JP2002255988A - Sugar mixture, method for producing the same and use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sugar mixture containing a cyclic tetrasaccharide and a sugar alcohol, to establish a method for producing the same and to provide a use of the sugar mixture. SOLUTION: This sugar mixture containing a cyclic tetrasaccharide and a sugar alcohol is obtained without having a bad influence upon the cyclic tetrasaccharide being a nonreducing saccharide by hydrogenating a cyclic tetrasaccharide together with a reducing saccharide-containing saccharide. This method for sugar mixture is provided. This use for the sugar mixture is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a saccharide mixture, a method for producing the same, and a use thereof. More specifically, the present invention relates to cyclo (｛)-6-α-D-glucopyranosyl- (1 → 3) -α.
-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 3) -α-D-glucopyranosyl-
A saccharide mixture comprising a saccharide having a structure of 1 →｝ (the saccharide may be hereinafter referred to as a cyclic tetrasaccharide after the structure) and a saccharide alcohol. And its manufacturing method and use.

[0002]

2. Description of the Related Art In recent years, a new tetrasaccharide has been reported as a non-reducing saccharide having glucose as a constituent sugar. That is, "European Journal of Bioc"
Chemistry) ", Vol. 226, 641-648.
Page (1994) states that the enzyme hydrolyze alternanase mainly acts on alternan, in which four glucose residues are alternately linked by α-1,3 bonds and α-1,6 bonds. Then, cyclo 、 → 6) -α-D-glucopyranosyl- (1 → 3) -α-D-glucopyranosyl- (1
→ 6) -α-D-glucopyranosyl- (1 → 3) -α-
It has been shown that a cyclic tetrasaccharide having a structure of D-glucopyranosyl- (1 →｝) is produced and crystallized in the presence of methanol which is a kind of organic solvent.

[0003] Since a cyclic tetrasaccharide has a cyclic structure,
It is expected that it has the ability to include various compounds, and because it is non-reducing, does not cause an aminocarbonyl reaction and can be processed and used in various applications without fear of browning and deterioration. However, it is difficult to obtain alternan as a raw material for producing a cyclic tetrasaccharide and alternanase, which is an enzyme necessary for producing a cyclic tetrasaccharide, and microorganisms producing them have not been readily available.

[0004] Under such circumstances, the present inventors have previously filed Japanese Patent Application No.
As disclosed in the specification of US Pat. No. 000-229557, α-isomaltosyltransferase, a novel enzyme for producing a cyclic tetrasaccharide from α-isomaltosyl glucosaccharide such as panose, was discovered, and this novel enzyme was utilized. Thus, a method for producing a cyclic tetrasaccharide from α-isomaltosyl glucosaccharide such as panose produced from a starch raw material was established. Further, as disclosed in Japanese Patent Application No. 2000-234937, the present inventors have made a starch raw material act by combining an α-isomaltosyl glucosidase and the α-isomaltosyltransferase. Thus, a method for efficiently producing a cyclic tetrasaccharide directly from an inexpensive starch raw material was established.

However, in the above-described method for producing a cyclic tetrasaccharide, since a starch raw material is used, a saccharide containing a reducing tetrasaccharide such as glucose, maltose, isomaltose, and panose together with the cyclic tetrasaccharide is obtained. . The produced saccharide containing a cyclic tetrasaccharide exhibits reducibility because of the coexisting reducing saccharide, even if the cyclic tetrasaccharide itself is non-reducing. Therefore, in order to make a product containing a cyclic tetrasaccharide non-reducing, it is necessary to remove the reducing saccharide through a number of purification steps to obtain a high-purity cyclic tetrasaccharide product.

[0006] Starch partially decomposed products produced from starch, such as starch liquefied products, various dextrins, various maltooligosaccharides, and the like, usually have a reducing group at the terminal of the molecule and have a reducing saccharide (hereinafter referred to as "saccharide"). In the present specification, the present reducing saccharide is sometimes referred to as reducing starch saccharide.). In general, reducing starch sugars reduce the magnitude of reducing power per solid substance by dextrose equivalent (D
exquisition (exhibit Equivalent, DE). A substance having a large value is, for example, when used for processed foods and the like, usually has a small molecule, a low viscosity and a strong sweetness, but has a high reactivity and a substance having an amino group such as an amino acid or a protein. It is known that it is liable to cause browning, to generate a bad smell, and to easily deteriorate the quality.

