JP5240480B2 - Trehalose-releasing enzyme, its production method and use - Google Patents

Description

  The present invention relates to trehalose-releasing enzyme, its production method and use, and more specifically, between a trehalose moiety of a non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more and other glycosyl moieties. Containing a novel trehalose-releasing enzyme that specifically hydrolyzes the binding of the trehalose to release trehalose, a method for producing the same, a microorganism producing the same, a trehalose produced using the novel trehalose-releasing enzyme, and the trehalose-containing enzyme It relates to a caulked composition.

  Trehalose (α, α-trehalose) has long been known as a non-reducing carbohydrate having glucose as a constituent sugar, and its presence is described in Non-Patent Document 1, Non-Patent Document 2, and the like. In addition, it has a wide range of microorganisms, mushrooms, insects, etc., even in small quantities. Trehalose is a non-reducing saccharide, so it does not cause aminocarbonyl reaction with substances having amino groups such as amino acids and proteins, and does not damage amino acid-containing substances, so it can be used and processed without concern for browning and deterioration. Therefore, establishment of the industrial manufacturing method is desired.

  As a method for producing trehalose, for example, a method using a microorganism reported in Patent Document 1 or a method of converting maltose by a combination of maltose phosphorylase and trehalose phosphorylase proposed in Patent Document 2 is known. It has been. However, in the method using microorganisms, microbial cells are used as starting materials, and the trehalose content contained therein is usually 15 w / w% per solid (hereinafter, unless otherwise specified, w / w % Is abbreviated as%.) In addition, the process of extracting and purifying this is complicated and unsuitable as an industrial production method. In addition, both the methods using maltose phosphorylase and trehalose phosphorylase are via glucose-1 phosphate, and it is difficult to increase the substrate concentration, and the reaction system of both enzymes is a reversible reaction. The product production rate is low, and furthermore, it is difficult to keep the reaction system of both enzymes stable and allow the reaction to proceed smoothly, and it has not yet been realized as an industrial production method.

  In relation to this, in the section of “Oligosaccharide” in Non-Patent Document 3, “A tremendous range of trehalose can be considered, but direct sugar transfer and hydrolysis reaction of this sugar from starch carbohydrates were used. Enzymatic production is said to be scientifically impossible at present. ”As described in the article, the production of trehalose using starch as a raw material by enzymatic reaction has traditionally been academic. Has been said to be impossible.

  On the other hand, it is known that starch partially decomposed products produced from starch as a raw material, such as starch liquefied products, various dextrins, various maltooligosaccharides, etc., usually have a reducing group at the end of the molecule and exhibit reducing properties. . Such a starch partial decomposition product is referred to as a reducing starch partial decomposition product in the present specification. Generally, the reduced starch partial decomposition product expresses the magnitude of the reducing power per solid as Dextrose Equivalent (DE). Those with large values usually have small molecules, low viscosity, and strong sweetness, but are highly reactive and easily cause aminocarbonyl reactions with substances having amino groups such as amino acids and proteins. Thus, it is known that the quality is easily deteriorated.

  Various characteristics of such a reduced starch partial decomposition product depend on the size of DE, and the relationship between the starch partial decomposition product and DE is extremely important. Traditionally, the industry has even believed that it was impossible to break this relationship.

  However, the present inventors have overturned this common sense in the industry, and as disclosed in Patent Document 3, the reduced starch partial decomposition product having a glucose polymerization degree of 3 or more produced from starch as a raw material is from now on. By directly reacting a non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure with a glucose polymerization degree of 3 or more and glucoamylase, trehalose is directly enzymatically decomposed from the partially degraded starch. Has succeeded in producing. In the trehalose production method using this non-reducing saccharide-forming enzyme and glucoamylase, the trehalose production rate from the reduced starch partial degradation product is about 30%, which is industrially sufficient. Therefore, there is concern about the high cost in trehalose production. Therefore, establishment of a new method for producing trehalose that further improves the production rate from the reduced starch partially decomposed product is desired.

JP 50-154485 A JP 58-216695 A Japanese Patent Application No. 4-362131

"Advances in Carbohydrate Chemistry", Vol. 18, pp. 201-225 (1963) Academic Press (USA) "Applied and Environmental Microbiology", Vol. 56, pp. 3213-3215 (1990) “Monthly Food Chemical”, August, 67-72 (1992), “Current Status and Issues of Starch Development”

  The present invention is to provide a novel method for producing trehalose having a high production rate from starch that can be stably supplied at low cost.

  In order to solve the above-mentioned problems, the present inventors hoped to realize a completely new enzyme that releases trehalose from a non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more. We have searched extensively for microorganisms that produce. As a result, the non-reducing carbohydrate-producing enzyme-producing microorganism M-11 belonging to the genus Rhizobium from the soil disclosed in Japanese Patent Application No. 4-362131, and Japanese Patent Application No. 5-265416. It was found that the non-reducing saccharide-forming enzyme-producing microorganism Q36 belonging to the genus Arthrobacter isolated from the soil disclosed in the specification also produces a novel trehalose-releasing enzyme. It was found that the desired high-reproduction trehalose production reaction can be easily performed by allowing the non-reducing saccharide-forming enzyme and this novel trehalose-releasing enzyme to act on the degradation product. Non-reducing saccharide-forming enzyme and novel trehalose-releasing enzyme are allowed to act on the product, followed by glucoamylase. By, it is possible to further obtain a high-purity trehalose-containing reaction solution, easily found that can produce trehalose, and have completed the present invention. Furthermore, microorganisms producing this trehalose-releasing enzyme were searched more widely than known bacteria. As a result, it was found that microorganisms belonging to the genus Brevibacterium and Micrococcus also produce the trehalose-free enzyme of the present invention, and trehalose derived from the microorganisms belonging to the genera Rhizobium and Arthrobacter. Similar to the free enzyme, it was found that trehalose was released from a non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more, thereby completing the present invention. At the same time, the present invention was completed by establishing compositions such as foods, drinks, cosmetics, and pharmaceuticals containing trehalose obtained as described above or a sugar containing the same.

  The novel trehalose-releasing enzyme of the present invention releases trehalose from a non-reducing saccharide having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more, and a reduced starch partial degradation product together with a non-reducing saccharide-forming enzyme. By acting, trehalose is produced in high yield. Therefore, the establishment of the present invention is an industrially large amount of trehalose and a saccharide containing the same that have not been easily obtained as desired from a partially decomposed starch derived from starch, which is an inexpensive and endless resource. It will open up a whole new way to supply cheaply.

It is a figure which shows the elution pattern of the trehalose releasing enzyme of this invention from a DEAE-Toyopearl, and a non-reducing carbohydrate production | generation enzyme. It is a figure which shows the influence of the temperature which acts on the enzyme activity of the trehalose free enzyme derived from Rhizobium sp. M-11 of this invention. It is a figure which shows the influence of pH which acts on the enzyme activity of the trehalose release enzyme derived from Rhizobium sp. M-11 of this invention. It is a figure which shows the influence of the temperature which acts on the stability of the trehalose release enzyme derived from Rhizobium sp. M-11 of this invention. It is a figure which shows the influence of pH which acts on the stability of the trehalose free enzyme derived from Rhizobium sp. M-11 of this invention. It is a figure which shows the influence of the temperature which acts on the enzyme activity of the trehalose free enzyme derived from Arthrobacter sp. Q36 of this invention. It is a figure which shows the influence of pH which acts on the enzyme activity of the trehalose free enzyme derived from Arthrobacter sp. Q36 of this invention. It is a figure which shows the influence of the temperature which acts on the stability of the trehalose free enzyme derived from Arthrobacter sp. Q36 of this invention. It is a figure which shows the influence of pH on the stability of the trehalose release enzyme derived from Arthrobacter sp. Q36 of this invention.

  Next, the identification test result of the microorganism M-11 belonging to the genus Rhizobium of the present invention is shown. The identification test was performed according to “Classification and Identification of Microorganisms” (Takeharu Hasegawa, Academic Publishing Center, 1985).

<A cell morphology>
Meat juice agar culture, 27 ℃
Usually 0.6-0.8 × 1.0-1.5 μm bacilli. A single, rarely paired, linear pair is also observed. No polymorphism. There is mobility. No spores. Flagella is periflagellate. Non-acidic. Gram negative. Capsule negative. Metachromatic granules positive. Accumulate Poly-β-hydroxybutyrate.

<B culture properties>
(1) Meat broth agar plate culture, 27 ° C
Shape: Circular Size is 1.5 mm in 24 hours.
Perimeter: All edges
Raised: flat or half-lens
Gloss: yes
Surface: Smooth
Color tone: Translucent, cream, no pink pigment formation (2) Dextrose / tryptone agar plate culture, 27 ° C
Colony is translucent, cream color, mucoid production (3) Yeast extract / mannitol agar plate culture, 27 ° C
Shape: Circular The size is about 3 mm in 5 days.
Color tone: Translucent, cream color, mucoid production (4) Congo-red containing yeast extract mannitol agar plate culture, 27 ° C
The colony is faint pink and hardly absorbs Congo-Red.
(5) Yeast extract / mannitol agar plate culture containing 2 w / v% NaCl, 27 ° C.
Grows.
(6) Meat broth agar slope culture, 27 ℃
Growth degree: Good
Shape: filamentous (7) gravy gelatin puncture culture, 27 ° C
Does not liquefy.

<C physiological properties>
(1) Reduction of nitrate: positive (2) Denitrification reaction: negative (3) Methyl red test: negative (4) VP test: negative (5) Indole production: negative (6) Hydrogen sulfide production: positive ( 7) Hydrolysis of starch: Negative (8) Use of citric acid: Positive (9) Use of inorganic nitrogen source: Both ammonium salt and nitrate can be used.
(10) Pigment production: None (11) Urease: Positive (12) Oxidase: Negative (13) Catalase: Positive (14) Growth range: pH 5.5 to 9.0
Temperature 4 ~ 35 ℃
(15) Attitude toward oxygen: aerobic (16) Use of carbon source and presence or absence of acid generation
Usability Acid generation
Use D-glucose positive
D-galactose use positive
D-fructose positive
L-arabinose use positive
D-xylose used positive
L-Rhamnose Use Positive
Maltose use negative
Sucrose use positive
Lactose use negative
Trehalose use negative
Raffinose use positive
Mannitol use negative
Dextrin use negative
Negative (17) Amino acid decarboxylation test: Negative for all of L-lysine, L-arginine and L-ornithine.
(18) Utilization of amino acids: L-sodium glutamate, sodium L-aspartate, L-histidine, and L-proline are all utilized.
(19) DNase: negative (20) 3-ketolactose production: negative (21) G-C content of DNA: 61%

  Based on the above bacteriological properties, with reference to "Bergey's Manual of Systematic Bacteriology", Volume 1 (1984) I examined the difference. As a result, it was found to be a microorganism belonging to the genus Rhizobium. Although this bacterium shows properties close to Rhizobium merillotti, it differs from this bacterium in that it does not produce acid from maltose, lactose, and mannitol, and it is also a reduced starch partial decomposition product. Specifically hydrolyzes the non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure from the enzyme and the bond between the trehalose part and the other glycosyl part of the non-reducing saccharide to release trehalose The novel trehalose-releasing enzyme that produces a novel trehalose releasing enzyme is described.

