortly obtained from the culture. Since it can be produced in a short time, it facilitates the industrial production of trehalose or a sugar containing the same. Further, as maltose, β-amylase or β-amylase was added to a liquefied starch solution.
Utilizing maltose obtained by allowing starch debranching enzyme to act together with amylase significantly enhances the yield of trehalose from starch and facilitates its industrial production. The trehalose or the sugar containing the trehalose thus obtained is excellent in stability, has a good quality and has an elegant sweetness. Also, it is digested and absorbed by ingestion and becomes a calorie source.
In particular, trehalose is also used parenterally and is often metabolically utilized. Therefore, the trehalose of the present invention, or a sugar containing the trehalose is used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, etc.
It can be advantageously used for various compositions such as pharmaceuticals.

The establishment of the present invention makes it possible to industrially provide a large amount of trehalose or a sugar containing the trehalose, which has not heretofore been easily obtained from starch, which is an inexpensive and unlimited resource, on an industrial scale. It opens up a new path, and its impact is not limited to the food, cosmetics and pharmaceutical industries, but also to the agricultural, aquatic and livestock industries, the chemical industry, and the industrial significance to these industries is immeasurable.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of temperature on the enzyme activity of maltose-trehalose converting enzyme of Pimobacter plaques species R48.

[Fig. 2] pH on the enzyme activity of maltose-trehalose converting enzyme of Pimelobacter species R48.
It is a figure which shows the influence of.

FIG. 3 is a graph showing the effect of temperature on the stability of maltose-trehalose converting enzyme of Pimobacter plaques species R48.

FIG. 4 is a diagram showing the effect of pH on the stability of maltose-trehalose-converting enzyme of Pimelobacter species R48.

FIG. 5 is a graph showing the effect of temperature on the enzyme activity of Pseudomonas putida H262 maltose-trehalose converting enzyme.

FIG. 6 is a graph showing the effect of pH on the enzyme activity of maltose trehalose converting enzyme of Pseudomonas putida H262.

FIG. 7 is a diagram showing the effect of temperature on the stability of Pseudomonas putida H262 maltose trehalose converting enzyme.

FIG. 8 shows the effect of pH on the stability of Pseudomonas putida H262 maltose trehalose converting enzyme.

FIG. 9: Thermus aquaticus ATCC3392
It is a figure which shows the influence of the temperature on the enzyme activity of the maltose trehalose converting enzyme of 3.

FIG. 10: Thermus Aquaticus ATCC339
It is a figure which shows the influence of pH on the enzyme activity of the maltose trehalose converting enzyme of 23.

FIG. 11: Thermus Aquaticus ATCC339
It is a figure which shows the influence of the temperature on the stability of 23 maltose trehalose converting enzyme.

FIG. 12: Thermus Aquaticus ATCC339
It is a figure which shows the influence of pH on the stability of 23 maltose trehalose converting enzyme.

Claims (13)

[Claims] 1. A nutrient medium containing maltose,
Trehalose obtained by culturing a microorganism capable of producing a maltose / trehalose converting enzyme, or a sugar containing the same. 2. Maltose is liquefied starch,
The trehalose according to claim 1, which is β-amylase or maltose obtained by reacting β-amylase with starch debranching enzyme, or a sugar containing the same. 3. The trehalose according to claim 1, wherein the microorganism is a microorganism selected from the genus Pimelobacter, the genus Pseudomonas, and the genus Thermus, or the saccharide containing the trehalose. 4. The trehalose according to claim 1, 2 or 3, which is a water-containing crystal or an anhydrous crystal.
Carbohydrate containing this. 5. A nutrient medium containing maltose,
A microorganism having the ability to produce maltose / trehalose converting enzyme is cultured, and trehalose or, from the obtained culture,
A method for producing trehalose or a saccharide containing the trehalose, which comprises collecting a saccharide containing the trehalose. 6. Maltose is added to the nutrient medium at 20 w / v%.
The method for producing trehalose according to claim 5, or a saccharide containing the trehalose, which is contained below. 7. The method for producing trehalose according to claim 5 or 6, or a carbohydrate containing the same, wherein the nutrient medium contains a surfactant together with maltose. 8. The trehalose according to claim 5, 6 or 7, wherein the microorganism is a microorganism selected from the genus Pimelobacter, the genus Pseudomonas and the genus Thermus.
Alternatively, a method for producing a sugar containing the same. 9. The method for producing trehalose according to claim 5, 6, 7 or 8, or a saccharide containing the trehalose, characterized in that the culture is carried out by a batch method, a continuous method or a semi-continuous method. 10. A culture or a carbohydrate obtained therefrom,
A method for producing trehalose according to claim 5, 6, 7, 8 or 9, or a saccharide containing the same, wherein glucoamylase or α-glucosidase is allowed to act. 11. A composition comprising trehalose obtained by culturing a microorganism having maltose / trehalose converting enzyme-producing ability in a nutrient medium containing maltose, or a saccharide containing the trehalose. 12. The composition according to claim 11, wherein trehalose is a water-containing crystal or an anhydrous crystal. 13. The composition according to claim 11 or 12, wherein the composition is a food or drink, a cosmetic product, or a pharmaceutical product.