[0007]

SUMMARY OF THE INVENTION The present invention is intended to solve the drawback of a saccharide comprising a cyclic tetrasaccharide and a reducing saccharide, and comprises a sugar alcohol in addition to the cyclic tetrasaccharide. It is intended to establish a saccharide mixture having reduced reducibility and a method for producing the same, and to provide a use of the saccharide mixture having reduced reducibility.

[0008]

Means for Solving the Problems In order to solve the above problems, the present inventors have focused on a method for reducing the reducing ability of a saccharide containing a reducing saccharide together with a cyclic tetrasaccharide, and have conducted various studies. I have continued. As a result, by reducing the reducing ability by hydrogenating the saccharide, the reducing saccharide is converted to the corresponding sugar alcohol without adversely affecting the cyclic tetrasaccharide which is a non-reducing saccharide, The present inventors have found that a saccharide mixture containing a sugar alcohol together with a tetrasaccharide is obtained, and the subsequent purification step is also easy, so that the above-mentioned problem can be solved. Thus, the present invention has been completed.

[0009]

DETAILED DESCRIPTION OF THE INVENTION First, the cyclic tetrasaccharide-forming enzyme used in the present invention is α-isomaltosylglucose such as panose or isomaltosylmaltose, which is an enzyme that produces a cyclic tetrasaccharide from α-isomaltosylglucosaccharide. Isomaltosyltransferase and α-isomaltosylglucosaccharide-forming enzyme, which is an enzyme that generates α-isomaltosylglucosaccharide from starch. These enzymes include, for example, Japanese Patent Application No.
No. 00-229557 and Japanese Patent Application No. 2000-23
Bacillus globisporus C9 disclosed in 4937
(FERM BP-7143), Bacillus globisporus C
Α-isomaltosyltransferase and α-isomaltosylglucosaccharide-forming enzyme derived from No. 11 (FERM BP-7144) and the like are preferably used.

The starch used in the present invention may be ground starch such as corn starch, rice starch, wheat starch, or underground starch such as potato starch, sweet potato starch, tapioca starch. In order to liquefy starch, usually, starch milk in which starch is suspended in water, desirably at a concentration of 10 w / w% (hereinafter, w / w% is abbreviated to% unless otherwise specified in this specification). ) Above, more preferably about 20-50%, which may be heated and mechanically liquefied or liquefied with acids or enzymes. Suitable for the degree of liquefaction is a relatively low DE of less than 20, preferably less than DE 15, more preferably less than DE 10. In the case of liquefaction with an acid, for example, it may be liquefied with hydrochloric acid, phosphoric acid, oxalic acid or the like, and then neutralized to a required pH with calcium carbonate, calcium oxide, sodium carbonate or the like before use. In the case of liquefaction with an enzyme, the use of α-amylase, in particular, liquefied α-amylase having heat resistance is suitable.

The thus obtained starch liquefied solution is reacted with α-isomaltosyltransferase and α-isomaltosylglucosaccharide-forming enzyme together with starch debranching enzyme and / or cyclomaltodextrin glucanotransferase. Has a pH at which these enzymes can act,
The reaction may be performed at a temperature of usually 4 to 10, preferably 5 to 8, and a temperature of about 10 to 80 ° C, preferably about 30 to 70 ° C. In addition, regardless of the order in which these enzymes are added to the liquefied starch solution, one of the enzymes may be added first, and then the other enzyme may be added and then acted on, or the enzymes may be added and acted on simultaneously. Is also optional.

The amount of the enzyme to be used may be appropriately selected depending on the working conditions and the reaction time. Usually, α-isomaltosyltransferase and α-isomaltosylglucosaccharide are used per gram of the solid substance per liquefied starch as a substrate. In the case of a synthesizing enzyme, each is selected from about 0.01 to 100 units, in the case of a starch debranching enzyme, it is selected from about 1 to 10,000 units, and in the case of cyclomaltodextrin / glucanotransferase, about 0.1 to 100 units. Selected from 05 to 500 units. The reducing saccharide containing starch sugars such as glucose, maltose, isomaltose, and panose, which exhibit reducing properties together with the non-reducing cyclic tetrasaccharide thus obtained, is advantageously used as the reducing saccharide for the raw material of the present invention. Available.

