JPH0866188A - Heat-resistant non-reducing carbohydrate-producing enzyme and its production and use - Google Patents

Abstract

PURPOSE: To obtain a new enzyme derived from Sulfolobus microbes, capable of producing a non-reducing carbohydrate having trehalose structure at the terminal from a reducing starch partially decomposed product, thus capable of giving heat-resistant non-reducing carbohydrates useful for foods/beverages, cosmetics, medicines etc. CONSTITUTION: Microbes having ability to produce an enzyme capable of producing a heat-resistant non-reducing carbohydrate having trehalose structure at the terminal from a reducing starch partially decomposed product >=3 in degree of polymerization of glucose (e.g. Sulfolobus acidocaldarius ATCC 33909) are cultured in a trophic medium, and the product is taken from the cultured product, thus obtaining the objective new enzyme having the above-mentioned activity and the following characteristics: molecular weight: about 69000-79000 dalton (SDS-PAGE); isoelectric point: pI about 5.4-6.4 (ampholine-contg. electrophoresis); optimal temperature: about 75 deg.C in a reaction at pH5.5 for 60min; optimal pH: 5.0-5.5 in a reaction at 60 deg.C for 60min.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a thermostable non-reducing saccharide-forming enzyme and a method for producing the same, and more specifically, one or more reducing starch partial decompositions having a glucose polymerization degree of 3 or more. Novel thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure from the product, a method for producing the same, and a terminal produced by using the novel thermostable non-reducing saccharide-forming enzyme The present invention relates to a non-reducing saccharide having a trehalose structure, a low reducing saccharide containing the trehalose structure, trehalose produced from the trehalose, and a composition containing the non-reducing saccharide.

[0002]

2. Description of the Related Art Trehalose (α, α-trehalose) has long been known as a non-reducing sugar containing 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, it covers a wide range of microorganisms, mushrooms, insects, etc., even in a small amount. Since trehalose does not cause aminocarbonyl reaction with substances having amino groups such as amino acids and proteins due to non-reducing sugar and does not damage amino acid-containing substances, it is expected that trehalose can be used and processed without fear of browning or deterioration. Therefore, it is desired to establish an industrial manufacturing method thereof.

As a method for producing trehalose, for example, a method using a microorganism reported in JP-A-50-154485 and maltose phosphorylase and trehalose phosphorylase proposed in JP-A-58-216695 are used. A method of converting maltose by a combination of is known. However, in the method using a microorganism, cells are used as a starting material, and the content of trehalose contained in the starting material is usually 15 w / w% per solid matter (hereinafter, in the present specification, unless otherwise specified, w / w. % Is abbreviated as%.), And the process of extracting and refining this is complicated, which is unsuitable as an industrial production method. In addition, the methods using maltose phosphorylase and trehalose phosphorylase are both via glucose phosphate, and it is difficult to increase the substrate concentration, and the reaction system of both enzymes is an equilibrium reaction, The production rate is low, and it is difficult to keep the reaction system of both enzymes stable and to proceed the reaction smoothly,
It has not yet been realized as an industrial manufacturing method.

In connection with this, in "Monthly Food Chemical", August issue, pp. 67-72 (1992), "Current status and problems of starch utilization development", "Oligosaccharide", "About trehalose" Although there are significant potential applications, it is said that the enzymatic production of this sugar by direct sugar transfer from starch sugar and hydrolysis reaction is currently not possible academically. ” As stated,
It has traditionally been considered academically impossible to produce trehalose using starch as a raw material by an enzymatic reaction.

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. In the present specification, such a partially decomposed product of starch is referred to as a partially decomposed product of reducing starch. In general, the reducing starch partially decomposed product expresses the magnitude of the reducing power per solid matter as Dextrose Equivalent (DE). A substance with a large value usually has a small molecule and low viscosity and a strong sweetness, but it is highly reactive and easily undergoes an aminocarbonyl reaction with a substance having an amino group such as amino acids and proteins, causing browning and producing a bad odor. It is known that there is a property that the quality is easily deteriorated.

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 requires advanced safety facilities and management from the viewpoint of disaster prevention. In addition, the sugar alcohol obtained by partially degrading the reducing starch is different in that it is composed of glucose and sorbitol, whereas the reducing starch partially degrading material of the raw material is composed only of glucose. However, there is a concern that it may cause transient indigestion and laxative symptoms. Therefore, it has been desired to establish a method for reducing or eliminating the reducing power of glucose, which is a constituent sugar of a partially decomposed product of reducing starch.

[0008] In view of such a situation, when the present inventor made an earnest search for an enzyme that produces a sugar having a trehalose structure from starch sugar, it was found from the soil previously disclosed in Japanese Patent Application No. 5-349216. Rhizobium isolate (Rhi
bacterium M-11 belonging to the genus Zodium and Arthrobacter isolated from soil
It is said that a microorganism such as the microorganism Q36 belonging to the genus r) produces a non-reducing sugar having a trehalose structure at the end from a partially decomposed product of reducing starch having a glucose polymerization degree of 3 or more.
It has been found to produce a completely new non-reducing saccharide-forming enzyme that has been unknown so far. Further, it was also found that trehalose can be easily produced by causing glucoamylase or α-glucosidase to act on a non-reducing sugar having a trehalose structure at the end obtained by using this enzyme.

However, the above-mentioned enzymes of the genus Rhizobium or Arthrobacter have poor thermostability, and if an attempt is made to produce a non-reducing sugar or trehalose having a trehalose structure at the end using these enzymes, it will be about 55 ° C. It is necessary to carry out an enzymatic reaction at the following temperature. In relation to this
"Knowledge of Enzyme Application", first edition, pp. 80-129 (1
986), "Carbohydrate-related enzymes and their applications" in the section "Carbohydrate-related enzymes", "in industrial saccharification conditions, 55 ℃
In the following, the risk of contamination by miscellaneous bacteria is accompanied, and the pH is lowered during the saccharification reaction. As described above, in the case of an enzyme reaction using starch as a raw material for a long time, under the reaction conditions of a temperature of 55 ° C. or less, the pH of the reaction solution decreases due to contamination of various bacteria,
There is a concern that the enzyme may be inactivated during the reaction, and it may be necessary to prevent contamination of various bacteria by adding lysozyme or the like and adjust the pH of the reaction solution. In addition, when the hydrolysis rate of the partially decomposed starch is low, formation of insoluble matter due to aging is also a concern. On the other hand, since the thermostable enzyme undergoes an enzymatic reaction even at a high reaction temperature, it is considered that the reaction using the thermostable enzyme is less likely to cause microbial contamination and that the partial degradation product of starch is less likely to age. Therefore, it is desired to establish a novel method for producing trehalose or a non-reducing saccharide having a trehalose structure at the terminal, which uses a thermostable non-reducing saccharide-forming enzyme capable of an enzymatic reaction at a temperature exceeding 55 ° C.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides a novel method for producing a non-reducing saccharide from a partially decomposed product of reducing starch using a thermostable non-reducing saccharide-forming enzyme, its non-reducing saccharide, and It is intended to provide its use.