  Based on these results, the present inventors named this bacterium Rhizobium sp. M-11. In addition, as of December 24, 1992, the bacterium has been registered at the Institute of Microbiological Technology, Institute of Industrial Technology, Ministry of International Trade and Industry, 1-3-3 Higashi 1-chome, Tsukuba, Ibaraki Prefecture. It was entrusted with the fine work laboratory No. 4130 (FERM BP-4130).

  In the present invention, not only the above-mentioned bacteria, but also belonging to the genus Rhizobium, the bond between the trehalose part of a non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more and the other glycosyl moiety is specifically Other strains that produce trehalose-releasing enzyme that hydrolyzes to release trehalose, and mutant strains of these strains are also used as appropriate.

  Next, the identification test result of microorganism Q36 belonging to the genus Arthrobacter of the present invention is shown. The identification test was performed according to “Classification and Identification of Microorganisms” (Takeharu Hasegawa, Society Publishing Center, 1985).

<A cell morphology>
(1) Meat juice agar culture, 27 ℃
Usually 0.5-0.7 × 0.8-1.6 μm bacilli. Alone.
There is polymorphism. No mobility. No spores. No flagella. Non-acidic. Gram positive. Capsule negative.
(2) EYG agar culture, 27 ° C
The growth cycle of Neisseria gonorrhoeae-cocci is shown.

<B culture properties>
(1) Meat broth agar plate culture, 27 ° C
Shape: Circular The size is 2 to 2.5 mm in 3 days.
Perimeter: All edges
Raised: half-lens
Gloss: wet light
Surface: Smooth
Color: Translucent, white to pale yellow (2) Meat broth agar slope culture, 27 ° C
Growth degree: Good
Shape: Filament (3) Yeast extract / pepton agar slope culture, 27 ° C
Growth degree: Good
Shape: filamentous (4) gravy gelatin puncture culture, 27 ° C
Liquefaction.

<C physiological properties>
(1) Nitrate reducibility: positive (2) Denitrification reaction: negative (3) Methyl red test: negative (4) VP test: positive (5) Indole production: negative (6) Hydrogen sulfide production: positive ( 7) Hydrolysis of starch: Negative (8) Degradation of cellulose: Negative (9) Use of citric acid: Positive (10) Use of inorganic nitrogen source: Both ammonium salt and nitrate can be used.
(11) Pigment production: None (12) Urease: Positive (13) Oxidase: Negative (14) Catalase: Positive (15) Range of growth: pH 5 to 10
Temperature 4 ~ 37 ℃
(16) Attitude toward oxygen: aerobic (17) Availability of carbon source and presence or absence of acid generation
Usability Acid generation
D-glucose use negative
D-galactose use negative
D-fructose use negative
L-arabinose use negative
D-xylose used negative
L-Rhamnose Use Negative
Maltose use negative
Sucrose use negative
Lactose use negative
Raffinose use negative
Mannitol use negative
Dextrin use negative
Dulcitol Utilization Negative (18) Utilization of amino acids: L-sodium glutamate, sodium L-aspartate, L-histidine, and L-proline are all utilized.
(19) DNase: positive (20) production of 3-ketolactose: negative (21) main diamino acid in cell wall: lysine (22) GC content of DNA: 63%

  Based on the above bacteriological properties, with reference to "Bergey's Manual of Systematic Bacteriology", Volume 2 (1984) The difference was examined. As a result, the present bacterium was found to be a microorganism belonging to the genus Arthrobacter. In addition, the present bacterium has a non-reducing sugar saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure from a reductive starch partial decomposition product, and between the trehalose part of the non-reducing sugar sugar and the other glycosyl part. It has a feature not yet described in the literature for producing a novel trehalose-releasing enzyme that specifically hydrolyzes the bond and releases trehalose.

  Based on these results, the present inventors named this bacterium the new microorganism Arthrobacter sp. Q36. In addition, as of June 3, 1993, this microbe is registered with the microbial depository number FERM at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture. Entrusted with BP-4316.

  In the present invention, not only the strains but also belonging to the genus Arthrobacter, the binding between the trehalose part of the non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more and the other glycosyl part is specific. Other strains that produce a trehalose-releasing enzyme that hydrolyzes and releases trehalose, and mutants of those strains are also used as appropriate.

  The microorganism used in the present invention may be any microorganism having the ability to produce the trehalose-releasing enzyme of the present invention. For example, the novel microorganism Rhizobium sp. M-11 FERM BP-4130 and Arthrobacter sp. Q36 FERM BP Not only -4316 but also known microorganisms such as Brevibacterium herovolum ATCC 11822, Micrococcus roseus ATCC 186 and the like can be advantageously used.

  The medium used for culturing the microorganism of the present invention may be any nutrient medium that can grow microorganisms and can produce the trehalose-free enzyme of the present invention, and may be either a synthetic medium or a natural medium. As the carbon source, any substance that can be assimilated by microorganisms may be used. For example, sugars such as glucose, fructose, lactose, sucrose, mannitol, sorbitol, molasses, and reduced starch partial degradation products, or citric acid, succinic acid An organic acid such as or a salt thereof 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 reduced starch partial decomposition product, usually 20% or less is desirable, and 5% or less is preferred from the growth and proliferation of bacteria. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing materials such as urea, corn steep liquor, casein, peptone, yeast extract, and meat extract. Moreover, as an inorganic component, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt etc. are used suitably, for example. Furthermore, amino acids, vitamins and the like are also used as necessary.

  Cultivation is usually carried out aerobically under conditions selected from a temperature of 4 to 40 ° C., preferably 20 to 37 ° C., pH 4 to 10, preferably 5 to 9. The culture time may be a time during which the microorganism can grow, and is preferably 10 hours to 100 hours. Further, 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 aeration amount, stirring, adding oxygen to the aeration, and increasing the pressure in the fermenter are adopted. The culture method may be either batch culture or continuous culture.

  Thus, after culturing the microorganism, the enzyme of the present invention is recovered. This enzyme activity is observed in both the microbial cells and the sterilization solution of the culture. The microbial cells and the sterilization 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 employed to remove the 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 or the like, a method of separating by membrane filtration such as a flat membrane or a hollow fiber membrane, etc. are suitably employed. The sterilizing solution can be used as an enzyme solution as it is, but generally it is used after being concentrated. As the concentration method, for example, an ammonium sulfate salting-out method, an acetone and alcohol precipitation method, a membrane concentration method such as a flat membrane or a hollow fiber membrane, or the like is employed.

  Furthermore, the disinfectant solution and the concentrate thereof can be immobilized by a known method. For example, a binding method to an ion exchanger, a covalent bond / adsorption method with a resin and a membrane, a comprehensive method using a polymer substance, and the like are employed. Moreover, although the microbial cell isolate | separated from the culture can be used as a crude enzyme as it is, you may fix | immobilize and use this. As an example, this is mixed with sodium alginate and dropped into a calcium chloride solution to be gelled and fixed in a granular form. This granulated product may be further fixed with polyethyleneimine or glutaraldehyde. It is also possible to extract an enzyme from the cells and use the extract as a crude enzyme solution. For example, it is possible to extract enzymes from bacterial cells by ultrasonic crushing, glass beads and alumina mechanical crushing, French press crushing, etc., and to obtain a clear crude enzyme solution by centrifugation or membrane filtration. it can.

  This enzyme solution can be used as it is, but can be further purified and used by a known method. As an example, after dialysis of a crude enzyme preparation concentrated by subjecting the treated product of the culture solution to salting out with ammonium sulfate, anion exchange column chromatography using DEAE-Toyopearl resin, followed by a hydrophobic column using butyl Toyopearl resin Chromatography, purified by gel filtration chromatography using Toyopearl HW-55 resin, a single enzyme can be obtained electrophoretically.

The trehalose-releasing enzyme of the present invention thus obtained has the following physicochemical properties.
(1) Action It specifically hydrolyzes the bond between the trehalose moiety of a non-reducing carbohydrate having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more and the other glycosyl moiety.
(2) Molecular weight About 57,000 to 68,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point pI of about 3.3 to 4.6 by an ampholine-containing electrophoresis method.
(4) Optimal temperature: pH 7.0, reaction for 30 minutes, around 35 to 45 ° C.
(5) Optimum pH
PH of about 6.0 to 7.5 after reaction at 40 ° C. for 30 minutes.
(6) Temperature stability Stable up to around 30 to 45 ° C at pH 7.0, held for 60 minutes.
(7) pH stability The pH is about 5.0 to 10.0 at 25 ° C. for 16 hours.

  The activity of the trehalose-free enzyme of the present invention is measured as follows. Add 1 ml of enzyme solution to 4 ml of maltotriosyl trehalose (also known as α-maltotetraosyl α-glucoside) as a substrate at 1.25 w / v% (50 mM phosphate buffer, pH 7.0) and react at 40 ° C. for 30 minutes. After that, the reaction is stopped by adding a somological copper solution, and the reducing power is measured by the somological Nelson method. As a control, the same measurement is performed using an enzyme solution inactivated by heating at 100 ° C. for 10 minutes in advance. Using the above measurement method, the amount of enzyme that increases the reducing power corresponding to 1 μmole of glucose per minute is defined as 1 unit.

  The substrate of the enzyme may be a non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more. For example, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltohepta. Glycosyl trehalose, such as glucosyl trehalose, maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, maltopentaosyl trehalose, which can be obtained by allowing a non-reducing saccharide-forming enzyme to act on aose or the like is used. In addition, a trehalose structure is formed at the end where a non-reducing saccharide-forming enzyme can act on a reducing starch partial degradation product that can be obtained by partially hydrolyzing starch such as starch, amylopectin, or amylose with amylase or acid. A low-reducing starch partial decomposition product containing a non-reducing sugar having a glucose polymerization degree of 3 or more is used.

  An amylase that partially hydrolyzes starch is described, for example, in “Handbook of Amylases and Related Enzymes” (1988) Pergamon Press (Tokyo). Α-amylase, maltopentaose producing amylase, maltohexaose producing amylase and the like are used. A combination of these amylases and debranching enzymes such as pullulanase and isoamylase can also be advantageously carried out.

  The substrate concentration is not particularly limited. For example, even when used as a 0.1% substrate solution or as a 50% substrate solution, the reaction of this enzyme proceeds to produce trehalose. Further, an insoluble substrate that cannot be completely dissolved in the substrate solution may be contained. The reaction temperature may be up to the temperature at which the enzyme reaction proceeds, that is, up to about 55 ° C., but preferably in the range of 40 to 50 ° C. The reaction pH is usually adjusted to a range of 5 to 10, but is preferably adjusted to a range of about pH 6 to 8. The reaction time may be appropriately selected depending on the progress of the enzyme reaction, and is usually about 0.1 to 100 hours at an enzyme usage of about 0.1 to 100 units per gram of substrate solid matter.