The reaction mixture is filtered, centrifuged, etc. to remove insolubles, then decolorized with activated carbon, H-form, OH
The product is desalted and purified with a type ion-exchange resin and concentrated to obtain a syrup-like product. Furthermore, it is optional to dry it into a powdery product. If necessary, further purification, for example, ion-exchange column chromatography, activated carbon column chromatography, fractionation by column chromatography such as silica gel column chromatography, fractionation with organic solvents such as alcohol and acetone, membrane with appropriate separation performance A carbohydrate mixture in which the content of non-reducing cyclic tetrasaccharides is increased and the content of remaining reducing starch sugars is reduced by purifying by combining one or more methods such as separation by In addition, any saccharide in which the content of reducing starch saccharide is increased and the remaining non-reducing cyclic tetrasaccharide is reduced can be advantageously used as the reducing saccharide for a raw material of the present invention.

In particular, industrial mass production methods include:
The use of ion-exchange column chromatography is preferable. For example, JP-A-58-23799 and JP-A-5-23799
Producing a reducing saccharide for a raw material by removing contaminating saccharides by column chromatography using a strongly acidic cation exchange resin disclosed in, for example, JP-A-8-72598 can also be advantageously carried out. At this time, any of the fixed bed system, the moving bed system, and the simulated moving bed system may be adopted. The reducing saccharide for a raw material thus obtained is usually 1: 9 by weight ratio of the cyclic tetrasaccharide to the reducing saccharide.
9 to 99: 1, preferably 10:90 to 90:10
In the range.

In order to hydrogenate a saccharide containing a reducing saccharide together with the cyclic tetrasaccharide thus obtained, the reducing saccharide contained in the saccharide can be replaced with a corresponding sugar alcohol without decomposing the cyclic tetrasaccharide. For example, the raw material saccharide concentration is made into an aqueous solution of about 30 to 70%, put into an autoclave, and as a catalyst, about 4 to 10% of Raney nickel per saccharide solid is added and stirred. 90 temperature while
To 150 ° C. to complete the hydrogenation, desirably hydrogenation until the DE, which is substantially non-reducing, is reduced to less than 0.5, then removes Raney nickel, and further activates the activated carbon in a conventional manner. After a purification step such as decolorization by ion exchange resin or desalting with an ion exchange resin, the syrup may be concentrated to a syrup-like product, or the syrup may be dried to an amorphous powdery product. It is optional to crystallize it into a mass kit and then pulverize it into a crystal-containing powder product. The saccharide mixture having reduced reducibility thus produced contains, in addition to the cyclic tetrasaccharide, one or more sugar alcohols selected from sorbitol, maltitol, isomaltitol and panitol. ing. The saccharide mixture containing a sugar alcohol together with the cyclic tetrasaccharide of the present invention is preferably in a weight ratio of the cyclic tetrasaccharide to the sugar alcohol, preferably 1:99 to 99: 1, and more preferably in order to further derive its characteristics. Preferably 10:90
A range of from 90:10 to 90:10 is preferred.

A saccharide mixture containing a sugar alcohol together with the cyclic tetrasaccharide of the present invention (hereinafter, the mixture may be referred to as the saccharide mixture of the present invention in the present specification) is also provided.
With a DE of less than 0.5, the reducibility is extremely low and stable, and even when mixed with other materials, particularly amino acids, oligopeptides and substances having amino acids such as proteins, they do not brown or generate off-flavors. There is little damage to other mixed materials. In addition, it has low viscosity despite its low reducing power, and has good quality and elegant sweetness. In addition, by oral ingestion, maltitol as well as the main component cyclic tetrasaccharide,
Oligosaccharide alcohols such as isomaltitol and panitol are relatively difficult to digest and absorb and are used as low-calorie sweeteners. Further, the saccharide mixture of the present invention is hardly fermented by caries-inducing bacteria or the like, and can be used as a sweetener that hardly causes dental caries.