[0011]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have prepared a heat-resistant non-reducing sugar which produces a non-reducing sugar having a trehalose structure at the terminal from a partially decomposed product of reducing starch. We have broadly searched for microorganisms that produce this enzyme, with the hope of realizing a quality-generating enzyme. As a result, the microorganism belonging to the genus Sulfolobus Sulfolobus acidocaldarius (Sulfolo)
bus acidocaldarius) ATCC33
909 and ATCC 49426, as well as Sulfolobus sol.
fataricus) ATCC35091 and ATCC
It was found that 35092 produces a non-reducing sugar having a trehalose structure at the end from a partially decomposed product of reducing starch and produces a novel thermostable non-reducing saccharide-forming enzyme that is stable up to around 85 ° C. By allowing a thermostable non-reducing saccharide-forming enzyme to act on the partially decomposed product of reducing starch, a non-reducing saccharide having a trehalose structure at the end can be easily produced under the intended action conditions exceeding 55 ° C. It is also found that the thermostable non-reducing saccharide-forming enzyme is allowed to act on the partially decomposed product of reducing starch, and then glucoamylase or α-glucosidase is allowed to act thereon.
The inventors have found that trehalose can be easily produced and completed the present invention. At the same time, the present invention was completed by establishing a composition for foods, drinks, cosmetics, and the like containing this non-reducing sugar, a low-reducing sugar containing the same, and / or trehalose. In this specification, unless otherwise specified,
A novel thermostable non-reducing sugar-forming enzyme capable of producing a non-reducing sugar having a trehalose structure at the terminal from a partially decomposed product of reducing starch and performing an enzymatic reaction at a temperature exceeding 55 ° C. It is referred to as a plasmogenic enzyme.

In the present invention, a thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the terminal from a reducing starch partial degradation product is produced in addition to the above-mentioned bacterium, and belongs to the genus Sulfolobus. Other strains of the above strains, and mutants of these strains are also appropriately used.

The medium used for culturing the microorganism of the present invention may be any nutrient medium in which the microorganism can grow and which can produce the thermostable non-reducing saccharide-forming enzyme of the present invention, and either a synthetic medium or a natural medium can be used. Good. The carbon source may be any substance that can be assimilated by microorganisms, for example, glucose, fructose, lactose, sucrose, mannitol, sorbitol, molasses, sugars such as starch partial degradation products, or citric acid, succinic acid, etc. Organic acids or their salts can also be used. The concentration of these carbon sources in the medium is appropriately selected depending on the type of carbon source. For example, in the case of a partially decomposed starch, it is usually desirable that the content is 20% or less, and preferably 5% or less from the viewpoint of the growth and growth of bacteria. 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 used. 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.

Culturing is usually carried out under conditions selected from a temperature of 40 to 95 ° C., preferably 50 to 90 ° C. and a pH of 2 to 7, preferably 2 to 6. The culture time may be any time as long as the microorganism can grow, and preferably 10 to 1
It's 00 hours. The dissolved oxygen concentration of the culture solution is not particularly limited, but usually 0.5 to 20 ppm is preferable. Therefore, means such as adjusting the amount of aeration, stirring, adding oxygen to the aeration, and increasing the pressure in the fermenter are adopted. Further, the culture method may be either batch culture or continuous culture.

After culturing the microorganism in this manner, the enzyme of the present invention is recovered. This enzyme activity is mainly found in the cells of the culture, and it is desirable to purify it by a known method before use. For example, after dialysis of a crude enzyme preparation obtained by salting out and extracting ammonium sulfate from the bacterial cell extract, gel "DEA" manufactured by Tosoh Corporation
Anion exchange column chromatography using "E-Toyopearl", etc., and purification using hydrophobic column chromatography using gel "Butyltoyopearl" manufactured by the same company, etc. Can be obtained. Furthermore, by subsequently purifying using gel filtration chromatography using a gel “Ultrogel AcA 44” manufactured by Sepracor, etc., and anion exchange column chromatography using a gel “Mono Q” manufactured by Pharmacia LCB. It is also possible to obtain an electrophoretically single enzyme.

The thermostable non-reducing saccharide-forming enzyme of the present invention thus obtained has the following physicochemical properties. (1) Action A non-reducing saccharide having a trehalose structure at the terminal is produced from one or more reducing starch partial decomposition products selected from the glucose polymerization degree of 3 or more. (2) Molecular weight By SDS-gel electrophoresis, about 69,000 to 7
9,000 Daltons. (3) pI of about 5.4 to 6.4 by electrophoresis including isoelectric point amphoraine. (4) Optimum temperature: Around 75 ° C in reaction at pH 5.5 for 60 minutes. (5) Optimum reaction at 60 ° C. for 60 minutes, pH around 5.0 to 5.5. (6) Temperature stability It is stable up to about 85 ° C by keeping it at pH 7.0 for 60 minutes. (7) pH stability pH is about 4.0 to 9.5 when kept at 25 ° C. for 16 hours.

The activity of the thermostable non-reducing saccharide-forming enzyme of the present invention is measured as follows. Maltopentaose 1.25 w / v% (20 mM acetate buffer, pH
5.5) Add 1.0 ml of enzyme solution to 4 ml and 60 at 60 ℃
After reacting for 30 minutes, the reaction is stopped by heating at 100 ° C. for 30 minutes, the reaction solution is exactly diluted 10 times with deionized water, and the reducing power of the diluted solution is measured by the Somogy Nelson method. . As a control, the same measurement is performed using an enzyme solution inactivated by heating at 100 ° C. for 30 minutes in advance. A copper solution is added to stop the reaction, and the reducing power is measured by the Somogy Nelson method. Using the above measurement method,
The amount of enzyme that reduces the reducing power corresponding to 1 μmole of maltopentaose per minute was defined as 1 unit.

As a substrate of the present enzyme, a partially decomposed product of reducing starch obtained by partially hydrolyzing starch such as starch, amylopectin and amylose with amylase or acid is used. Examples of the partially decomposed products of reducing starch decomposed with amylase include, for example, "Handbook of Amylases and Related Enzymes (H
andbook of Amylases and R
[Elated Enzymes] ”(1988) Pergamon Press (Tokyo), α-amylase, maltotriose-producing amylase, maltotetraose-forming amylase, maltopentaose-forming amylase, maltohexaose-forming amylase, etc. The partially decomposed product of reducing starch decomposed with amylase is used. Furthermore, it is optional to act a debranching enzyme such as pullulanase and isoamylase in preparing the partially decomposed product of reducing starch. Further, malto-oligosaccharides such as maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like 1
It is also advantageous to use one species or two or more species.

The substrate concentration is not particularly limited. For example,
Even when used as a 0.1% substrate solution, the reaction of this enzyme proceeds, but industrially 2% or more, preferably 5% or more.
A high-concentration reaction of from 50% to 50% is preferable, and a non-reducing sugar having a trehalose structure at the end can be advantageously produced. The reaction temperature may be a temperature at which the enzyme is stable, that is, up to about 85 ° C, but a temperature of about 55 to 70 ° C is preferably used. The reaction pH may be usually adjusted in the range of 3 to 9, but is preferably adjusted in the range of about pH 4 to 7. The reaction time may be appropriately selected depending on the progress of the enzymatic reaction, and is usually about 0.1 to 100 hours when the amount of enzyme used is about 0.1 to 100 units per gram of the solid substrate.

The reaction liquid containing the non-reducing sugar obtained by the above reaction has remarkably reduced reducing power as compared with the partially decomposed product of the reducing starch used as the substrate. For example, when maltopentaose is used as the substrate, about 75% of the initial reducing power of the substrate maltopentaose solution disappears and the reducing power of the reaction solution due to this enzymatic reaction disappears to about 25%.

The reaction solution is filtered and centrifuged to remove insoluble materials by a conventional method, and then decolorized with activated carbon, H type, OH
It is desalted with type ion exchange resin and concentrated to give a syrup-like 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. By separation, further, by a method such as fermentation treatment with yeast, decomposition and removal of the remaining reducing sugar by alkali treatment, etc., or by purification in combination of two or more,
It is also easy to obtain the highest purity non-reducing sugar product.