  Regarding the production rate of trehalose from the raw material substrate, when producing trehalose from a relatively low DE high molecular weight reducing starch partial decomposition product, that is, a reduced starch partial decomposition product having a high degree of glucose polymerization, the method according to the present invention comprises: Compared with the method described in Japanese Patent Application No. 4-362131 using a non-reducing saccharide-forming enzyme and glucoamylase, the production rate is significantly increased. While the trehalose production rate obtained by the reaction of the non-reducing saccharide-forming enzyme and glucoamylase of the prior application is about 30%, the non-reducing saccharide-forming enzyme of the present invention and trehalose-free enzyme act together. In the reaction, the trehalose production rate is as high as about 60% or more.

  The principle of this action is as follows. That is, first, a reduced starch partial degradation product having a relatively high degree of glucose polymerization is converted into a non-reducing sugar having the same degree of glucose polymerization of one molecule having a trehalose structure at its end by a non-reducing sugar-forming enzyme. The non-reducing saccharide produces one molecule of trehalose and one molecule of a reduced starch partial degradation product having a glucose polymerization degree reduced by 2 by hydrolysis reaction of trehalose-releasing enzyme. If the newly produced reduced starch partial degradation product has a glucose polymerization degree of 3 or more, this reduced starch partial degradation product is further reduced to a non-reducing property having a trehalose structure at its end by a non-reducing carbohydrate-forming enzyme. One molecule of trehalose and one molecule of a reduced starch partial degradation product having a degree of glucose polymerization reduced by 2 are generated by trehalose-releasing enzyme while being converted into a saccharide. In this way, by repeating the reaction of the non-reducing saccharide-forming enzyme and the reaction of trehalose-releasing enzyme, a plurality of trehalose and glucose polymerization degrees are generated from one molecule of a reduced starch partial degradation product. It is possible to produce a non-reducing starch partial decomposition product with a reduced equivalent of double.

  In this method of action, a non-reducing saccharide-forming enzyme and the trehalose-releasing enzyme of the present invention can be allowed to act simultaneously on a reduced starch partial decomposition product having a glucose polymerization degree of 3 or more. A non-reducing saccharide-forming enzyme can be allowed to act on a product, and then trehalose-releasing enzyme can be allowed to act. Moreover, after making both the enzymes act, glucoamylase can be acted on to further increase the trehalose production rate.

  The reaction solution is filtered, centrifuged, etc., to remove insoluble matters, then decolorized with activated carbon, desalted with H-type and OH-type ion exchange resins, and concentrated to obtain a syrup-like product. Furthermore, it is optional to dry it into a powdered product.

  If necessary, further purification, for example, fractionation by ion exchange column chromatography, fractionation by activated carbon column chromatography, fractionation by silica gel column chromatography, fractionation with organic solvents such as alcohol and acetone, reducing sugar by alkali treatment It is also easy to obtain the highest purity trehalose product by purifying it by a method such as quality degradation and removal.

  The saccharide containing the trehalose of the present invention thus obtained is hydrolyzed with α-amylase, β-amylase, glucoamylase, α-glucosidase, trehalase or the like, if necessary, or cyclomaltodextrin / glucanotransferase. It is also possible to adjust the sweetness, reducing power, etc. by reducing sugar transfer by using glycosyltransferase or glucosyltransferase, etc., or reducing the viscosity by hydrogenating the reducing sugar into a sugar alcohol. It is optional to perform further processing such as extinction. From this, glucose is removed by the purification method described above, for example, ion exchange column chromatography, and a fraction containing high trehalose is collected. This can be purified and concentrated to obtain a syrup-like product, or further concentrated to be supersaturated and crystallized to obtain trehalose hydrous crystals or anhydrous crystalline trehalose.

  As ion exchange column chromatography, contaminating saccharides are removed by column chromatography using a salt type strongly acidic cation exchange resin disclosed in JP-A-58-23799 and JP-A-58-72598. Thus, a method of collecting a fraction containing high trehalose can be advantageously implemented. At this time, it is optional to adopt any of a fixed floor method, a moving floor method, and a simulated moving floor method.

  In order to produce a water-containing trehalose crystal, for example, a trehalose-containing 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 the temperature is 95 ° C. or less in the presence of 0.1 to 20% seed crystals. Desirably, it is slowly cooled with stirring in the range of 10 to 90 ° C. to produce a mass kit containing trehalose hydrated crystals. As a method for producing a trehalose hydrated crystal or a honey-containing crystal containing the same from a mass kit, a known method such as a honey mash method, a block pulverization method, a fluid granulation method, or a spray drying method may be employed.

  In the case of the honey method, the mass kit is usually applied to a basket-type centrifuge to separate the trehalose hydrated crystals from the nectar (mother liquor), and if necessary, the crystals can be easily washed by spraying a small amount of cold water. And is suitable for producing higher-purity water-containing trehalose crystals. In the case of the spray drying method, usually, a mass kit having a concentration of 60 to 85% and a crystallization rate of 20 to 60% is sprayed from a nozzle with a high-pressure pump, and hot air at a temperature at which the crystal powder does not dissolve, for example, 60 to 100 ° C. And then aged for about 1 to 20 hours with warm air of 30 to 60 ° C., non-hygroscopic or hardly hygroscopic honey-containing crystals can be easily produced. 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 about 10 to 60% is allowed to stand for several hours to 3 days to crystallize and solidify the whole into a block shape. If powdered and dried by a method such as pulverization or cutting, non-hygroscopic or hardly hygroscopic honey-containing crystals can be easily produced. In order to produce anhydrous crystalline trehalose, it is possible to dry and convert the trehalose hydrated crystal, but generally, a high concentration trehalose-rich solution with a moisture content of less than 10% is placed in an auxiliary crystal can and coexists with seed crystals. A mass kit containing anhydrous crystalline trehalose is produced while stirring at a temperature of 50 to 160 ° C., preferably 80 to 140 ° C., and this is dried under relatively high temperature drying conditions, for example, a block pulverization method, a fluid granulation method, etc. It is produced by crystallization and powdering by a method such as spray drying.

  The trehalose of the present invention thus produced has no reducing power, is stable, and can be mixed and processed with other materials, particularly amino acids, oligopeptides, proteins and other substances containing amino groups or amino groups. It is less likely to turn brown, generate off-flavors, or damage other mixed materials. Moreover, it has a good quality and elegant sweetness. Furthermore, since trehalose is easily decomposed into glucose by trehalase, it is digested and absorbed by ingestion and used as a calorie source. It can be used as a sweetener that is not easily fermented by caries-inducing bacteria and that is unlikely to cause caries. In addition, the trehalose of the present invention is used parenterally as a tube feeding agent, an infusion agent, etc., is free of concern about toxicity and side effects, is frequently used for metabolism, and can be advantageously used for energy supply to the living body. .

  Moreover, since it is a stable sweetener, in the case of a crystal product, it can be advantageously used as a sugar coating for tablets in combination with a binder such as pullulan, hydroxyethyl starch, polyvinylpyrrolidone and the like. In addition, it has properties such as osmotic pressure controllability, shaping, irradiating properties, moisture retention, viscosity, anti-crystallization of other sugars, difficult fermentation, and anti-aging properties of gelatinized starch.

  Therefore, the trehalose of the present invention and the saccharides containing the same are used as sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. as various compositions such as foods, foods, foods, cosmetics, and pharmaceuticals. Can be advantageously used.

  The trehalose of the present invention and a saccharide containing the same can be used as a seasoning for sweetening as it is. If necessary, for example, flour, glucose, fructose, maltose, sucrose, isomerized sugar, honey, maple sugar, erythritol, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L -May be used in admixture with an appropriate amount of one or more of other sweeteners such as aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, etc., and if necessary, dextrin, starch, It can also be used by mixing with a bulking agent such as lactose.

  In addition, the trehalose of the present invention and the saccharide powder or crystalline product containing the same may be used as they are or mixed with a bulking agent, an excipient, a binder or the like to form granules, spheres, and short rods. It is also optional to use it in various shapes such as plate, cube, and tablet.

  In addition, the sweetness of trehalose of the present invention and saccharides containing the same well matches various substances having other tastes such as acidity, salt to taste, astringency, umami, and bitterness, and has high acid resistance and heat resistance. Therefore, it can be advantageously used for sweetening and taste improvement of general foods and drinks and quality improvement.

  For example, amino acids, peptides, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hishio, sprinkle, mayonnaise, dressing, vinegar, three cups of vinegar, sushi vinegar, Chinese soup, tempura soup, noodle soup, sauce, ketchup, yakiniku It can be advantageously used as various seasonings such as sauce, curry roux, stew, soup, dashi, nucleic acid-based seasoning, compound seasoning, mirin, new mirin, table sugar, coffee sugar.

  In addition, for example, Japanese rice cakes such as rice crackers, hail, rice cakes, rice cakes, manju, oirou, chanterelles, sheep mushrooms, water sheep rice cakes, brocade balls, jelly, castella, rice cakes, bread, biscuits, crackers, cookies, pie , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy and other Western confectionery, ice cream, sorbet and other ice confectionery, fruit syrup pickled, syrup such as ice honey, flower Paste such as paste, peanut paste, fruit paste, spread, fruit such as jam, marmalade, syrup pickles, sugar cane, processed foods of vegetables, pickles such as Fukujin pickles, bedara pickles, thousand pieces pickles, pickled pickles, takan pickles, Pickles such as Chinese cabbage pickles Meat products such as fish, ham and sausage, fish meat ham, fish sausage, fish products such as kamaboko, chikuwa, tempura, sea urchin, squid salt, vinegared konbu, suki-meme, various delicacies Tsukudani, made with seaweed, wild vegetables, seaweed, small fish, shellfish, boiled beans, potato salad, side dish foods such as konbu rolls, dairy products, fish meat, livestock meat, fruits, bottled vegetables, canned foods, synthetic sake, Alcoholic beverages such as western liquor, tea, coffee, cocoa, juice, carbonated drinks, soft drinks such as lactic acid drinks, lactic acid bacteria drinks, pudding mixes, hot cake mixes, instant foods such as instant soups, baby food, treatment It can be advantageously used for sweetening various foods such as foods, drinks, peptide foods, frozen foods, and dried foods, for improving taste, and for improving quality.

  Moreover, it can also be used for the purpose of improving the palatability of feed, feed, etc. for domestic animals, poultry, and other domestic animals such as bees, cormorants and fish. In addition, tobacco, toothpaste, lipstick, lip balm, oral solution, tablets, troches, liver oil drops, mouth fresheners, mouth fragrances, gargles, etc. It can be advantageously used as a sweetener for various compositions, as a taste improver, a taste corrector, and as a quality improver.

  As the quality improver and stabilizer, it can be advantageously applied to various physiologically active substances that easily lose their active ingredients and activities, health foods containing them, pharmaceuticals, and the like. For example, interferon-α, -β, -γ, Tsumore necrosis factor-α, -β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, lymphokine such as interleukin 2, insulin, growth hormone, prolactin, erythropoietin , Hormones such as egg cell stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, polio vaccine, seedling, tetanus toxoid, hub antitoxin, human immunoglobulin and other biologics, penicillin, erythromycin, chloramphenicol, tetracycline, Antibiotics such as streptomycin and kanamycin sulfate, thiamine, riboflavin, L-ascorbic acid, liver oil, carotenoids, ergosterol, tocopherol and other vitamins, lipases, Enzymes such as elastase, urokinase, protease, β-amylase, isoamylase, glucanase, lactase, ginseng extract, suppon extract, placenta extract, chlorella extract, aloe extract, propolis extract, viruses, lactic acid bacteria, yeast, etc. Various physiologically active substances such as live bacteria and royal jelly can easily produce stable and high-quality health foods and pharmaceuticals without losing their active ingredients and activities.