Further, since the saccharide mixture of the present invention is a stable sweetener, in the case of a product having a high crystal content, a sugar coating of tablets is used in combination with a binder such as pullulan, hydroxyethyl starch and polyvinylpyrrolidone. Use as an agent can also be carried out advantageously. In addition, osmotic pressure adjustment, shaping, shine imparting, moisturizing, viscosity, anti-crystallization of other sugars,
It has properties such as poor fermentability and anti-aging properties of gelatinized starch.

Further, the saccharide mixture of the present invention may be used as a sweetener, a taste improver, a flavor improver, a quality improver, a stabilizer, an excipient, etc. for foods and drinks, feeds, feeds, cosmetics, pharmaceuticals and the like. It can be advantageously used for various compositions.

The saccharide mixture of the present invention can be used as it is as a seasoning for sweetening. If necessary, for example, powdered candy, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, isomaltooligosaccharide, galactooligosaccharide, fructooligosaccharide, lactosucrose, sorbitol, maltitol, lactitol, palatinit, dihydrochalcone, stevioside ,
α-glycosyl stevioside, rebaudioside, α-
Glycosyl rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, sucralose, acesulfame K, saccharin, glycine, mixed with an appropriate amount of one or more other sweeteners such as alanine and the like And if necessary,
It can also be used in admixture with bulking agents such as dextrin, starch, lactose and the like.

The carbohydrate mixture comprising a sugar alcohol together with the cyclic tetrasaccharide of the present invention, in particular, a powdery product may be used as it is or, if necessary, with a bulking agent, an excipient,
Mixing with a binder or the like and optionally shaping into various shapes such as granules, spheres, short rods, plates, cubes, and tablets may be used.

The sweetness of the carbohydrate mixture containing a sugar alcohol in addition to the cyclic tetrasaccharide of the present invention is often the same as that of various substances having other tastes such as sourness, salty taste, astringency, umami, and bitterness. Since it is in harmony and has high acid resistance and heat resistance, it can be advantageously used for sweetening and taste improvement of general foods and drinks, quality improvement, and the like.

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

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

Further, it can be used for raising livestock, poultry, other honeybees, silkworms, fish, and other breeding animals for the purpose of improving the palatability of feed and feed. In addition, various solids such as tobacco, toothpaste, lipstick, lip balm, oral liquid, tablets, troches, liver oil drops, mouth fresheners, mouth flavors, gargles, etc. Can be advantageously used as a sweetener for various compositions, as a taste improver, a flavor enhancer, and further as a quality improver, a stabilizer and the like.

As a quality improving agent and a stabilizer, an active ingredient,
The present invention can be advantageously applied to various physiologically active substances that easily lose their activity and the like, or health foods and pharmaceuticals containing the same. For example, interferon-α, interferon-β, interferon-γ, Tsumore necrosis factor-α, Tsumore necrosis factor-β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, lymphokines such as interleukin II, insulin Hormones such as growth hormone, prolactin, erythropoietin, and egg cell stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, pox seedling, tetanus toxoid, hub antitoxin, biological products such as human immunoglobulin, penicillin, erythromycin, Antibiotics such as chloramphenicol, tetracycline, streptomycin, kanamycin sulfate,
Thiamine, riboflavin, L-ascorbic acid, liver oil,
Vitamin such as carotenoid, ergosterol, tocopherol, lipase, elastase, urokinase,
Enzymes such as protease, β-amylase, isoamylase, glucanase, lactase, extracts such as ginseng extract, turtle extract, chlorella extract, aloe extract, propolis extract, viruses, lactic acid bacteria, live bacteria such as yeast, royal jelly, etc. Bioactive substances,
Without losing its active ingredient and activity, it can be easily produced into a stable, high-quality liquid, paste-like or solid health food or medicine.

The method for incorporating the above-described various compositions into a saccharide mixture containing a sugar alcohol together with the cyclic tetrasaccharide of the present invention may be included in the steps until the product is completed. For example, mixing, dissolving, melting, dipping,
Known methods such as penetration, spraying, coating, coating, spraying, pouring, crystallization, and solidification are appropriately selected. The amount is usually 0.1% or more, preferably 1% or more.