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.
Non-reducing saccharides having an improved content of the target substance can be advantageously produced by removing contaminating saccharides by column chromatography using a strongly acidic cation exchange resin disclosed in, for example, JP-A 8-72598. At this time, any of the fixed bed system, the moving bed system, and the simulated moving bed system may be adopted.

The non-reducing saccharide having a trehalose structure at the terminal of the present invention thus obtained or a low-reducing saccharide containing the same is optionally used for amylase such as α-
Amylase, β-amylase, glucoamylase and the like, or by decomposing with α-glucosidase to adjust sweetness, reducing power, etc., reduce viscosity, and reducing sugar remaining after hydrogenation. Further processing such as changing the quality to sugar alcohol to eliminate the reducing power is optional.

Particularly, for the non-reducing sugar of the present invention or the low reducing sugar containing the same, glucoamylase or α
-Trehalose can be easily produced by allowing glucosidase to act. That is, glucoamylase or α-glucosidase is allowed to act on these non-reducing or low-reducing sugars to give a mixed solution of trehalose and glucose, which is purified by the above-mentioned purification method, for example, by ion exchange column chromatography. , Glucose is removed, and a trehalose-rich fraction is 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-containing crystals, for example, a trehalose-containing solution having a purity of 60% or more and a concentration of 65 to 90% is placed in an auxiliary crystal can and, if necessary, coexisting with 0.1 to 20% of seed crystals. At a temperature of 95 ° C or lower, preferably
The mixture is gradually cooled in the range of 10 to 90 ° C. with stirring to produce a mass kit containing trehalose hydrous crystals. Also,
It is also advantageous to employ a continuous crystallization method in which crystallization is performed while concentrating under reduced pressure. The method for producing a trehalose hydrous crystal or a honey crystal containing the trehalose from Muskit is
For example, honey method, block crushing method, fluidized granulation method,
A known method such as 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 from 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 60 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. 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 several hours to 3 days to crystallize and solidify the whole in a block form. A non-hygroscopic or hardly hygroscopic syrup-containing crystal can be easily produced by pulverizing and drying by a method such as crushing or cutting.

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, and this was prepared under relatively high-temperature drying conditions, for example, a block pulverizing method, a flow method. It is produced by crystallization by a method such as a granulation method or a spray drying method and then pulverized.

The non-reducing sugar, low-reducing sugar and trehalose containing the same of the present invention thus produced are
Compared to the reducing starch partially decomposed material of the raw material, it has low reducibility and is stable, and even when mixed and processed with other materials, particularly substances containing amino acids or amino groups such as amino acids, oligopeptides and proteins, It does not brown, does not give off an offensive odor, and does not damage other mixed materials.
Further, unlike the case of the partially decomposed product of reducing starch, it has a low reducing power but a low viscosity, and a low average glucose polymerization degree has a good and elegant sweetness. .

Further, amylase, for example, α derived from pancreas
-Decomposed by amylase to produce low-molecular non-reducing oligosaccharides and low-molecular maltooligosaccharides, and these oligosaccharides are also easily decomposed by α-glucosidase and small intestinal enzyme,
Glucose and trehalose are produced, and the produced trehalose is easily decomposed into glucose by trehalase. Therefore, it is digested and absorbed by ingestion and used as a calorie source. Further, it can be used as a sweetener that is hard to be fermented by caries-inducing bacteria and is unlikely to cause caries. Also, osmotic pressure controllability, shapeability,
It has properties such as luster-imparting property, moisturizing property, viscosity, anti-crystallization property of other sugars, difficulty in fermentation, and anti-aging property of 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 crystalline product, 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 also be advantageously used as a dehydrating agent for foods, pharmaceuticals, cosmetics, raw materials thereof, or hydrous substances such as processing intermediates, and can easily produce stable, high-quality solid materials such as powders, granules, and tablets. .

Therefore, the non-reducing sugar of the present invention, or the low reducing sugar containing the same and trehalose produced from the same are used as sweeteners, taste improvers, quality improvers, stabilizers and excipients. As a dehydrating agent, etc., it can be advantageously used for various compositions such as foods and drinks, favorite foods, feeds, feeds, cosmetics, and pharmaceuticals.

The non-reducing sugar of the present invention, the low reducing sugar containing the same, and the trehalose produced from them can be used as they are as a seasoning for sweetening. If necessary, for example, starch syrup, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, isomalt oligosaccharide, galactooligosaccharide, fructooligosaccharide,
One of the other sweeteners such as lactose, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, etc. Alternatively, they may be used by mixing with an appropriate amount of two or more kinds, and if necessary, they may be used by mixing with a bulking agent such as dextrin, starch, lactose and the like.

Further, the non-reducing saccharide of the present invention, the low reducing saccharide containing the same, and the trehalose powder or crystalline product produced from the non-reducing saccharide may be used as they are or, if necessary, as a bulking agent or excipient. It may optionally be mixed with a shaping agent, a binder and the like and molded into various shapes such as granules, spheres, short rods, plates, cubes and tablets for use.

The sweetness of the non-reducing sugar of the present invention, the low-reducing sugar containing the same, and the trehalose produced from the same are not limited to acidity, saltiness, taste, astringency, umami, bitterness and other characteristics. Since it is well harmonized with various substances having a taste and has a large acid resistance and heat resistance, it can be advantageously used for sweetening, improving the taste and improving the quality of general food and drink.

For example, amino acids, peptides, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hishio, sprinkle,
Mayonnaise, dressing, vinegar, three cups vinegar, powdered sushi vinegar, Chinese sauce, tempura soup, noodle soup, sauce, ketchup,
Sauce of roasted meat, curry roux, stew base, soup base,
It can be advantageously used as various cooking ingredients 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 jam, yokan, water yoyo, nishikidama, jelly, castella, hard candy, etc.,
Bread, biscuits, crackers, cookies, pies, puddings, butter cream, custard cream, cream puffs, waffles, sponge cakes, donuts, chocolate, chewing gum, caramel, candy and other Western confectionery, ice cream, sherbet, and other frozen desserts,
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 Betara-zuke, Senmai-zuke, and Rakkyo-zuke, pickle-based pickles, pickled pickles such as Chinese cabbage pickles, livestock products such as ham and sausages, fish ham, fish sausages,
Fish products such as kamaboko, chikuwa, and tempura, sea urchin, salted squid, vinegared konbu, simmered sardines, and other delicacies such as dried sushi, fugu mirin, seaweed, sardines, small fish, and boiled beans made from shellfish. , Side dishes such as potato salad, kelp rolls, dairy products such as yogurt and cheese, fish meat,
Alcoholic beverages such as meat, fruits, vegetables bottled, canned goods, sake, synthetic liquor, liquor, coffee, tea, cocoa,
Soft drinks such as juices, carbonated drinks, lactic acid drinks, lactic acid bacteria drinks, pudding mixes, hot cake mixes, instant foods such as instant shiruko, instant soup, and even baby food,
It can be advantageously used for sweetening various foods and drinks such as therapeutic foods, drinks, peptide foods and frozen foods, for improving taste, and for improving quality.