  The method of adding trehalose to the various compositions as described above may be included in the process until the product is completed, for example, mixing, dissolving, melting, dipping, infiltration, spraying, coating, coating, spraying. Well-known methods such as injection, 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 production, purification and properties of trehalose-releasing enzyme from the novel microorganism Rhizobium species M-11 will be explained, and then the trehalose-releasing enzyme from Arthrobacter species Q36 will be explained in the same manner. Furthermore, trehalose releasing enzyme from known microorganisms will be described.

<Experiment 1: Production of trehalose-releasing enzyme from Rhizobium species M-11>
Paindex # 4 (manufactured by Matsutani Chemical Co., Ltd.) 2.0 w / v%, peptone 0.5 w / v%, yeast extract 0.1 w / v%, disodium phosphate 0.1 w / v%, monophosphate A liquid medium consisting of potassium 0.1 w / v% and water was adjusted to pH 7.0. About 100 ml of this medium is placed in a 500 ml Erlenmeyer flask, sterilized by autoclaving at 120 ° C. for 20 minutes, cooled, inoculated with Rhizobium species M-11 (FERM BP-4130), and incubated at 27 ° C. and 130 rpm for 24 hours. The culture was used as a seed culture solution.

  A fermenter with a volume of 30 l is charged with about 20 l of medium having the same composition as in the case of seed culture, sterilized and cooled to a temperature of 27 ° C., then inoculated with 1 w / v of the seed culture, at a temperature of 27 ° C. and a pH of 6. While maintaining at 0 to 8.0, the culture was aerated and stirred for about 72 hours.

  The enzyme activity of the non-reducing saccharide-forming enzyme in the culture solution was about 1.5 units / ml, and the enzyme activity of the trehalose-free enzyme of the present invention was about 2 units / ml. A portion of the culture solution is collected and centrifuged to separate the cells and the culture supernatant, and the cells are suspended in 50 mM phosphate buffer (pH 7.0) in the same volume as the original culture solution. Then, the enzyme activity of the bacterial cell suspension and the culture supernatant was measured. The bacterial cell suspension had an enzyme activity of about 0.6 units / ml of non-reducing saccharide-forming enzyme, trehalose. The enzyme activity of the free enzyme is found to be about 0.8 unit / ml, and the culture supernatant has about 0.9 unit / ml of the enzyme activity of the non-reducing saccharide-forming enzyme and about 1 unit of trehalose free enzyme. 2 units / ml were observed.

  The non-reducing saccharide-forming enzyme activity was measured by adding 1 ml of enzyme solution to 4 ml of maltopentaose 1.25 w / v% (50 mM phosphate buffer, pH 7.0) as a substrate and reacting at 40 ° C. for 60 minutes. After that, the reaction is stopped by heating at 100 ° C. for 10 minutes, the reaction solution is diluted exactly 10 times, 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 10 minutes in advance. 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.

<Experiment 2: Purification of enzyme>
About 18 l of the culture solution obtained by the method of Experiment 1 was treated with an ultrahigh-pressure cell disruption device minilab (manufactured by Dainippon Pharmaceutical Co., Ltd.) to disrupt the contained cells. The treatment liquid was centrifuged (10,000 rpm, 30 minutes) to obtain about 16 l of a centrifugal supernatant. Ammonium sulfate was added to the solution so that the degree of saturation was 0.2, dissolved, and allowed to stand at 4 ° C. for 1 hour, and then centrifuged (10,000 rpm, 30 minutes) to recover the supernatant.

  Further, ammonium sulfate was dissolved in the solution so as to have a saturation degree of 0.6, and allowed to stand at 4 ° C. for 24 hours, and then centrifuged (10,000 rpm, 30 minutes) to recover ammonium sulfate salted-out. The obtained ammonium sulfate salted-out product was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against the same buffer for 24 hours, and centrifuged (10,000 rpm, 30 minutes) to remove insoluble matter. Excluded. The dialysate (360 ml) was divided into two portions and subjected to ion exchange column chromatography (gel amount: 300 ml) using DEAE-Toyopearl gel (manufactured by Tosoh Corporation).

  Both the trehalose-free enzyme and the non-reducing saccharide-forming enzyme of the present invention were adsorbed on DEAE-Toyopearl gel and eluted from the column at different salt concentrations with the same buffer containing salt. The elution pattern from DEAE-Toyopearl gel is shown in FIG. The non-reducing saccharide-forming enzyme was eluted at a salt concentration of about 0.2M, and the trehalose-free enzyme was eluted at a salt concentration of about 0.3M. The respective enzyme activity fractions were collected, and both enzymes were purified separately below.

  The nonreducing carbohydrate-forming enzyme active fraction is dialyzed against the same buffer containing 2M ammonium sulfate, the dialyzed solution is centrifuged (10,000 rpm, 30 minutes) to remove insoluble matters, and the resulting supernatant is butyl. Hydrophobic column chromatography (gel amount: 300 ml) using Toyopearl 650 gel (manufactured by Tosoh Corporation) was performed. The adsorbed enzyme was eluted from the column with a linear gradient of 2M to 0M ammonium sulfate, and the enzyme activity fraction was recovered. Subsequently, gel filtration chromatography (gel amount: 300 ml) using Toyopearl HW-55 resin (manufactured by Tosoh Corporation) was performed to recover a non-reducing carbohydrate-forming enzyme active fraction.

  For the purification of trehalose-free enzyme, the trehalose-free enzyme active fraction eluted from the DEAE-Toyopearl column was used for a buffer solution containing 2M ammonium sulfate in the same manner as the above-described method for purifying non-reducing saccharide-forming enzyme. Dialysis was performed, followed by hydrophobic column chromatography and gel filtration chromatography.

  The amount of enzyme activity, specific activity, and yield in each step of purification are shown in Table 1 for the non-reducing saccharide-forming enzyme and Table 2 for the trehalose-free enzyme of the present invention.

  The purified non-reducing saccharide-forming enzyme preparation and the purified trehalose-free enzyme preparation obtained as gel filtration eluates in the steps of Table 1 and Table 2, respectively, are used on polyacrylamide gel (gel concentration 7.5%). When the purity was tested by electrophoresis, it was shown that the protein band was single, and the obtained enzyme preparation was a single sample having high purity by electrophoresis.

<Experiment 3: Properties of trehalose-releasing enzyme>
The purified trehalose-free enzyme preparation obtained by the method of Experiment 2 was subjected to electrophoresis using SDS-polyacrylamide gel (gel concentration: 10%) and simultaneously migrated molecular weight marker (manufactured by Nippon Bio-Rad Laboratories) The molecular weight of the present enzyme was measured in comparison with the molecular weight of about 58,000 to 68,000 daltons.

  The purified enzyme preparation was subjected to isoelectric focusing using a polyacrylamide gel (containing 2% ampholine, manufactured by Pharmacia El Kavy, Sweden). After electrophoresis, the pH of the gel was measured to determine the isoelectricity of the enzyme. When the points were determined, the isoelectric point was about 3.3 to 4.3.

  The effects of temperature and pH on the enzyme activity were examined according to the activity measurement method. The results are shown in FIG. 2 (effect of temperature) and FIG. 3 (effect of pH). The optimum temperature of the enzyme was about 45 ° C. when reacted at pH 7.0 for 30 minutes, and the optimum pH was about 6.0 to 7.5 after reacting at 40 ° C. for 30 minutes. The temperature stability of the enzyme was determined by measuring the remaining enzyme activity after holding the enzyme solution (containing 50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water. The pH stability was determined by maintaining the enzyme in a 50 mM buffer solution at each pH at 25 ° C. for 16 hours, adjusting the pH to 7, and measuring the remaining enzyme activity. The respective results are shown in FIG. 4 (temperature stability) and FIG. 5 (pH stability). The thermal stability of the enzyme was up to about 40 ° C., and the pH stability was about 5 to 10.

<Experiment 4: Preparation of trehalose from a non-reducing carbohydrate having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more>
A non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more used as a substrate was prepared according to the method described in Japanese Patent Application No. 4-362131. That is, the production of a purified non-reducing carbohydrate obtained by the method of Experiment 2 in a 20% aqueous solution of a reducing starch partial degradation product selected from maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose Enzyme preparations were added at a rate of 2 units per gram of substrate solids and allowed to act at 40 ° C. and pH 7.0 for 48 hours, followed by heat deactivation, filtration, decolorization, desalting, concentration, Ion exchange column chromatography using an alkali metal type strongly acidic cation exchange resin (XT-1016, Na ⁺ type, cross-linking degree 4%, manufactured by Tokyo Organic Chemical Industry Co., Ltd.) was performed. The resin is packed into three jacketed stainless steel columns with an inner diameter of 2.0 cm and a length of 1 m, connected in series, and the reaction sugar solution is added to the resin at 5 v / v% while maintaining the column temperature at 55 ° C. This was fractionated by flowing warm water at 55 ° C. with SV0.13 to prepare a high-purity sample of a non-reducing saccharide having a trehalose structure at the end and a glucose polymerization degree of 3 or more. Among the obtained high purity samples, the purity of glucosyl trehalose in the glucosyl trehalose sample is 97.6%, the purity of maltosyl trehalose in the maltosyl trehalose sample is 98.6%, and maltotriosyl trehalose. The purity of maltotriosyl trehalose in the sample is 99.6%, the purity of maltotetraosyl trehalose in the sample is 98.3%, and the malto in the maltopentaosyl trehalose sample is The purity of pentaosyl trehalose was 98.1%.

  A 20% aqueous solution of the above five non-reducing carbohydrates (glycosyl trehalose) was prepared, and each of the purified trehalose-free enzymes obtained in Experiment 2 was added at a rate of 2 units per gram of substrate solids, and the temperature was 40 ° C., pH 7 The reaction product was analyzed by high performance liquid chromatography using a Wakobead WB-T-330 column (manufactured by Wako Pure Chemical Industries, Ltd.). As a control, purified trehalose-free enzyme was similarly applied to maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose, and analyzed by high performance liquid chromatography. The results are shown in Table 3.

As is clear from the results in Table 3,
1. Trehalose-free enzyme specifically hydrolyzes the bond between the trehalose part and glycosyl part of a non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more, and the degree of polymerization of trehalose and glucose is 1 or more. To produce reducing carbohydrates.
2. Maltooligosaccharides are not affected at all by trehalose-releasing enzyme.

  From these results, the trehalose-releasing enzyme of the present invention hydrolyzes the bond between the trehalose moiety of a non-reducing carbohydrate having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more and other glycosyl moieties very specifically. It is judged to be an enzyme with a completely new mechanism of action that decomposes and releases trehalose.