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

[0028]

[Experiment 1] <Preparation of cyclic tetrasaccharide from culture> Partially degraded starch (trade name "Paindex # 1", manufactured by Matsutani Chemical Co., Ltd.) 5 w / v%, yeast extract (trade name "Asahi Mist", Asahi Breweries, Ltd.) 1.5 w / v%,
Liquid culture medium 100 consisting of dipotassium phosphate 0.1 w / v%, monosodium phosphate / 12 hydrate 0.06 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, and water
of the Bacillus globisporus C9 (FERM BP-7143) in a 500 ml Erlenmeyer flask, sterilized in an autoclave at 121 ° C. for 20 minutes, cooled.
After incubating at 27 ° C. and 230 rpm for 48 hours, the cells were centrifuged to remove the cells and obtain a culture supernatant. Furthermore, the culture supernatant was autoclaved (120 ° C., 15 ° C.).
After cooling, insolubles were removed by centrifugation and the supernatant was recovered.

In order to examine the saccharides in the obtained supernatant, a mixture of n-butanol, pyridine and water (volume ratio 6: 4: 1) as a developing solvent and “Kieselgel 60” (aluminum) manufactured by Merck as a thin layer plate were used. A silica gel thin layer chromatography (hereinafter, abbreviated as “TLC”) was performed twice using a plate (20 × 20 cm) to separate saccharides in the supernatant. As a detection method, the separated whole saccharide is colored by a sulfuric acid-methanol method, and the reduced saccharide is diphenylamine-
When the color was examined by the aniline method, the Rf value was about 0.3.
At position 1, a non-reducing saccharide that was positive by the sulfuric acid-methanol method and negative by the diphenylamine-aniline method was detected.

About 90 ml of the supernatant obtained above was adjusted to pH 5.0 and a temperature of 45 ° C., and then 1 g of α-glucosidase (trade name “Transglucosidase L“ Amano ”, manufactured by Amano Pharmaceutical Co., Ltd.) was used. Glucoamylase (available from Nagase Seikagaku Co., Ltd.) at a rate of 1,500 units per gram and 75 units per gram of the solid substance, and the mixture is treated for 24 hours, then adjusted to pH 12 with sodium hydroxide and boiled for 2 hours. The remaining reducing sugar was decomposed. After removing insoluble matter by filtration, decolorization and desalting were performed using Mitsubishi Chemical's ion exchange resins "Diaion PK218" and "Diaion WA30", and further, Mitsubishi Chemical's cation exchange resin "Diaion SK-1B". Was desalted again with an anion exchange resin "IRA411" manufactured by Organo, decolorized with activated carbon, finely filtered, concentrated by an evaporator, and freeze-dried under vacuum to obtain about 0.6 g of a saccharide powder as a solid.

When the composition of the obtained saccharide was examined by high performance liquid chromatography (hereinafter abbreviated as HPLC), as shown in FIG. 1, only a single peak was detected at an elution time of 10.84 minutes. As a result, the purity was found to be extremely high at 99.9% or more. In addition, HPLC is "Shodex KS-801 column"
The detection was performed using a differential refractometer “RI-8012” (manufactured by Tosoh Corporation) under the conditions of a column temperature of 60 ° C. and a flow rate of 0.5 ml / min using a (manufactured by Showa Denko KK).

When the reducing power was measured by the Somogi-Nelson method, the reducing power was below the detection limit, and it was determined that the present sample was substantially a non-reducing saccharide.

[0033]

[Experiment 2] <Structural analysis of non-reducing saccharide> The non-reducing saccharide obtained by the method of Experiment 1 was subjected to mass spectrometry (commonly called “FAB-MS”) by a high-speed atom bombardment method. Was significantly detected, and the mass number of the present saccharide was found to be 648.

Further, when the constituent sugars are hydrolyzed using sulfuric acid according to a conventional method and the constituent sugars are examined by gas chromatography, only D-glucose is detected, and the constituent sugars of the present saccharide may be D-glucose. As a result, it was found that the present saccharide was a cyclic saccharide composed of four D-glucose molecules in consideration of the mass number.