Further, it can also be used for the purpose of improving the palatability of feed, feed and the like 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. The quality improver and stabilizer can be advantageously applied to various physiologically active substances that easily lose their active ingredients, activities, etc. or health foods, pharmaceuticals, etc. containing them. 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, lymphokine such as interleukin II, insulin, growth hormone, prolactin, erythropoietin, hormones such as egg cell stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, Smallpox, tetanus toxoid, hub antitoxin, biologics such as human immunoglobulin, penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin, kanamycin sulfate and other antibiotics, thiamine, riboflavin, L-ascorbic acid, liver oil, carotenoid, ergo Vitamin such as sterol and tocopherol, lipase, elastase, urokinase, protease, β-amylase, isoamylase, glucose Kinase, an enzyme such as lactase,
Medicinal ginseng extract, soft-shelled turtle extract, chlorella extract, aloe extract, propolis extract and other extracts, virus, lactic acid bacteria, live bacteria such as yeast, various physiologically active substances such as royal jelly, the active ingredient, without losing the activity, The stable, high-quality liquid, paste, or solid health foods and pharmaceuticals can be easily manufactured.

The product is completed by the method of incorporating the non-reducing sugar having a trehalose structure at the terminal, the low reducing sugar containing the same, and the trehalose produced from the various compositions as described above. Any known method such as mixing, dissolving, melting, dipping, permeating, spraying, coating, coating, spraying, pouring, crystallization, and solidifying may be appropriately selected. The amount is usually 0.1% or more, and preferably 1% or more.

Next, the present invention will be described more specifically by experiments.

[0041]

[Experiment 1 Preparation of thermostable non-reducing sugar-forming enzyme derived from Sulfolobus acidocaldarius ATCC33909] Peptone 0.1 w / v%, yeast extract 0.1 w / v%, ammonium sulfate 0.2 w / v%, monophosphate Liquid medium consisting of 0.05 w / v% potassium, 0.02 w / v magnesium sulphate heptahydrate, 0.02 w / v% potassium chloride and about 100 ml of liquid medium was placed in a 500 ml Erlenmeyer flask and autoclaved at 120 ° C. for 20 minutes.
After sterilizing for 1 minute and cooling, the pH was adjusted to 3.0 with sulfuric acid. This liquid medium was inoculated with Sulfolobus acidocaldarius ATCC33909, and the temperature was 75 ° C and 130 rpm.
What was cultured for 24 hours was used as the primary seed culture solution.

A fermenter having a volume of 10 liters was charged with about 5 liters of a medium having the same composition as in the case of the primary seed culture, sterilized and cooled to pH 3.0 and a temperature of 75 ° C.
/ V% was inoculated and subjected to aeration culture at a temperature of 75 ° C. and an aeration rate of 500 ml / min for about 48 hours to obtain a second seed culture solution.

About 250 l of a medium having the same composition as in the case of the primary seed culture was put into a fermenter having a volume of 300 l, sterilized and cooled to pH 3.0 and a temperature of 75 ° C., and then the secondary seed culture solution 1 v / v% inoculated, temperature 75 ℃, aeration 100l /
The culture was aerated for about 42 hours. About 170 of the obtained culture solution
By subjecting 1 to an SF membrane and centrifuging, 258 g of the microbial cell was collected as a wet weight. 300 ml of 10 mM phosphate buffer (pH 7.0) was added to and suspended in the cells, and then ultrasonic disrupter model “US30” manufactured by Nippon Seiki Seisakusho Co., Ltd.
The cells were crushed with "0". Centrifuge the disrupted solution (10,000
At 0 rpm for 30 minutes), about 300 ml of the centrifugal supernatant was obtained. Ammonium sulfate was added to the solution to a saturation degree of 0.7 to dissolve the solution, and the solution was left standing at 4 ° C. for 24 hours and then centrifuged to collect a salted out product. The salted-out product thus obtained was dissolved in 10 mM Tris / hydrochloric acid buffer (pH 8.5), dialyzed against the same buffer for 24 hours, and centrifuged to remove insoluble materials. The dialysate (about 600 ml) was divided into two portions and subjected to ion exchange column chromatography using DEAE-Toyopearl (gel amount about 350 ml). The adsorbed present enzyme was eluted from the column with a linear gradient of 0M to 0.3M sodium chloride concentration,
The enzyme active fraction eluted near a concentration of 0.1 M sodium chloride was collected. The obtained enzyme-active fraction was mixed with 10 mM Tris / hydrochloric acid buffer (pH 8.
5) It was dialyzed against, the dialysate was centrifuged to remove insoluble matter, and the resulting supernatant was subjected to hydrophobic column chromatography (gel amount 350 ml) using gel "Butyl Toyopearl 650" manufactured by Tosoh Corporation. I served. The adsorbed present enzyme was eluted from the column with a linear gradient of 1M to 0M ammonium sulfate concentration, and about 440 units of the enzyme active fraction eluted near 0.8M ammonium sulfate concentration was recovered. The partially purified preparation obtained showed a specific activity of about 20 units / mg protein.

The partially purified sample was dialyzed against 10 mM Tris / hydrochloric acid buffer (pH 8.5) containing 0.2 M sodium chloride, the dialysate was centrifuged to remove insoluble matter, and the resulting supernatant was collected. Subjected to gel filtration chromatography using Ultrogel AcA 44 (gel amount 350 ml),
After recovering the enzyme active fraction, it was dialyzed against 10 mM Tris / hydrochloric acid buffer (pH 8.5), the dialyzed solution was centrifuged to remove insoluble matter, and the resulting supernatant was subjected to ionization using Mono Q. Exchange column chromatography (gel amount 10
The adsorbed enzyme was eluted from the column with a linear gradient of 0 M to 0.2 M sodium chloride concentration, and the thermostable non-reducing saccharide-forming enzyme active fraction eluted near 0.1 M sodium chloride concentration was collected. About 40 units were collected.

The purified thermostable non-reducing saccharide-forming enzyme preparation thus obtained showed a specific activity of about 81 units / mg protein and SD
When the purity was assayed by an electrophoresis method using S-polyacrylamide gel (gel concentration 10%), the protein band was shown to be single, and the sample was electrophoretically single and had a high purity. .

[0046]

[Experiment 2 Physicochemical properties of thermostable non-reducing saccharide-forming enzyme]

[Experiment 2-1 Action] As a substrate, 10% aqueous solutions of glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were prepared and obtained by the method of Experiment 1, respectively. The purified thermostable non-reducing saccharide-forming enzyme was added at a ratio of 2 units per gram of substrate solid content and the pH was adjusted to 60 ° C.
After acting at 5.5 for 48 hours, it was desalted and the reaction product was analyzed by high performance liquid chromatography using a column "Wako beads WB-T-330" manufactured by Wako Pure Chemical Industries, Ltd. High performance liquid chromatography is performed at room temperature,
Water was flown as an eluent at a flow rate of 0.5 ml / min, and analyzed with a differential refractometer "RI-8012" manufactured by Tosoh Corporation. The results are shown in Table 1.

[0047]

[Table 1]

As is clear from the results shown in Table 1, the purified enzyme was obtained from maltotriose or maltoheptaose, which is a partial degradation product of starch having a glucose polymerization degree of 3 or more, and a non-reducing sugar having a trehalose structure at the end. It was found to produce α-glucosyltrehalose to α-maltopentaosyltrehalose. In addition to the non-reducing sugars produced in the reactants without changing the degree of polymerization of glucose and glucose, glucose, which is a hydrolyzate of a relatively small amount of substrate, and low molecular weight maltooligosaccharides, and produced from it It was found that non-reducing sugars are present, and in addition to the non-reducing sugar forming effect, they have a weak hydrolysis effect. The production rates of non-reducing sugars and reducing sugars produced by hydrolysates from the respective substrates by this purified enzyme were 30.2% and 27.6% from maltotriose. To 65.4% and 18.4%, from maltopentaose to maltoheptaose to about 74 to 75% and about 2 to 3%,
It was found that malto-oligosaccharides having a glucose polymerization degree of 5 or more produced a non-reducing sugar at a high production rate and produced only a little hydrolyzate. It was found that glucose and maltose do not produce new sugars.