  Subsequently, in order to purify trehalose from each reaction product, decolorization, desalting and concentration were performed, and column fractionation using an alkali metal type strongly acidic cation exchange resin (XT-1016) was carried out to obtain a high trehalose content of 97% or more. Purity fractions were collected. The obtained high-purity fraction is concentrated to a concentration of about 65%, and left at 25 ° C. for 2 days to crystallize hydrous trehalose crystals, honey, vacuum-dried, and high-purity with a trehalose content of 99% or more A sample was prepared. The respective yields relative to the raw material substrate are 9.5% from glucosyl trehalose, 14.9% from maltosyl trehalose, 16.0% from maltotriosyl trehalose, and 18.5 from maltotetraosyl trehalose in terms of solids. %, 17.7% from maltopentaosyl trehalose. Using each of the obtained high-purity trehalose preparations, a commercially available reagent trehalose (sold by Wako Pure Chemical Industries, Ltd.) as a standard product, melting point, heat of fusion, specific rotation, infrared absorption spectrum, powder X-ray diffraction pattern As compared with the degradability of the product and the porcine kidney-derived trehalase (sold by Sigma), all the high-purity trehalose preparations prepared had a melting point of 97.0 ± 0.5 ° C. and a heat of fusion of 57.8 ± 1.2 KJ / Mole, specific rotation + 182 ± 1.1 °, which was in good agreement with the measured value of the reagent trehalose, and the infrared absorption spectrum and the powder X-ray diffraction pattern were also in good agreement with the spectrum or pattern of the reagent trehalose. Furthermore, the high purity trehalose preparation was decomposed into glucose by the porcine kidney-derived trehalase (sold by Sigma) in the same manner as the reagent trehalose. As is clear from the above results, it was confirmed that the sugar produced by the action of the trehalose-free enzyme of the present invention on a non-reducing sugar having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more was trehalose.

<Experiment 5: Preparation of trehalose from partially reduced starch degradation product>
5% waxy corn starch suspension was gelatinized by heating, then adjusted to pH 4.5 and temperature 50 ° C, and isoamylase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) was added at a rate of 4,000 units per gram starch. And allowed to react for 20 hours. The reaction solution was autoclaved (120 ° C., 10 minutes), then cooled to 60 ° C., and this was subjected to glucose polymerization by gel filtration chromatography (gel amount 750 ml) using Toyopearl HW-50S resin (manufactured by Tosoh Corporation). A reduced starch partial decomposition product having a degree of 35 to 10 was prepared.

  The resulting reduced starch partial degradation product or maltotriose having a glucose polymerization degree of 3 was adjusted to a concentration of 1% with 10 mM phosphate buffer (pH 7.0), and the purified non-purified product prepared by the method of Experiment 2 was used. A reducing sugar-producing enzyme preparation and a purified trehalose-free enzyme preparation are added at a rate of 4 units per substrate solid, respectively, and allowed to act at 40 ° C. for 24 hours. The reaction product was analyzed graphically.

  The remaining reaction solution was further adjusted to 50 ° C. and pH 4.5, glucoamylase (manufactured by Seikagaku Corporation) was added at a rate of 50 units per substrate solid, allowed to act for 24 hours, and desalted in the same manner. Then, the reaction product was analyzed by high performance liquid chromatography. The results are shown in Table 4.

  As shown in Table 4, the production rate of trehalose after the action of non-reducing saccharide-forming enzyme and trehalose-releasing enzyme was somewhat low at 4.2% for maltotriose with a glucose polymerization degree of 3. However, in the starch partial decomposition product having a glucose polymerization degree of 10 to 34.1, a high value of 66.1 to 80.8% was obtained. It has also been found that the higher the degree of glucose polymerization of the reduced starch partial decomposition product, the higher the trehalose purity obtained. Furthermore, glucoamylase is allowed to act on the reaction solution in which the both enzymes are allowed to act, and a non-reducing carbohydrate having a trehalose structure at the remaining terminal and having a glucose polymerization degree of 3 or more is decomposed into trehalose and glucose. It has also been found that the purity of trehalose increases.

<Experiment 6: Maillard reaction>
A solution containing 10% of the high-purity trehalose preparation (purity 99.5%) obtained by the method of Experiment 4 and 1% glycine and 50 mM phosphate buffer (pH 7.0) is kept at 100 ° C. for 90 minutes. After cooling, the absorbance of this solution at 480 nm and 1 cm cell was measured. As a control, glucose and maltose were used in the same manner, and the absorbance at 480 nm was measured. The results are shown in Table 5.

  As is clear from the results in Table 5, the trehalose preparation has a slight coloration due to the Maillard reaction and is only about 0.4 to 0.6% of the coloration degree of glucose or maltose. The product was found to be a carbohydrate showing little Maillard reaction. Therefore, even if this carbohydrate is mixed with an amino acid, it is a carbohydrate that hardly damages the amino acid.

<Experiment 7: In vivo use test>
In accordance with the method reported by Koji et al. In “Clinical Nutrition”, Vol. 41, No. 2, pages 200 to 208 (1972), a high-purity trehalose preparation obtained by the method of Experiment 4 ( (99.5% purity) 30 g of 20 w / v% aqueous solution, which was orally administered to 6 volunteers (healthy 26-year-old, 27-year-old, 28-year-old, 29-year-old, 30-year-old man, 31-year-old man) Blood was collected and blood glucose level and insulin level were measured. As a control, glucose was used. As a result, trehalose behaved in the same manner as glucose, and both the blood glucose level and the insulin level showed a maximum value after about 0.5 to 1 hour after administration. Trehalose has been found to be easily digested, absorbed, and metabolized to become an energy source.

<Experiment 8: Acute toxicity test>
Using mice, the high-purity trehalose preparation (purity 99.5%) obtained by the method of Experiment 4 was orally administered to conduct an acute toxicity test. As a result, trehalose is a low-toxic substance, and no death was observed even at the maximum dose. Therefore, although it cannot be said to be an accurate value, its LD50 value was 50 g / kg or more.

<Experiment 9: Production of trehalose-releasing enzyme from Arthrobacter sp. Q36>
The cells were cultured in a fermenter for about 72 hours in the same manner as in Experiment 1 except that Arthrobacter species Q36 (FERM BP-4316) was used instead of Rhizobium species M-11 (FERM BP-4130). The enzyme activity of the non-reducing saccharide-forming enzyme in the culture solution was about 1.3 units / ml, and the enzyme activity of the trehalose-free enzyme of the present invention was about 1.8 units / ml. The enzyme activity of the bacterial cell suspension and the culture supernatant was measured in the same manner as in Experiment 1. As a result, the enzymatic activity of the non-reducing saccharide-forming enzyme was about 0.5 units / ml in the bacterial cell suspension. The enzyme activity of trehalose-free enzyme is found to be about 0.5 unit / ml, and the culture supernatant has about 0.8 unit / ml of non-reducing saccharide-forming enzyme activity and trehalose-free enzyme activity. About 1.3 units / ml were observed.

<Experiment 10: Purification of enzyme>
Using about 18 l of the culture solution obtained by the method of Experiment 9, purification was performed in the same manner as in Experiment 2. The results of each purification step are summarized in Table 6 for non-reducing saccharide-forming enzymes and Table 7 for trehalose-free enzymes.

  Purity of the purified non-reducing saccharide-forming enzyme and purified trehalose-free enzyme obtained as gel filtration eluates in Table 6 and Table 7 was examined by electrophoresis in the same manner as in Experiment 2. The protein band was shown to be single, and both purified enzymes obtained were single samples with high purity by electrophoresis.

<Experiment 11: Properties of enzyme>
When the molecular weight of the purified trehalose-free enzyme obtained by the method of Experiment 10 was measured by SDS-polyacrylamide gel electrophoresis in the same manner as in Experiment 3, it was about 57,000 to 67,000 daltons. Further, when the isoelectric point of the enzyme was determined by isoelectric focusing as in the case of Experiment 3, the isoelectric point was 3.6 to 4.6. Further, the effects of temperature and pH on the enzyme activity, and the temperature stability and pH stability of the enzyme were determined in the same manner as in Experiment 3. As a result, the influence of temperature is shown in FIG. 6, the influence of pH is shown in FIG. 7, the temperature stability is shown in FIG. 8, and the pH stability is shown in FIG.

  As is apparent from the figure, the optimum temperature of the enzyme is around 45 ° C., and the optimum pH is about 6.0 to 7.5. The temperature stability is up to around 45 ° C., and the pH stability is about 5.0 to 10.0.

<Experiment 12: Preparation of trehalose from a non-reducing carbohydrate having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more>
Using the purified enzyme obtained by the method of Experiment 10, an experiment for the preparation of trehalose from a non-reducing carbohydrate having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more was performed according to the method of Experiment 4. Similar to the case of trehalose-releasing enzyme derived from Rhizobium sp. M-11, it was found that trehalose from a non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more was released.

<Experiment 13: Production and properties of trehalose-releasing enzyme from known microorganisms>
Among known microorganisms, Brevibacterium heroborum ATCC 11822 and Micrococcus roseus ATCC 186, which were confirmed to be capable of producing the trehalose-releasing enzyme of the present invention, were cultured at 27 ° C. for 72 hours in a fermenter as in Experiment 1. Using about 18 l of each culture solution, the culture solution is treated with a crushing apparatus in the same manner as in Experiment 2, and the centrifuged supernatant is recovered, followed by ammonium sulfate precipitation, dialysis, and ion exchange column chromatography. The properties of the partially purified enzyme preparation thus obtained were examined. These results are summarized in Table 8 together with the above-mentioned cases of Rhizobium species M-11 and Arthrobacter species Q36.

  In addition, using these partially purified enzymes derived from known microorganisms, an experiment for the preparation of trehalose from a non-reducing carbohydrate having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more was conducted according to the method of Experiment 12. As in the case of Trehalose-releasing enzyme derived from Rhizobium sp. M-11, it was found that trehalose is released from a non-reducing carbohydrate having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more.

<Experiment 14: Partial amino acid sequence of trehalose-releasing enzyme>
(1) N-terminal amino acid sequence One of the purified enzyme preparation derived from Rhizobium sp. M-11 obtained by the method of Experiment 2 and the purified enzyme preparation derived from Arthrobacter sp. Q36 obtained by the method of Experiment 10 After each part was dialyzed against distilled water, about 80 μg of protein 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 using a protein sequencer model 473A (manufactured by Applied Biosystems, USA). The obtained N-terminal sequences are shown in Table 9.

  As is apparent from Table 9, the N-terminal amino acid is alanine in the case of the enzyme derived from Rhizobium sp. M-11, and the subsequent amino acid sequence is lysine-proline-valine-glutamine-glycine-alanine-glycine-arginine-phenylalanine. It turned out to be. On the other hand, in the case of the enzyme derived from Arthrobacter sp. Q36, the N-terminal amino acid is threonine, and the subsequent amino acid sequence is found to be proline-threonine-tyrosine-proline-arginine-glutamic acid-arginine-alanine-lysine. did.