Further, when a nuclear magnetic resonance method (commonly known as NMR) was performed using the present saccharide, a ¹ H-NMR spectrum shown in FIG. 2 and a ¹³ C-NMR spectrum shown in FIG. 3 were obtained. Was compared with those of known carbohydrates to find that "European Journal of Biochemistry (European Journal of Biochemistry)"
f Biochemistry) ”, 641 to 648
Non-reducing cyclic carbohydrate cyclo ｛(6) -α-D-glucopyranosyl- (1 →) described on page (1994).
3) -α-D-glucopyranosyl- (1 → 6) -α-D
-Glucopyranosyl- (1 → 3) -α-D-glucopyranosyl- (1 → ス ペ ク ト ル), and the structure of the present saccharide is a cyclic tetrasaccharide shown in FIG. 4, ie, cyclo ｛→ 6) -α-
D-glucopyranosyl- (1 → 3) -α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl-
It was found that (1 → 3) -α-D-glucopyranosyl- (1 →｝).

[0036]

[Experiment 3] <α from Bacillus globisporus C9
-Production of isomaltosyl glucosaccharide-forming enzyme> Partial degraded starch (trade name “Paindex # 4”, manufactured by Matsutani Chemical Co., Ltd.) 4.0 w / v%, yeast extract (trade name “Asahi Mist” (Asahi 1.8w /
v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate / 12-hydrate 0.06 w / v%, magnesium sulfate / 7-hydrate 0.05 w / v%, and a liquid medium comprising water, Put 100ml each in 500ml Erlenmeyer flask,
The solution was sterilized in an autoclave at 121 ° C. for 20 minutes, cooled, and subjected to Bacillus globisporus C9 (FERM BP).
-7143) and inoculated at 27 ° C. and 230 rpm for 48 hours as a seed culture.

A fermenter having a capacity of 30 liters is charged with about 20 liters of a medium having the same composition as that for seed culture, sterilized by heating, cooled to a temperature of 27 ° C., and inoculated with 1 v / v% of a seed culture solution.
While maintaining the temperature at 27 ° C. and the pH at 6.0 to 8.0, the cells were cultured with aeration and stirring for 48 hours. After the cultivation, the α-isomaltosylglucosaccharide-forming enzyme activity in the culture solution is about 0.45 units / ml.
And the activity of α-isomaltosyltransferase is about 1.5 units /
In ml, the cyclic tetrasaccharide-forming activity was about 0.95 units / ml, and the enzyme activity of about 18 l of the supernatant collected by centrifugation (10,000 rpm, 30 minutes) was measured.
Isomaltosyl glucosaccharide-forming enzyme is about 0.45 units /
ml of activity (total activity of about 8,110 units), α-isomaltosyltransferase has an activity of about 1.5 units / ml (total activity of about 26,900 units), and a cyclic tetrasaccharide-forming activity of about 0.9.
5 units / ml (total activity about 17,100 units).

The enzyme activity was measured as follows. That is, α-isomaltosylglucosaccharide-forming enzyme activity was measured by dissolving maltotriose in a 100 mM acetate buffer (pH 6.0) to a concentration of 2 w / v% to obtain a substrate solution. 0.5 ml of the enzyme solution was added to 0.5 ml, and the enzyme reaction was carried out at 35 ° C. for 60 minutes.
After boiled for a minute to stop the reaction, the amount of maltose among isomaltosyl maltose and maltose mainly produced in the reaction solution was quantified by the HPLC method described in Experiment 1. One unit of the activity of the α-isomaltosylglucosaccharide-forming enzyme was defined as the amount of the enzyme that produced 1 μmol of maltose per minute under the above conditions.

The activity of α-isomaltosyltransferase was measured by measuring the concentration of panose in 100 m
M acetate buffer (pH 6.0) to give a substrate solution, and 0.5 ml of the enzyme solution was added to 0.5 ml of the substrate solution.
After the reaction is stopped for 30 minutes by boiling the reaction solution for 10 minutes, the amount of glucose is quantitatively determined by the glucose oxidase method among the cyclic tetrasaccharide and glucose mainly produced in the reaction solution. By doing that. α-
One unit of the activity of the isomaltosyltransferase was defined as the amount of the enzyme that produces 1 μmol of glucose per minute under the above conditions.