[0049]

[Experiment 2-2 Molecular Weight] U.K. Laemli's "Nature", Volume 227, 680-6
SD according to the method reported on page 85 (1970)
When subjected to S-polyacrylamide gel electrophoresis, the enzyme showed a single band at a position corresponding to a molecular weight of about 69,000 to 79,000 daltons. The molecular weight markers used at this time were myosin (200,000 daltons), β-galactosidase (116,250 daltons), phosphorylase B (97,400 daltons), serum albumin (66,200 daltons) and ovalbumin. (45,000 Dalton) was used.

[0050]

[Experiment 2-3 Isoelectric point] The purified thermostable non-reducing saccharide-forming enzyme was subjected to an isoelectric focusing method using a polyacrylamide gel (containing 2% ampholine, manufactured by Pharmacia LCB), and after electrophoresis, When the pH of the gel was measured and the isoelectric point of this enzyme was determined, the isoelectric point was about 5.4 to 6.
It was 4.

[0051]

[Experiment 2-4 Optimum temperature] According to a conventional method, a test was carried out under the conditions of incubating in a 20 mM acetate buffer (pH 5.5) for 60 minutes, and as shown in FIG.
The optimum temperature was shown at around 5 ° C.

[0052]

[Experiment 2-5 Optimum pH] When tested in a McKilvein's buffer solution having different pHs under the condition of incubating at 60 ° C. for 60 minutes by a conventional method, as shown in FIG. The optimum pH was shown in the vicinity of 0.0 to 5.5.

[0053]

[Experiment 2-6 Temperature Stability] When tested in a usual manner under the condition of incubating in 10 mM phosphate buffer (pH 7.0) for 60 minutes, as shown in FIG.
It was stable up to around 85 ° C.

[0054]

[Experiment 2-7] pH Stability According to a conventional method, a test was conducted in a McKilvein buffer solution or a sodium carbonate-sodium hydrogencarbonate buffer solution having different pHs under the condition of incubating at 25 ° C for 16 hours. As shown in
The enzyme was stable up to pH 4.5 to around 9.5.

[0055]

[Experiment 2-8 N-terminal amino acid sequence] A portion of the purified thermostable non-reducing saccharide-forming enzyme preparation obtained by the method of Experiment 1 was dialyzed against distilled water.
μg was used as a sample for N-terminal amino acid sequence analysis. The N-terminal amino acid sequence was analyzed from the N-terminal to 10 residues by using a gas phase protein sequencer "473A type" sold by Applied Biosystems Japan. The N-terminal sequence obtained is shown below.

[0056]

[0057]

[Experiment 3 Preparation of thermostable non-reducing sugar-forming enzyme derived from other Sulfolobus microorganisms] Sulfolobus acidocaldarius ATCC49426, Sulfolobus solfataricus ATCC35091 and sulfolobus solfatarica were used instead of sulfolobus acidocaldarius ATCC33909. Su ATCC350
The culture was performed for 42 hours in a fermenter in the same manner as in Experiment 1 except that 92 was used. The cells were collected from about 170 l of each culture solution, sonicated, and the supernatant was subjected to ammonium sulfate salting out, dialyzed, and further subjected to ion exchange column chromatography and hydrophobic column chromatography to obtain a partially purified enzyme preparation, I investigated its nature. The results are summarized in Table 2.

[0058]

[Table 2]

Further, the production of non-reducing sugars was examined by using these partially purified enzymes according to the method of Experiment 2-1. As a result, all of the enzymes were heat-resistant non-reducing from Sulfolobus acidocaldarius ATCC33909. In the same manner as in the case of the sugar-forming enzyme, a non-reducing sugar having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more can be produced from a reducing starch partial degradation product selected from a glucose polymerization degree of 3 or more. found.

The method for producing a non-reducing sugar, a low-reducing sugar containing the same and trehalose of the present invention will be described below in Example A.
Example B shows a composition containing a non-reducing sugar, a low-reducing sugar containing the same, and / or trehalose.

[0061]

Example A-1 Sulfolobus acidocaldarius ATCC33909 was cultured in a fermenter for about 42 hours according to the method of Experiment 1. After culturing, the bacterial cells were concentrated using an SF membrane and further centrifuged to recover the bacterial cells. According to the method of Experiment 3, the cells were sonicated, the supernatant was salted out with ammonium sulfate, dialyzed, further subjected to ion exchange column chromatography and hydrophobic column chromatography, and the specific activity was about 20 units / mg. Purified enzyme solution (18.0 units / m
l) was obtained. After the potato starch milk having a concentration of 6% is heated to gelatinize, the pH is adjusted to 4.5 and the temperature is adjusted to 50 ° C., and isoamylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) is added at a ratio of 2500 units per gram of starch. And added, and reacted for 20 hours. The reaction solution was adjusted to pH 6.5, autoclaved (120 ° C.) for 10 minutes, and then cooled to 60 ° C., and α-amylase “Termamir 60 manufactured by Novo Co.”
L ”at a rate of 30 units per gram of starch,
The reaction was carried out for 20 hours. The reaction solution was autoclaved (1
(20 ° C.) for 20 minutes, then cooled to 65 ° C. and adjusted to pH 5.5, so that the thermostable non-reducing saccharide-forming enzyme prepared above is in a ratio of 1 unit per gram of starch. In addition, the reaction was performed for 96 hours. The reaction solution is kept at 97 ° C. for 30 minutes, then cooled and filtered to obtain a filtrate, which is decolorized with activated carbon according to a conventional method, desalted with H-type and OH-type ion exchange resins, and purified. After further concentration, syrup having a concentration of about 70% was obtained at about 90% per solid content. This product is DE
24.6, non-reducing sugars per solid content, α-glucosyltrehalose 12.0%, α-maltosyltrehalose 5.5%, α-maltotriosyltrehalose 29.9%, α-malto Containing 1.5% of tetraosyltrehalose and 2.2% of α-maltopentaosyltrehalose, it has a mild and elegant sweetness, moderate viscosity and moisturizing property, and is a sweetener and a taste improver. , Various foods and drinks, cosmetics, as quality improvers, stabilizers, excipients, etc.
It can be advantageously used for various compositions such as pharmaceuticals.

[0062]

Example A-2 The sugar solution obtained by the method of Example A-1 was used as a raw sugar solution, and sodium organic strong acid cation exchange manufactured by Tokyo Organic Chemical Industry Co., Ltd. was used to increase the content of non-reducing sugars. Column fractionation was performed using the resin "XT-1016" (crosslinking degree 4%). The resin was filled in four jacketed stainless steel columns having an inner diameter of 5.4 cm and connected in series to give a total resin layer length of 20 m. While maintaining the temperature in the column at 55 ℃,
A sugar solution was added to the resin in an amount of 5 v / v%, and warm water at 55 ° C was flown at SV 0.13 to fractionate the fraction to remove glucose and maltose-rich fractions, and non-reducing sugar-rich fractions. Was collected. Further purification, concentration, vacuum drying and crushing gave a powder with high content of non-reducing sugars at about 64% based on solids. This product has a DE of 4.8 and contains non-reducing sugars in a solid content of α-glucosyltrehalose of 18.2%,
It contains 7.9% of α-maltosyltrehalose, 46.6% of α-maltotriosyltrehalose, 2.3% of α-maltotetraosyltrehalose, and 3.4% of α-maltopentaosyltrehalose. , Similar to Example A-1, having a mild and elegant sweetness, moderate viscosity and moisturizing property, a sweetener, a taste improver, a quality improver, a stabilizer,
As an excipient or the like, it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