(2) Internal partial amino acid sequence One of the purified enzyme preparation derived from Rhizobium sp. M-11 obtained by the method of Experiment 2 and the purified enzyme preparation derived from Arthrobacter sp. Q36 obtained by the method of Experiment 10 Each portion was dialyzed against 10 mM Tris / HCl buffer (pH 9.0), and then diluted with the same buffer to a concentration of about 1 mg / ml. To each of these sample solutions (1 ml), 10 μg of lysyl endopeptidase (available from Wako Pure Chemical Industries, Ltd.) was added and reacted at 30 ° C. for 22 hours for peptide formation. Reverse phase HPLC was performed to isolate the resulting peptide. In the case of an enzyme derived from Rhizobium sp. M-11, a capsule pack C18 column (diameter: 4.6 mm × length: 250 mm, manufactured by Shiseido Co., Ltd.) is used. The test was carried out under a linear gradient condition of 60 minutes from a fluoroacetic acid-16 v / v% acetonitrile solution to a 0.1 v / v% trifluoroacetic acid-48 v / v% acetonitrile solution. In the case of the enzyme derived from Arthrobacter sp. Q36, a microbonder pack C18 column (diameter 2.1 mm × length 150 mm, manufactured by Waters, USA) was used, and the flow rate was 0.9 ml / min at room temperature, 0.1 v / v. The test was carried out under a linear gradient condition of 60% from 30% trifluoroacetic acid-30 v / v% acetonitrile solution to 0.1 v / v% trifluoroacetic acid-55 v / v% acetonitrile solution. The peptide eluted from the column was detected by measuring the absorbance at a wavelength of 210 nm. 3 peptides each well separated from other peptides [Rhizobium enzyme-derived peptide, RT41 (retention time approximately 41 minutes), RT46 (retention time approximately 46 minutes), RT54 (retention time approximately 54 minutes); Arthrobacter enzyme-derived peptide , AT7 (retention time of about 7 minutes), AT30 (retention time of about 30 minutes), AT48 (retention time of about 48 minutes)], and after vacuum drying, 200 μl of 0.1 v / v% trifluoro Dissolved in an acetic acid-50 v / v% acetonitrile solution. These peptide samples were subjected to a protein sequencer and analyzed for amino acid sequences up to 10 residues each. The obtained partial internal sequences are shown in Table 10.

As is clear from Table 10, the sequence of Rhizobium sp. M-11 enzyme peptide RT41 and the Arthrobacter sp. Q36 enzyme peptide A30 match 9 of the 10 residues analyzed, and peptide RT46 9 of the 10 residues analyzed for the peptide AT48 and the peptide AT48 matched, and 8 residues for the peptide RT54 and peptide AT7 matched. From this, it was judged that the internal partial amino acid sequence had high homology between the enzyme derived from the microorganism belonging to the genus Rhizobium and the enzyme derived from the microorganism belonging to the genus Arthrobacter. Asparagine-tryptophan-alanine-glutamic acid-alanine-X ₁ -X ₂ -glycine-aspartic acid (where X ₁ means serine or alanine, X ₂ means alanine or glutamic acid), and aspartic acid -Glutamic acid-arginine-alanine-valine-histidine-isoleucine-leucine-glutamic acid-X ₃ (wherein X ₃ means glutamic acid or aspartic acid), and further X ₄ -glycine-glutamic acid-glycine-asparagine-threonine- Tryptov Down - glycine - asparagine - serine (where, X ₄ means histidine or glutamine.) Can be expressed as.

  Hereinafter, the production method of the trehalose-releasing enzyme of the present invention and the production method of trehalose and a saccharide containing the same using Examples 1 to 8 will be described, and the composition containing trehalose and the saccharide containing the same will be described as Examples. 9 to 31 are shown.

  Rhizobium sp. M-11 (FERM BP-4130) was cultured in a fermenter for about 80 hours according to the method of Experiment 1. After culturing, sterilization filtration is performed using an SF membrane, and about 18 liters of the culture filtrate is collected, and the filtrate is further concentrated in a UF membrane to give a non-reducing carbohydrate-forming enzyme (17.2 units / ml). And about 1 liter of concentrated enzyme solution containing trehalose-free enzyme (20.8 units / ml) was recovered.

After adding calcium carbonate to 15% corn starch milk to a final concentration of 0.1% by weight, the pH is adjusted to 6.0, and α-amylase (manufactured by Novo, trade name Termamil 60L) is added per gram of starch. The mixture was added to 0.2% by weight and reacted at 95 ° C. for 15 minutes. The reaction solution was autoclaved (2 kg / cm ² ) 30
After cooling to 45 ° C., pullulanase (produced by Hayashibara Biochemical Laboratories Co., Ltd.), 1,000 units per gram starch, non-reducing saccharide-forming enzyme and trehalose-free enzyme prepared by the above method The concentrated solution containing was added at a rate of 0.2 ml per gram starch and allowed to react for 48 hours. The reaction solution is kept at 95 ° C. for 10 minutes, then cooled and filtered, and the obtained filtrate is decolorized with activated carbon and purified by desalting with H-type and OH-type ion exchange resins according to a conventional method. Further concentration gave syrup having a concentration of 60% at about 92% per solid.

  This product contains 70.2% trehalose, 2.4% glucosyl trehalose, 3.3% maltosyl trehalose, 0.7% glucose, 10.1% maltose and 12.1% maltotriose per solid. It contains 9% and 0.4% malto-oligosaccharide or higher malto-oligosaccharide, and has a mellow and elegant sweetness, low reducibility, low viscosity, moderate moisture retention, sweetener, taste improver, quality As an improver, stabilizer, excipient, etc., it can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals.

In order to increase the trehalose content by using the sugar solution obtained by the method of Example 1 as a raw sugar solution, an alkali metal type strongly acidic cation exchange resin (XT-1016, Na ⁺ type,
Ion exchange column chromatography using a degree of cross-linking of 4%, manufactured by Tokyo Organic Chemical Industry Co., Ltd.). The resin was packed into four jacketed stainless steel columns with an inner diameter of 5.4 cm and connected in series to a total resin layer length of 20 m. While maintaining the temperature in the column at 55 ° C., a sugar solution is added to the resin at 5 v / v%, and 55 ° C. warm water is flowed at SV0.13 to perform fractionation, and maltose such as maltose and maltotriose. Were removed, and a fraction containing trehalose was collected. Further purification, concentration, vacuum drying, and pulverization gave a trehalose-rich powder with a yield of about 56% per solid. This product contains about 97% trehalose, has extremely low reducibility, mellow and elegant sweetness, and as a sweetener, taste improver, quality improver, stabilizer, excipient, etc. It can be advantageously used in various compositions such as products, cosmetics, and pharmaceuticals.

The trehalose-rich fraction obtained by the method of Example 2 was concentrated to a concentration of about 70% by decolorizing with activated carbon and desalting with an ion exchange resin according to a conventional method. About 2% of a trehalose hydrate crystal was added as a seed crystal and cooled slowly to obtain a mass kit having a crystallization rate of about 45%. This mass kit was sprayed at a high pressure of 150 kg / cm ² from a nozzle on a drying tower. At the same time, hot air of 85 ° C. is blown from the top of the drying tower, the crystal powder is collected on a transfer wire mesh conveyor provided at the bottom, and hot air of 45 ° C. is sent from the bottom of the conveyor while the powder is dried. It was gradually moved out and removed. The crystal powder is filled in an aging tower and aged for 10 hours while sending warm air, crystallization and drying are completed, and the trehalose-containing crystal powder is about 90 per solid with respect to the raw trehalose-rich sugar solution. % Yield.

  This product does not substantially exhibit hygroscopicity and is easy to handle. As sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc., various compositions such as various foods, cosmetics, pharmaceuticals, etc. It can be used to advantage.

  The trehalose-rich fraction obtained by the method of Example 2 was purified in the same manner as in Example 3, then taken into an evaporation kettle and boiled under reduced pressure to obtain syrup having a water content of about 3.0%. Next, it was transferred to an auxiliary crystallizer, to which 1% of anhydrous crystalline trehalose was added as a seed crystal per syrup solid, stirred at 120 ° C for 5 minutes, then taken out into an aluminum vat and crystallized at 100 ° C for 6 hours. A block was prepared by aging.

  Next, this block is pulverized with a cutting machine and fluidly dried to obtain an anhydrous crystalline trehalose powder having a moisture content of 0.3% in a yield of about 85% per solid relative to the raw trehalose-rich sugar solution. It was. This product is not only used as a dehydrating agent for water-containing products such as food, cosmetics, pharmaceuticals, raw materials, or processed intermediates, but also as a white powder sweetener with elegant sweetness. It can be advantageously used in the composition.

  According to the method of Example 1, a mutant strain of Rhizobium sp. M-11 (FERM BP-4130) was cultured in a fermenter for about 70 hours. After culturing, sterilization filtration is performed using an SF membrane, and about 100 liters of the culture liquid is collected. Further, the liquid is concentrated in a UF membrane to obtain a non-reducing carbohydrate-forming enzyme (about 410 units / ml). About 5 liters of concentrated enzyme solution containing trehalose-free enzyme (about 490 units / ml) was recovered.

  6% potato starch milk is gelatinized by heating, then adjusted to pH 4.5, temperature 50 ° C, and isoamylase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) to 500 units per gram starch. In addition, the reaction was allowed to proceed for 20 hours. The reaction solution was adjusted to pH 6.5, autoclaved (120 ° C.) for 10 minutes, then cooled to 95 ° C., and α-amylase (manufactured by Novo, trade name Termamil 60 L) was added to the starch to 0.1% per gram of starch. The mixture was added at a rate of% by weight and reacted for 15 minutes. The reaction solution was autoclaved (130 ° C.) for 30 minutes, then cooled to 45 ° C., and a concentrated solution containing the non-reducing saccharide-forming enzyme and trehalose-free enzyme prepared by the above method was added per gram of starch. It added so that it might become a ratio of 0.01 ml, and it was made to react for 64 hours. After maintaining the reaction solution at 95 ° C. for 10 minutes, the reaction solution is adjusted to 50 ° C. and pH 5.0, and 10 units of glucoamylase (manufactured by Nagase Seikagaku Corporation, trade name Glucozyme) is added per gram starch for 40 hours. And then heated to inactivate the enzyme. This solution was decolorized with activated carbon according to a conventional method, desalted with an ion exchange resin, and concentrated to a concentration of about 60%. The sugar solution contained 80.5% trehalose per solid. After concentrating the solution to a concentration of about 84%, take it to an auxiliary crystal machine, add about 2% of trehalose-containing crystals as seed crystals, stir the auxiliary crystal, then take it out into a plastic vat and leave it at room temperature for 3 days for crystallization. A block was prepared by aging. Next, this block was pulverized with a cutting machine to obtain a trehalose hydrous crystal powder in a yield of about 90% per solid with respect to the raw starch.

  This product does not substantially exhibit hygroscopicity and is easy to handle. As sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc., various compositions such as various foods, cosmetics, pharmaceuticals, etc. It can be used to advantage.

  Arthrobacter sp. Q36 (FERM BP-4316) was cultured in a fermenter for about 72 hours according to the method of Experiment 9. The culture solution was sterilized by centrifugation (10,000 rpm, 30 minutes), then concentrated on a UF membrane, and non-reducing carbohydrate-forming enzyme (16.3 units / ml) and trehalose-free enzyme (25.1 units). / Ml) was recovered.