The cyclic tetrasaccharide-forming activity was determined by measuring the partial decomposition of starch (trade name “Paindex # 100”, manufactured by Matsutani Chemical Co., Ltd.) in a 50 mM acetate buffer (pH 6.0) so as to have a concentration of 2 w / v%. To a substrate solution, 0.5 ml of the substrate solution was added to 0.5 ml of the enzyme solution, the enzyme reaction was performed at 35 ° C. for 60 minutes, and the reaction solution was heat-treated at 100 ° C. for 10 minutes to stop the reaction. Further, it contains 70 units / ml of α-glucosidase (trade name “transglucosidase L“ Amano ”” manufactured by Amano Pharmaceutical Co., Ltd.) and 27 units / ml of glucoamylase (sold by Nagase Seikagaku Corporation).
Add 1 ml of 0 mM acetate buffer (pH 5.0) and add 50 ml.
After treatment at 60 ° C. for 60 minutes and heat treatment of the solution at 100 ° C. for 10 minutes to inactivate the enzyme, the amount of cyclic tetrasaccharide was determined by the HPLC method described in Experiment 1. One unit of the cyclic tetrasaccharide-forming activity was defined as the amount of the enzyme capable of producing 1 μmol of the cyclic tetrasaccharide per minute under the above conditions.

[0041]

[Experiment 4] <Preparation of Bacillus globisporus C9-derived enzyme>

<Experiment 4-1><Purification of Bacillus globisporus C9-derived enzyme> About 18 l of the culture supernatant obtained in Experiment 3 was
After salting out with a saturated ammonium sulfate solution and leaving at 4 ° C. for 24 hours, the salted out precipitate is collected by centrifugation (10,000 rpm, 30 minutes) and dissolved in a 10 mM phosphate buffer (pH 7.5). Then, the buffer was dialyzed and the crude enzyme solution was
1 was obtained. This crude enzyme solution has 8,110 units of α-isomaltosylglucosaccharide-forming enzyme activity, 24,700 units of α-isomaltosyltransferase activity, and about 15,600 units of cyclic tetrasaccharide-forming activity. I was This crude enzyme solution was used as a "Sepabead" (manufactured by Mitsubishi Chemical Corporation).
s) The sample was subjected to ion exchange chromatography using FP-DA13 "gel (gel volume: 1,000 ml). At this time, all of α-isomaltosylglucosaccharide-forming enzyme, α-isomaltosyltransferase and cyclic tetrasaccharide were not adsorbed on the “Sepabeads FP-DA13” gel but eluted in the non-adsorbed fraction. . 1M of this enzyme solution
The solution was dialyzed against a 10 mM phosphate buffer (pH 7.0) containing ammonium sulfate, and the dialysate was centrifuged to remove impurities, and a “Sephacryl HR S-200” gel manufactured by Amersham Pharmacia Biotech Co., Ltd. was used. Affinity chromatography (gel amount 500
ml). The enzyme active ingredient is “Sephacryl (S
ephacryl) HR S-200 ”gel,
A linear gradient from ammonium sulfate 1M to 0M, followed by
When eluted with a linear gradient of maltotetraose from 0 mM to 100 mM, α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase were separated and eluted, and α-isomaltosyltransferase activity was determined by the linear gradient of ammonium sulfate. At a concentration of about 0M,
The activity of α-isomaltosyl glucosaccharide-forming enzyme was found to be about 30 at a linear gradient of maltotetraose.
It eluted around mM. Therefore, the α-isomaltosyltransferase active fraction and the α-isomaltosyl glucosaccharide-forming enzyme active fraction were separately collected and collected. In addition, it was found that the cyclic tetrasaccharide-forming activity was not observed in any of the fractions of the present column chromatography, and the obtained α-isomaltosylglucosaccharide-forming enzyme fraction and α-isomaltosyltransferase It has also been found that the enzyme solution obtained by mixing the α-isomaltosyltransferase and the α-isomaltosylglucosylcarboxylase has an activity of producing a cyclic tetrasaccharide from a partially degraded starch. It was found that the enzyme was exerted by the synergistic action of both enzyme activities.

Hereinafter, a method for separately purifying α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase will be described.