[0063]

Example A-3: 33% corn starch milk was added with calcium carbonate to a final concentration of 0.1%, and then the pH was adjusted.
The mixture was adjusted to 6.5, and 60 L of Termamile was added to this so that the concentration was 0.2% per gram of starch, and the mixture was reacted at 95 ° C. for 15 minutes. The reaction solution was autoclaved (120 ° C) for 10 minutes and then cooled to 55 ° C.
-240783 disclosed by Hayashibara Biochemical Research Laboratories, Inc., maltoteoraose-producing amylase at a ratio of 5 units per gram of starch, and allowed to react for 6 hours. Amylase "α-amylase 2A" was added in an amount of 30 units per gram of starch and further reacted at 65 ° C for 4 hours. The reaction solution is autoclaved (120 ° C) for 10 minutes and then 65 ° C.
After cooling to pH and adjusting the pH to 5.5, it was added to Example A
The thermostable non-reducing saccharide-forming enzyme prepared by the method No. 1 was added at a ratio of 2 units per gram of starch and reacted for 48 hours. The reaction solution is kept at 97 ° C. for 30 minutes, then cooled and filtered to obtain a filtrate, which is decolorized with activated carbon according to a conventional method, desalted with H-type and OH-type ion exchange resins, and purified. After further concentration, syrup having a concentration of about 70% was obtained with a yield of about 90% based on the solid matter. This product has DE 17.1 and contains non-reducing sugars in a solid content of α-glucosyltrehalose 8.9%, α-maltosyltrehalose 2
9.3%, α-maltotriosyltrehalose 0.8
%, Α-maltotetraosyltrehalose 0.7%,
It also contains 0.7% of α-maltopentaosyltrehalose and has a mild and elegant sweetness, moderate viscosity, and moisturizing property, and has a sweetener, a taste improver, a quality improver, a stabilizer, and an additive. As a shaping agent, etc., it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

[0064]

Example A-4 The sugar solution obtained by the method A-3 of Example was used as a raw sugar solution, and in order to increase the content of α-maltosyltrehalose in this solution, it was used as a fractionation resin by Dow Chemical Company. Column chromatography was carried out according to the method of Example A-2 except that the sold magnesium-type strongly acidic cation exchange resin "Dowex 50W x 4" was used, and the α-maltosyltrehalose-rich fraction was collected. Further, it was purified, concentrated, and spray-dried to obtain a powder having a high content of non-reducing sugar in a yield of about 41% based on the solid content. This product is a non-reducing sugar per solid matter, α-glucosyl trehalose 10.9%,
It contains 61.3% of α-maltosyltrehalose and 1.0% of α-maltotriosyltrehalose, and has a DE of 2.5, which is extremely low in reducing property and is similar to that of Example A-3. It has a mild and elegant sweetness, moderate viscosity and moisturizing properties, and is a sweetener, taste improver, quality improver, stabilizer,
As an excipient or the like, it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

[0065]

[Example A-5] 40 parts by weight of partially decomposed starch "Paindex # 4" manufactured by Matsutani Chemical Industry Co., Ltd. was dissolved in 60 parts by weight of water by heating, and the solution was adjusted to 65 ° C and pH 5.5. The thermostable non-reducing saccharide-forming enzyme prepared by the method of Example A-1 was added at a ratio of 1 unit per gram of partially decomposed starch, and the mixture was reacted for 96 hours and then at 97 ° C. for 3 hours.
The enzyme was inactivated by heating for 0 minutes. This reaction solution was diluted to a concentration of about 20%, 10 units of glucoamylase "glucozyme" manufactured by Nagase Seikagaku Co., Ltd. was added per gram of partially decomposed product of starch and reacted for 10 hours, and then heated to inactivate the enzyme. It was This solution was decolorized with activated carbon, desalted with an ion exchange resin, and concentrated to a concentration of about 60% according to a conventional method. This sugar solution contained 30.1% trehalose per solid matter. As a fractionation resin, sodium type strong acid cation exchange resin "CG6" sold by Organo Co., Ltd.
Column chromatography was carried out according to the method of Example A-2 except that "000" was used to collect a trehalose-rich fraction. This high content liquid contained about 97% trehalose per solid matter. After this solution was concentrated to a concentration of about 75%, it was taken in an auxiliary crystal can, and about 2% of trehalose hydrous crystals was added as a seed crystal and gradually cooled to obtain a mass kit with a crystallization rate of about 45%. Use this mass kit from the nozzle on the drying tower for 15
It was sprayed at a high pressure of 0 kg / cm ² . At the same time 85
While sending hot air of ℃, warm air of 45 ℃ from the top of the drying tower,
The powder was gradually moved to the outside of the drying tower and taken out. The crystal powder was filled in an aging tower and aged for 10 hours while sending warm air to complete crystallization and drying to obtain trehalose hydrous crystal powder. This product has virtually no hygroscopicity, is easy to handle, and has a sweetener, taste improver, quality improver,
As a stabilizer, an excipient, etc., it can be advantageously used in various compositions such as various foods, drinks, cosmetics and pharmaceuticals.

[0066]

[Example A-6] Sulfolobus solfataricus
According to the method of Experiment 3, ATCC35091 was cultured in a fermenter for about 42 hours. After culturing, the cells were concentrated using an SF membrane, further centrifuged to collect the cells, sonicated, the supernatant was salted out with ammonium sulfate, dialyzed, further subjected to ion exchange column chromatography and a hydrophobic column. Chromatography was performed to obtain a partially purified enzyme solution (19.0 units / ml) having a specific activity of about 18 units / mg. Using 30% corn starch milk, according to the method of Example A-3, 60 L of tarmamir, then maltotetraose-forming amylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) and α-amylase 2A were allowed to act, and the autoclave ( 120 ° C.), then cooled to 65 ° C., to which a thermostable non-reducing saccharide-forming enzyme prepared by the above method was added per 2 g of starch.
It was added as a unit and reacted for 64 hours. Then
The enzyme was inactivated by heating at 97 ° C for 30 minutes. According to the method of Example A-5, this reaction liquid was allowed to act with glucozyme, decolorized and desalted, and concentrated to a concentration of about 60%. This sugar solution contained about 23% trehalose per solid matter. This sugar solution was subjected to column chromatography using a salt-type strongly acidic cation exchange resin according to the method of Example A-5 to collect a trehalose-rich fraction. This high content liquid contained about 95% trehalose per solid matter. This solution was placed in an evaporator and boiled under reduced pressure to obtain syrup having a water content of 4.0%, which was then transferred to an auxiliary crystallizer and anhydrous crystal trehalose as a seed crystal was added to the syrup solid content at 1% per solid content.
In addition, the mixture was agitated at 95 ° C. for 5 minutes, then taken out in an aluminum vat and aged at 100 ° C. for 6 hours to prepare a block. Next, this block was crushed with a cutting machine and fluidized and dried to obtain anhydrous crystalline trehalose powder having a water content of 0.3%. The product can be advantageously used not only as a dehydrating agent for water-containing substances such as foods, pharmaceuticals, cosmetics, raw materials thereof, or processed intermediates, but also as a white powder sweetener having an elegant sweetness.