To 1 part by weight of potato starch, 6 parts by weight of water and 0.01 part by weight of α-amylase (manufactured by Nagase Seikagaku Co., Ltd., trade name Neospitase) are added and mixed by stirring. The pH of this suspension is 6.2. The starch was gelatinized and liquefied after being adjusted to 85 to 90 ° C, and the liquefied solution was heated at 120 ° C for 10 minutes to inactivate α-amylase, and then cooled to 45 ° C. Of isoamylase (produced by Hayashibara Biochemical Laboratories) at 500 units per gram of starch, and 0.2 ml per gram of concentrated solution containing non-reducing saccharide-forming enzyme and trehalose-free enzyme prepared by the above method. The reaction was continued for 48 hours. The reaction solution was heated at 95 ° C. for 10 minutes to inactivate the enzyme, then adjusted to 50 ° C. and pH 5.0, and glucoamylase (manufactured by Nagase Seikagaku Corporation, trade name Glucoteam) per gram of starch. Ten units were added and allowed to react for 40 hours, then heated to inactivate the enzyme. This solution was decolorized with activated carbon according to a conventional method, desalted with an ion exchange resin, and concentrated to a concentration of about 60%. The sugar solution contained 78.3% trehalose per solid. Except for using an alkali metal type strongly acidic cation exchange resin (Sold by Organo Co., Ltd., trade name: CG6000, Na ⁺ type) as the ion exchange resin, ion exchange column chromatography was performed according to the method of Example 2, and high trehalose content was obtained. Fractions were collected. This high-content liquid contained about 95% trehalose per solid. After concentrating this solution to a concentration of 75%, add it to an auxiliary crystallizer, add about 2% of trehalose-containing crystals as seed crystals, stir the auxiliary crystal, then take it out into a plastic vat and leave it at room temperature for 3 days to crystallize and mature. To prepare a block. Next, the block was pulverized with a cutting machine to obtain a trehalose hydrous crystal powder in a yield of about 70% per solid with respect to the raw material starch.

  This product does not substantially exhibit hygroscopicity and is easy to handle. As sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc., various compositions such as various foods, cosmetics, pharmaceuticals, etc. It can be used to advantage.

  Brevibacterium heroborum ATCC 11822 was cultured for about 72 hours with a fermenter according to the method of Experiment 13, the culture solution was treated with a crushing device, and the treatment solution was centrifuged (10,000 rpm, 30 minutes) to obtain a residue. Then, the solution was concentrated with a UF membrane, and about 700 ml of a concentrate containing non-reducing saccharide-forming enzyme (about 8 units / ml) and trehalose-free enzyme (about 12 units / ml) was recovered.

After adding calcium carbonate to 33% tapioca starch milk to a final concentration of 0.1%, the pH was adjusted to 6.0, and α-amylase (manufactured by Novo, trade name Termamir 60L) was added to this per gram of starch. The mixture was added to 3% and reacted at 95 ° C. for 20 minutes. The reaction solution was autoclaved (2 kg / cm ² ) for 30 minutes, then cooled to 40 ° C., and isoamylase (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) was prepared by the above method at 200 units per gram starch. A concentrated solution containing a non-reducing saccharide-forming enzyme and trehalose-free enzyme was added at a rate of 0.2 ml per gram of starch and allowed to react for 48 hours. The reaction solution is kept at 95 ° C. for 10 minutes, then cooled and filtered, and the obtained filtrate is decolorized with activated carbon and purified by desalting with H-type and OH-type ion exchange resins according to a conventional method. Further concentration gave 60% concentration of syrup at about 90% solids.

  This product contains 60.1% trehalose, 1.4% glucosyl trehalose, 1.5% maltosyl trehalose, 1.0% glucose, 6.5% maltose, and 8. 8% maltotriose per solid. 3% and 21.2% malto-oligosaccharide of maltotetraose, mellow and elegant sweetness, low reducibility, moderate moisturizing, sweetener, taste improver, quality improver, As stabilizers and excipients, it can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals.

  The trehalose-rich solution having a trehalose content of about 95% obtained by the method of Example 6 was decolorized and desalted according to a conventional method. After concentrating this solution to a concentration of about 75%, take it into an auxiliary crystal can and add about 2% of water-containing trehalose crystals as seed crystals to 50 ° C. The honey was separated by a separator, and the crystals were sprayed with a small amount of water and washed to obtain high-purity trehalose hydrous crystals having a purity of 99% or more in a yield of about 50%.

<Sweetener>
To 1 part by mass of the water-containing trehalose crystal powder obtained by the method of Example 5, 0.01 part by mass of α-glycosyl stevioside (trade name αG sweet) and L-aspartyl-L-phenylalanine methyl ester (product) Nominal aspartame) 0.01 parts by mass was uniformly mixed and subjected to a granulating machine to obtain a granular sweetener. This product has excellent sweetness quality and has a sweetness level about 2.5 times that of sucrose, and the calorie per sweetness level is reduced to about 1 / 2.5 of sucrose.

  This sweetener has no degradation of high-intensity sweetener blended in it, has excellent stability, and as a low-calorie sweetener, low-calorie food and drink for obese people, diabetics, etc. who restrict caloric intake Suitable for sweetening products.

  In addition, the present sweetener is suitable for sweetening foods and the like that suppress dental caries because it produces less acid by caries-inducing bacteria and produces less insoluble glucan.

<Hard candy>
30 parts by mass of trehalose-containing syrup obtained by the method of Example 7 was heated and mixed with 100 parts by mass of a 55% sucrose solution, and then heated and concentrated under reduced pressure until the water content was less than 2%, to which 1 part by mass of citric acid was added. A proper amount of lemon flavor and colorant were mixed and molded according to a conventional method to obtain a product.

  This product is a high quality hard candy that is crisp, tastes good and does not cause sucrose crystallization.

<Chewing gum>
Heat and melt 3 parts by weight of the gum base, add 4 parts by weight of sucrose and 3 parts by weight of water-containing trehalose crystals obtained by the method of Example 3, and mix an appropriate amount of fragrance and colorant. According to a conventional method, the product was obtained by kneading with a roll, molding and packaging.

  This product is a chewing gum with good texture and flavor.

<Sweetened condensed milk>
3 parts by mass of trehalose-containing syrup and 1 part by mass of sucrose obtained by the method of Example 1 are dissolved in 100 parts by mass of raw milk, sterilized by heating with a plate heater, then concentrated to a concentration of 70%, and canned in aseptic conditions. Got the product.

  This product has a mild sweet taste and good flavor, and can be advantageously used for seasoning infant foods, fruits, coffee, cocoa, tea, and the like.

<Lactic acid bacteria beverage>
175 parts by weight of skim milk powder, 130 parts by weight of trehalus-containing syrup obtained by the method of Example 1 and 50 parts by weight of high lactosucrose-containing powder disclosed in JP-A-4-281955 are dissolved in 1,150 parts by weight of water. Then, after sterilizing at 65 ° C. for 30 minutes and cooling to 40 ° C., 30 parts by mass of a starter of lactic acid bacteria was inoculated in accordance with a conventional method, and cultured at 37 ° C. for 8 hours to obtain a lactic acid bacteria beverage.

  This product is a lactic acid bacteria beverage with good flavor. In addition, this product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacteria growth promoting action.

<Powder juice>
50 parts by mass of the trehalose high content powder obtained by the method of Example 2, 10 parts by mass of sucrose, 0.65 parts by mass of anhydrous citric acid, 0.1 parts of malic acid with respect to 33 parts by mass of the orange juice powder produced by spray drying. Mass part, 0.1 part by weight of L-ascorbic acid, 0.1 part by weight of sodium citrate, 0.5 part by weight of pullulan, and appropriate amount of powdered fragrance are mixed and stirred, pulverized into a fine powder, and fluidized bed granulated. The product was charged into a machine, the exhaust air temperature was set to 40 ° C., and the trehalose-containing syrup obtained by the method of Example 1 was sprayed thereon 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 off-flavors and off-flavors and was stable for a long time.

<Custard cream>
100 parts by mass of corn starch, 100 parts by mass of trehalose-containing syrup obtained by the method of Example 7, 80 parts by mass of maltose, 20 parts by mass of sucrose and 1 part by mass of sodium chloride, and 280 parts by mass of chicken egg were added and stirred. Gradually add 1,000 parts by weight of boiled milk to this, and continue to stir it over fire. When cornstarch is completely gelatinized and the whole becomes translucent, stop the fire and cool it down to the appropriate amount. Of vanilla flavoring, weighed, filled and packed to obtain a product.

  This product has a smooth luster, mild sweetness and deliciousness.

<Uiro no Moto>
Rice bran was produced by uniformly mixing 90 parts by mass of rice flour with 20 parts by mass of corn starch, 40 parts by mass of sucrose, 80 parts by mass of the hydrous crystal powder containing trehalose obtained by the method of Example A3, and 4 parts by mass of pullulan. Ui-no-Moto, an appropriate amount of Matcha and water were kneaded, and the mixture was placed in a container and steamed for 60 minutes to produce Matcha Uiro.

  This product has good shine, mouthfeel, and good flavor. Moreover, the aging of starch is suppressed and the shelf life is good.

<An>
In accordance with a conventional method, water was added to 10 parts by mass of raw material azuki and boiled, astringent and extracted, and water-soluble impurities were removed to obtain about 21 parts by mass of azuki bean paste. Add 14 parts by weight of sucrose, 5 parts by weight of trehalose-containing syrup obtained by the method of Example 1 and 4 parts by weight of water to this raw sauce, boil it, and add a small amount of salad oil to this so as not to break the crumbs. After kneading, about 35 parts by mass of the product was obtained.

  This product has no color burn, has a good texture, has a good flavor, and is suitable as an ingredient for anpan, bun, dumpling, peach and ice confectionery.

<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-containing powder obtained by the method of Example 3 and 0.1 part by weight of inorganic food are kneaded with water according to a conventional method, Fermented at 26 ° C. for 2 hours, then aged for 30 minutes and baked.

  This product is a high quality bread with good hue and sudoku, moderate elasticity and mild sweetness.

<Ham>
A pork thigh of 1,000 parts by mass is uniformly rubbed with 15 parts by mass of sodium chloride and 3 parts by mass of potassium nitrate, and deposited in a cold room for a whole day and night. This was dipped in a salted solution consisting of 500 parts by mass of water, 100 parts by mass of sodium chloride, 3 parts by mass of potassium nitrate, 40 parts by mass of the water-containing trehalose crystal powder obtained by the method of Example 6 and spices in a cold room for 7 days. And washed with cold water, wrapped with string, smoked, cooked, cooled and packaged to obtain the product.

  This product is a high quality ham with good color and good flavor.

<Powder peptide>
1 part by weight of 40% food-grade soybean peptide solution (manufactured by Fuji Oil Co., Ltd., trade name High Newt S) is mixed with 2 parts by weight of the trehalose hydrous crystal powder obtained by the method of Example 6 and placed in a plastic vat. , Dried under reduced pressure at 50 ° C., and pulverized to obtain a powdered peptide.

  This product has a good flavor and can be advantageously used not only as a confectionery material such as premix and frozen confectionery but also as a baby food such as oral liquid food and tube liquid food, and a therapeutic nutrient.