[0067]

[Example B-1 Sweetener] To 1 part by weight of the powder containing a large amount of non-reducing sugar obtained by the method of Example A-4, α-glycosyl stevioside "αG suite" 0.0 sold by Toyo Seika Co., Ltd. was added.
1 part by weight and L-Aspartyl-L- manufactured by Ajinomoto Co., Inc.
Phenylalanine methyl ester "Aspartame"
Evenly mix 0.01 parts by weight and apply to a granule molding machine,
A granular sweetener was obtained. This product has an excellent sweetness quality and has a sweetness that is about twice that of sucrose, and the calorie per sweetness is reduced to about 1/2 of that of sucrose. This sweetener is excellent in stability without decomposition of the high-intensity sweetness compounded in it,
As a low-calorie sweetener, it is suitable for sweetening low-calorie foods and drinks for obese people, diabetics, etc. who have restricted caloric intake. 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.

[0068]

[Example B-2 Hard candy] 100 parts by weight of a 55% sucrose solution were mixed with 30 parts by weight of the non-reducing sugar-containing syrup obtained by the method of Example A-3, and then the water content was reduced to 2% under reduced pressure. The mixture is heated and concentrated until the amount becomes less than 1, and 1 part by weight of citric acid and an appropriate amount of a lemon flavor and a colorant are mixed therein,
A product was obtained by molding according to a conventional method. This product is a high quality hard candy that has crispness, good taste and does not cause sucrose to crystallize.

[0069]

[Example B-3 Chewing Gum] 3 parts by weight of a gum base were heated and melted to such an extent that it became soft, 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-5 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.

[0070]

[Example B-4 Sweetened condensed milk] In 100 parts by weight of raw milk, 3 parts by weight of the non-reducing sugar-containing syrup obtained by the method of Example A-1 and 1 part by weight of sucrose were dissolved and sterilized by heating with a plate heater. Then, the product was concentrated to a concentration of 70% and then aseptically canned to obtain a 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.

[0071]

[Example B-5 Lactic acid bacterium beverage] 175 parts by weight of skim milk powder,
Non-reducing sugar-rich powder 80 obtained by the method of Example A-2
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 bacterium beverage with a good flavor. In addition, this product contains oligosaccharides and not only stably holds lactic acid bacteria, but also has a bifidobacteria growth promoting action.

[0072]

[Example B-6 Powdered juice] 33 parts by weight of orange juice powder produced by spray drying was used in Example A-2.
50 parts by weight of non-reducing sugar-rich powder obtained by the method of above, 10 parts by weight of sucrose, 0.65 parts by weight of anhydrous citric acid, 0.1 part by weight of malic acid, 0.1 part by weight of L-ascorbic acid, citric acid 0.1 part by weight of acid soda, 0.5 part by weight of pullulan, and an appropriate amount of powdered flavor are well mixed and stirred, pulverized into a fine powder and charged into a fluidized bed granulator, and the exhaust air temperature is 40 ° C. and the air volume is 150 m.
³ , the non-reducing sugar-containing syrup obtained by the method of Example A-1 was sprayed as a binder, granulated for 30 minutes, weighed and packaged to obtain a product. This product is a powdered juice with a fruit juice content of about 30%. In addition, this product had no offensive taste or odor and was stable for a long period of time.

[0073]

Example B-7 Custard Cream 100 parts by weight of corn starch, 100 parts by weight of the non-reducing sugar-containing syrup obtained by the method of Example A-3, 80 parts by weight of maltose, 20 parts by weight of sucrose and 1 part by weight of salt were added. Mix thoroughly and roast 2 eggs
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.

[0074]

[Example B-8 Anan] To 10 parts by weight of raw azuki bean, according to a conventional method, water was added and boiled, then astringent, and dried.
Water-soluble contaminants were removed to obtain about 21 parts by weight of red bean paste. This raw bean paste, 14 parts by weight of sucrose, Example A-4
5 parts by weight of syrup containing a high content of non-reducing sugar and 4 parts by weight of water were added to the mixture and boiled, and a small amount of salad oil was added to the mixture to knead it so that the bean paste could not be broken. Parts by weight were obtained. This product has no color burn, has a good texture, and has a good flavor, and is suitable as a material for bean paste such as bean paste, manju, dango, monaka, and frozen dessert.

[0075]

Example B-9 Bread 100 parts by weight wheat flour, 2 parts by weight yeast, 5 parts by weight sugar, 1 part by weight non-reducing sugar-rich powder obtained by the method of Example A-2 and 0.1% inorganic hood. According to a conventional method, parts by weight were kneaded with water, the medium seeds were fermented at 26 ° C. for 2 hours, then aged for 30 minutes and baked. This product is a high-quality bread with good hue, good texture, moderate elasticity, and mild sweetness.

[0076]

[Example B-10 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 24 hours. 500 parts by weight of water, 100 parts by weight of salt, 3 parts by weight of potassium nitrate, 40 parts by weight of the powder containing a high content of non-reducing sugar obtained by the method of Example A-4 and a salting solution consisting of spices were kept in a cold room for 7 days. Immerse, then wash with cold water according to the usual method, tighten with a string,
The product was smoked, cooked and cold packed. This product is a high quality ham with good color and flavor.

[0077]

Example B-11 Powder Peptide 1 part by weight of Fuji Oil Co., Ltd. 40% soybean peptide solution for food "High New S" was added with 2 parts by weight of trehalose hydrous crystal powder obtained by the method of Example A-5. The mixture was 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.

[0078]

Example B-12 Powdered Egg Yolk Egg yolk prepared from raw eggs is sterilized by a plate 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-6. 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.

[0079]

[Example B-13 Cosmetic cream] Polyoxyethylene glycol monostearate 2 parts by weight, self-emulsifying type glyceryl monostearate 5 parts by weight, non-reducing sugar-rich powder obtained by the method of Example A-2 2 parts by weight, 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 preservative were dissolved by heating according to a conventional method, and 2 parts by weight of L-lactic acid, 1,
5 parts by weight of 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 by stirring to produce a cream. The product has antioxidant properties and high stability, and can be advantageously used as a high-quality sunscreen, skin-refining agent, skin-whitening agent and the like.

[0080]

[Example B-14 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. Trehalose obtained by the method of Example A-5 by adsorbing human natural interferon-α and removing bovine serum albumin as a stabilizer by passing it through, and then changing the pH. Elution was performed using physiological saline containing 5% of water-containing crystal powder. This solution is subjected to microfiltration and added to about 20 times the amount of anhydrous crystalline maltose powder "Finetose" sold by Hayashibara Shoji Co., Ltd., dehydrated and powdered, and then tableted with a tablet 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, maltose acts as a stabilizer together with trehalose, and maintains its activity well for a long time even when left at room temperature.

[0081]

Example B-15 Sugar-coated tablet A plain tablet having a weight of 150 mg was used as a core, to which 40 parts by weight of trehalose hydrous crystal powder obtained by the method of Example A-5 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. This product has excellent impact resistance and maintains high quality for a long time.

[0082]

[Example B-16 Liquid edible solid preparation] 500 parts by weight of trehalose hydrous crystalline powder produced by the method of Example A-5, 270 parts by weight of egg yolk, 209 parts by weight of skim milk powder, 4.4 parts by weight of sodium chloride, chloride 1.8 parts by weight of potassium, 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.

[0083]

Example B-17 Trauma Treatment Ointment To 200 parts by weight of trehalose hydrous crystal powder and 300 parts by weight of maltose produced by the method of Example A-5, 50 parts by weight of methanol in which 3 parts by weight of iodine were dissolved were 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.

[0084]

As is clear from the above, the novel thermostable non-reducing saccharide-forming enzyme of the present invention can easily carry out an enzymatic reaction at a temperature above 55 ° C., so that there is no fear of microbial contamination and starch The partially decomposed product is converted into a non-reducing sugar having the same degree of glucose polymerization in a high yield. The non-reducing sugar can be easily separated and purified, and the non-reducing sugar thus obtained, the low-reducing sugar containing the same and the trehalose produced from the non-reducing sugar are excellent in stability and of good quality. Has a sweetness. Non-reducing sugars, low-reducing sugars and trehalose containing them are used as sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. in various compositions such as food and drink, cosmetics and pharmaceuticals. It can be used to advantage.