<Powder miso>
3 parts by mass of crystalline anhydrous trehalose powder obtained by the method of Example 4 is mixed with 1 part by mass of red miso and poured into a metal plate provided with a number of hemispherical recesses, which is left to stand overnight at room temperature to solidify. Then, the mold was released to obtain about 4 g of solid miso per piece, which was pulverized to obtain a powder miso.

  This product can be advantageously used as a seasoning for instant ramen and instant soup.

  The solid miso can be used not only as a solid seasoning but also as a miso confectionery.

<Powdered egg yolk>
Egg yolk prepared from raw eggs is sterilized with a plate-type heat sterilizer at 60 to 64 ° C., and 1 part by mass of the liquid egg yolk obtained is 4 parts by mass of anhydrous crystalline trehalose powder obtained by the method of Example 4. After mixing, the mixture was transferred to a vat and allowed to stand overnight to convert into trehalose hydrous crystals to prepare a block. This block was pulverized with a cutting machine to obtain powdered egg yolk.

  This product can be advantageously used not only as a confectionery material such as premix, frozen confectionery, and emulsifier, but also as a baby food such as oral liquid food and tube liquid food, and a therapeutic nutrient. Moreover, it can be advantageously used as a skin beautifying agent, hair restorer and the like.

<Cosmetic cream>
2 parts by mass of polyoxyethylene glycol monostearate, 5 parts by mass of self-emulsifying glyceryl monostearate, 2 parts by mass of trehalose-rich powder obtained by the method of Example 2, 1 part by mass of α-glycosyl rutin, 1 mass of liquid paraffin Parts, 10 parts by mass of glycerin trioctanoate and an appropriate amount of preservative are dissolved by heating in accordance with a conventional method. To this, 2 parts by mass of L-lactic acid, 5 parts by mass of 1,3-butylene glycol and 66 parts by mass of purified water are added, and then applied to a homogenizer. After emulsification, an appropriate amount of a fragrance was added and mixed with stirring to produce a cream.

  This product has antioxidant properties, high stability, and can be advantageously used as a high-quality sunscreen, skin beautifying agent, whitening agent, and the like.

<Powdered ginseng extract>
After mixing 1.5 parts by mass of anhydrous crystalline trehalose powder obtained by the method of Example 4 with 0.5 parts by mass of medicinal carrot extract, the mixture was transferred to a vat and left to stand for 2 days to convert to trehalose hydrous crystals to prepare a block. . The block was pulverized with a cutting machine and classified to obtain a powdered ginseng extract.

  This product was applied to a granule molding machine together with appropriate amounts of vitamin B1 and vitamin B2 powder to obtain a vitamin-containing granular ginseng extract.

  This product can be advantageously used as a fatigue recovery agent, tonic, toughening agent and the like. It can also be used as a hair restorer.

<Solid formulation>
Human natural interferon-α preparation (manufactured by Hayashibara Biochemical Laboratories Co., Ltd., sold by Cosmo Bio Co., Ltd.) is applied to an immobilized anti-human interferon-α antibody column according to a conventional method, and human natural contained in the preparation Type interferon-α is adsorbed, the bovine serum albumin as a stabilizer is passed through and removed, and then the pH is changed to obtain a human trehalose-rich powder obtained by the method of Example 2 by using human interferon-α. Elution was performed using 5% physiological saline. This solution is microfiltered, added to about 20 times the amount of anhydrous crystalline maltose powder (Hayashibara Shoji Co., Ltd., trade name: Finetose), dehydrated and powdered, and tableted with a tableting machine. Tablets containing about 150 units of human natural interferon-α per about 200 mg) were obtained.

  This product can be used advantageously as a sublingual tablet for the treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors, etc. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., whose number of patients is rapidly increasing 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.

<Sugar-coated tablets>
An uncoated tablet having a weight of 150 mg is used as a core agent, and 40 parts by mass of trehalose-containing crystal powder obtained by the method of Example 3, 2 parts by mass of pullulan (average molecular weight 200,000), 30 parts by mass of water, 25 parts by mass of talc, and oxidation Sugar coating is carried out using an undercoat liquid consisting of 3 parts by mass of titanium until the tablet weight reaches about 230 mg, and then an overcoat liquid consisting of 65 parts by mass of the same trehalose-containing hydrated crystal powder, 1 part by mass of pullulan and 34 parts by mass of water is used. Then, the sugar-coated tablet was further coated with a wax solution to obtain a sugar-coated tablet with an excellent glossy appearance.

  This product has excellent impact resistance and maintains high quality for a long time.

<Toothpaste>
Formulation Dicalcium phosphate 45.0%
Pullulan 2.95%
Sodium lauryl sulfate 1.5%
Glycerin 20.0%
Polyoxyethylene sorbitan laurate 0.5%
Preservative 0.05%
Trehalose hydrous crystal powder obtained by the method of Example 3 12.0%
Maltitol 5.0%
Water 13.0%
The above materials were mixed according to a conventional method to obtain a toothpaste.

  This product has moderate sweetness and is particularly suitable as a toothpaste for children.

<Food edible solid formulation>
Trehalose hydrous crystal powder produced by the method of Example 6 500 parts by weight, powdered egg yolk 270 parts by weight, skim milk powder 209 parts by weight, sodium chloride 4.4 parts by weight, potassium chloride 1.8 parts by weight, magnesium sulfate 4 parts by weight, A blend comprising 0.01 parts by weight of thiamine, 0.1 parts by weight of sodium ascorbate, 0.6 parts by weight of vitamin E acetate and 0.04 parts by weight of nicotinamide is prepared. The sachet was filled and heat sealed to obtain a product.

  This product is dissolved in about 150 to 300 ml of water to make a liquid food, and it is used orally or by tube use to the nasal cavity, stomach, intestine, etc., and is advantageous for supplementing energy to the living body. Available to:

<Infusion>
The high-purity trehalose-containing crystal produced by the method of Example 8 is dissolved in water to a concentration of about 10 w / v%, then subjected to microfiltration according to a conventional method to make pyrogen-free, and aseptically filled into a plastic bottle and plugged And got the product.

  This product is a stable infusion that does not change over time and is suitable for intravenous, intraperitoneal and other administration. This product is isotonic with blood at a concentration of 10 w / v%, and can be replenished with energy at twice the concentration of glucose.

<Infusion>
The high-purity trehalose hydrated crystals produced by the method of Example 8 and the amino acid compound having the following composition were mixed and dissolved in water so that the concentrations were 5 w / v% and 30 w / v%, respectively, and then the same as in Example 10. The product was purified to be pyrogen-free, and further filled into a plastic bag and plugged to obtain a product.

Composition of amino acid compound (mg / 100ml)
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 with no changes over time because trehalose does not exhibit reducing properties despite the complex infusion of carbohydrates and amino acids, and is suitable for administration intravenously, intraperitoneally, etc. It is. This product can be advantageously used not only for energy supply to the living body but also for amino acid supply.

<Treatment for trauma>
50 parts by mass of methanol in which 3 parts by mass of iodine was added and mixed with 200 parts by mass of the trehalose-rich powder produced by the method of Example 2 and 300 parts by mass of maltose, and further 200 parts by mass of a 10 w / v% pullulan aqueous solution were added. To obtain a plaster for the treatment of trauma showing moderate elongation and adhesion.

  This product not only acts as a bactericidal action by iodine, but also acts as an energy replenisher to cells by trehalose, shortening the healing period and healing the wound surface cleanly.

  As is apparent from the above, the novel trehalose-releasing enzyme of the present invention releases trehalose from a non-reducing saccharide having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more, and is also non-reducing to the reduced starch partial degradation product. Trehalose is produced in high yield by acting together with a reducing saccharide-forming enzyme. The trehalose can be easily separated and purified, and the trehalose thus obtained and the saccharides containing the trehalose are excellent in stability, and have a high quality and elegant sweetness. Moreover, it is digested and absorbed by ingestion and becomes a calorie source. It is also used parenterally as an infusion and is often metabolized. Trehalose and carbohydrates containing it can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals as sweeteners, taste improvers, quality improvers, stabilizers, excipients, and the like.

  Therefore, the establishment of the present invention is an industrially large amount of trehalose and a saccharide containing the same that have not been easily obtained as desired from a partially decomposed starch derived from starch, which is an inexpensive and endless resource. It will open up a whole new way to supply cheaply, and the impact of this will not be limited to academic fields such as starch science, enzyme science, biochemistry, etc., but industry, especially food, cosmetics and pharmaceutical fields Of course, it extends to the agricultural, aquatic and livestock industries and the chemical industry, and the industrial significance given to these industries is immeasurable.

Claims (4)

It has an action of specifically hydrolyzing the bond between the trehalose part of a non-reducing carbohydrate having a trehalose structure at the terminal and a degree of glucose polymerization of 3 or more and the other glycosyl part,
(1) leucine - aspartic acid - tryptophan - alanine - glutamic acid - alanine _-X 1 -X ₂ - glycine - aspartic acid (where, _{X 1} is means serine or alanine, _{X 2} means alanine or glutamic acid)
(2) Aspartic acid-glutamic acid-arginine-alanine-valine-histidine-isoleucine-leucine-glutamic acid-X ₃ (where X ₃ means glutamic acid or aspartic acid)
(3) X ₄ -glycine-glutamic acid-glycine-asparagine-threonine-tryptophan-glycine-aspartic acid-serine (where X ₄ means histidine or glutamine)
A method for measuring the activity of trehalose-free enzyme having a partial amino acid sequence selected from 1 and 2 or more and having a molecular weight of about 57,000 to 68,000 daltons in SDS-gel electrophoresis. An aqueous solution containing a non-reducing saccharide having a trehalose structure at the terminal as a substrate solution and having a glucose polymerization degree of 3 or more is used as a substrate solution, and trehalose releasing enzyme is allowed to act on this to release trehalose from the terminal of the non-reducing saccharide. A method for measuring the activity of trehalose-free enzyme, which comprises measuring the amount of reducing carbohydrate produced by the method. The trehalose releasing enzyme is derived from a microorganism selected from the genus Rhizobium, Arthrobacter, Brevibacterium and Micrococcus, and is an enzyme having the following physicochemical properties: Method for measuring the activity of trehalose-free enzyme :
(1) Action Specifically hydrolyzes the bond between the trehalose moiety of a non-reducing carbohydrate having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more and the other glycosyl moiety ;
(2) Molecular weight About 57,000 to 68,000 daltons by SDS-gel electrophoresis ;
(3) Isoelectric point pI of about 3.3 to 4.6 by an ampholine-containing electrophoresis method ;
(4) Optimal temperature pH 7.0, reaction for 30 minutes, around 35 to 45 ° C . ;
(5) Optimum pH
PH of about 6.0 to 7.5 after reaction at 40 ° C. for 30 minutes ;
(6) Temperature stability pH 7.0, holding for 60 minutes, stable to around 30 to 45 ° C ;
(7) pH stability The pH is about 5.0 to 10.0 at 25 ° C. for 16 hours.   A non-reducing saccharide having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more is selected from glucosyl trehalose, maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose or The method for measuring the activity of trehalose-free enzyme according to claim 1 or 2, wherein there are two or more kinds.   The method for measuring the activity of trehalose-free enzyme according to any one of claims 1 to 3, wherein the amount of reducing sugar produced by measuring the reducing power of the enzyme reaction solution is measured.

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