Therefore, the establishment of the present invention was achieved by a non-reducing sugar having a trehalose structure at the terminal, which was not easily obtained by a conventional method from a partially decomposed product of starch derived from starch, which is an inexpensive and unlimited resource. Quality, low-reducing sugars containing it, and trehalose easily produced from it will open up a whole new way to industrially supply in large quantities and at low cost. , Of course not only in the pharmaceutical field, but also in the agriculture, water, livestock and chemical industries,
The industrial significance given to these industries is immeasurable.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of temperature on the enzyme activity of a thermostable non-reducing saccharide-forming enzyme of the present invention.

FIG. 2 is a graph showing the effect of pH on the enzyme activity of the thermostable non-reducing saccharide-forming enzyme of the present invention.

FIG. 3 is a graph showing the effect of temperature on the stability of the thermostable non-reducing saccharide-forming enzyme of the present invention.

FIG. 4 is a graph showing the effect of pH on the stability of the thermostable non-reducing saccharide-forming enzyme of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A23G 3/00 101 106 3/30 A23L 1/19 1/236 A 1/305 1/31 A 1 / 32 AD 2/39 A61K 7/00 J F 31/70 C07H 1/00 1/08 3/04 3/06 C08B 37/00 Z 7433-4C C12P 19/14 Z 7432-4B 19/16 7432- 4B 19/20 7432-4B // (C12N 9/24 C12R 1:01)

Claims (19)

[Claims] 1. A thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the end from a partially decomposed product of reducing starch. 2. The reducing starch partially decomposed product is one or more reducing starch partial decomposition products selected from glucose polymerization degree of 3 or more, and the non-reducing sugar has a trehalose structure at the terminal. The thermostable non-reducing saccharide-forming enzyme according to claim 1, which is a non-reducing saccharide. 3. The thermostable non-reducing saccharide-forming enzyme according to claim 1 or 2, which has a thermostability which is stable up to around 85 ° C. under the conditions of pH 7.0 and holding for 60 minutes. 4. The thermostable non-reducing saccharide-forming enzyme according to claim 1, 2 or 3, wherein the thermostable non-reducing saccharide-forming enzyme is a microorganism-derived enzyme. 5. The thermostable non-reducing saccharide-forming enzyme according to claim 4, wherein the microorganism is a microorganism belonging to the genus Sulfolobus. 6. A thermostable non-reducing saccharide-forming enzyme having the following physicochemical properties. (1) Action One or more reducing starch partial decomposition products selected from glucose polymerization degree of 3 or more, wherein the non-reducing sugar is
It produces a non-reducing sugar having a trehalose structure at the end. (2) Molecular weight By SDS-gel electrophoresis, about 69,000 to 7
9,000 Daltons. (3) pI of about 5.4 to 6.4 by electrophoresis including isoelectric point amphoraine. (4) Optimum temperature: Around 75 ° C in reaction at pH 5.5 for 60 minutes. (5) Optimum pH 60 ° C., reaction for 60 minutes, pH around 5.0 to 5.5. (6) Temperature stability It is stable up to about 85 ° C by keeping it at pH 7.0 for 60 minutes. (7) pH stability pH is about 4.0 to 9.5 when kept at 25 ° C. for 16 hours. 7. A microorganism having the ability to produce a heat-resistant non-reducing saccharide-forming enzyme that produces a non-reducing sugar having a trehalose structure at the terminal from a partially decomposed product of reducing starch is obtained by culturing in a nutrient medium. The heat-resistant non-reducing sugar which produces a non-reducing sugar having a trehalose structure at the terminal from a partially decomposed product of reducing starch, characterized in that the heat-resistant non-reducing sugar-forming enzyme is collected from the culture Method for producing produced enzyme. 8. The method for producing a thermostable non-reducing saccharide-forming enzyme according to claim 7, wherein the microorganism is a microorganism belonging to the genus Sulfolobus. 9. A glucose polymerization degree of 1 or more selected from 3 or more.
A microorganism having the ability to produce a heat-resistant non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the end from one or more partially decomposed products of reducing starch is cultivated in a nutrient medium, The thermostable non-reducing saccharide-forming enzyme is collected from the resulting culture, and a trehalose structure is added to the end from one or more reducing starch partial degradation products selected from the glucose polymerization degree of 3 or more. The method for producing a thermostable non-reducing saccharide-forming enzyme which produces a non-reducing saccharide having 10. A thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the terminal from one or more reducing starch partial degradation products selected from glucose polymerization degree of 3 or more. A non-reducing sugar having a trehalose structure at the terminal obtained by reacting with, or a low reducing sugar containing the same. 11. A solution containing one or two or more reducing starch partially decomposed products selected from glucose polymerization degree of 3 or more obtained by partially hydrolyzing starch, and selected from glucose polymerization degree of 3 or more. A thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the end is caused to act from one or more partially decomposed products of reducing starch to have a trehalose structure at the obtained end. A non-reducing sugar or a low reducing sugar containing the same. 12. A solution containing one or more reducing starch partial decomposition products selected from glucose polymerization degree of 3 or more, and one or two or more reducing starch partial decomposition of glucose polymerization degree of 3 or more. A non-reducing saccharide having a trehalose structure at the end and a contaminated saccharide-containing solution are reacted with a thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the end from the product, A non-reducing sugar having a trehalose structure at the terminal, which is obtained by subjecting it to column chromatography using a strongly acidic cation exchange resin, and which has an improved content. 13. A solution containing one or more reducing starch partial decomposition products selected from glucose polymerization degrees of 3 or more, and one or two or more reducing starch partial decompositions of glucose polymerization degree of 3 or more. A non-reducing saccharide having a trehalose structure at the end obtained by reacting a thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the end from a product, or low reducing property containing the same A composition containing a sugar. 14. The composition according to claim 13, which is a food or drink, a cosmetic product, or a pharmaceutical product. 15. A solution containing one or more reducing starch partial decomposition products selected from glucose polymerization degrees of 3 or more, to one or two or more reducing starch partial decompositions of glucose polymerization degree of 3 or more. A trehalose obtained by reacting a thermostable non-reducing sugar-forming enzyme that produces a non-reducing sugar having a trehalose structure at the terminal from the product, and then allowing glucoamylase or α-glucosidase to act. 16. The trehalose according to claim 15, which is a hydrous crystal or an anhydrous crystal. 17. A solution containing one or more reducing starch partial decomposition products selected from glucose polymerization degree of 3 or more, and one or two or more reducing starch partial decomposition of glucose polymerization degree of 3 or more. A thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the end from the product is allowed to act, and then glucoamylase or α-glucosidase is allowed to act to give a trehalose and a contaminant saccharide-containing solution, This is subjected to column chromatography using a strongly acidic cation exchange resin to obtain trehalose with improved content. 18. A solution containing one or more reducing starch partial decomposition products selected from glucose polymerization degrees of 3 or more, and one or two or more reducing starch partial decompositions of glucose polymerization degree of 3 or more. A composition containing trehalose obtained by allowing a thermostable non-reducing sugar-forming enzyme that produces a non-reducing sugar having a trehalose structure to act at the terminal from the product to act, and then allowing glucoamylase or α-glucosidase to act. . 19. The composition according to claim 18, which is a food or drink, a cosmetic or a pharmaceutical product.