[0043]

[Experiment 4-2] <Purification of α-isomaltosylglucosaccharide-forming enzyme> The α-isomaltosylglucosaccharide-forming enzyme fraction obtained in Experiment 4-1 was subjected to a 10 mM phosphate buffer containing 1 M ammonium sulfate ( The dialysate was centrifuged to remove insolubles, and subjected to hydrophobic chromatography (gel amount: 350 ml) using "Butyl-Toyopearl 650M" gel manufactured by Tosoh Corporation. did. This enzyme is referred to as “Butyl-Toyopearl (Butyl).
-Toyopearl) 650M ”gel was adsorbed and eluted with a linear gradient of 1M to 0M ammonium sulfate. The adsorbed enzyme was eluted at a concentration of about 0.3M ammonium sulfate,
Fractions showing this enzyme activity were collected and collected. Again, this recovered solution was added to a 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate.
0), the dialysate is centrifuged to remove insolubles, and “Sephacryl HR S-
200 "gel was used for affinity chromatography. Α in each step of this purification
-Isomaltosyl glucosaccharide-forming enzyme activity, specific activity,
The yield is shown in Table 1.

[0044]

[Table 1]

The purity of the purified α-isomaltosylglucosaccharide-forming enzyme preparation was assayed by gel electrophoresis containing 7.5% w / v polyacrylamide. It was a high standard.

[0046]

[Experiment 4-3] <Purification of α-isomaltosyltransferase>
The α-isomaltosyltransferase fraction separated from the α-isomaltosylglucosaccharide-forming enzyme fraction by the affinity chromatography described in Experiment 4-1 was placed in a 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. Dialysis,
The dialysate was centrifuged to remove insolubles, and “Butyl-Toyopearl” manufactured by Tosoh Corporation was used.
arl) 650M "gel (350 ml of gel). This enzyme is referred to as "Butyl-Toyopearl 65".
The enzyme was adsorbed on a “0M” gel and eluted with a linear gradient of ammonium sulfate from 1M to 0M. The adsorbed enzyme was eluted at a concentration of about 0.3M ammonium sulfate, and fractions showing this enzyme activity were collected and collected. Again, this recovered solution was dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialysate was centrifuged to remove insolubles.
ryl) HR S-200 "gel was used for purification by affinity chromatography. The amount of α-isomaltosyltransferase activity in each step of this purification,
Table 2 shows the specific activity and yield.

[0047]

[Table 2]

[0048]

[Experiment 5] <Properties of α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase>

[Experiment 5-1] <Properties of α-isomaltosylglucosaccharide-forming enzyme> The purified α-isomaltosylglucosaccharide-forming enzyme sample obtained by the method of Experiment 4-2 was subjected to SDS-polyacrylamide gel electrophoresis. The enzyme was subjected to the method (gel concentration 7.5 w / v%), and the molecular weight of the enzyme was measured in comparison with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.), which was simultaneously electrophoresed.
It was 000 Dalton.

The purified α-isomaltosylglucosaccharide-forming enzyme preparation was subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2 w / v% ampholine (manufactured by Amersham-Pharmacia Biotech). And the pH of the gel was measured to determine the isoelectric point of the enzyme.
The isoelectric point was pI about 5.2 ± 0.5.

The effects of temperature and pH on the activity of the enzyme were examined according to the method for measuring the activity. The effect of temperature was measured in the absence of Ca ^{2+ and in the} presence of 1 mM. FIG. 5 (effect of temperature) and FIG. 6 (effect of pH)
showed that. Optimum temperature of the enzyme is about 40 ° C. In reaction pH6.0,60 minutes ^{(Ca 2+} absence), about 45 ℃ ^{(Ca 2 +}
The optimal pH was about 6.0 to 6.5 in the reaction at 35 ° C. for 60 minutes. The temperature stability of the enzyme was determined by adding the enzyme solution (20 mM acetate buffer, pH 6.0) to Ca ²⁺
Each temperature was maintained for 60 minutes in the absence or presence of 1 mM, and after cooling with water, the remaining enzyme activity was measured. In addition, the pH stability of the present enzyme was measured at each pH of 50 m.
After holding at 4 ° C. for 24 hours in M buffer, the pH was increased to 6.0.
And determined by measuring the remaining enzyme activity. FIG. 7 (temperature stability) and FIG. 8 (pH
Stability). The temperature stability of the enzyme was up to about 35 ° C. (without Ca ²⁺ ) and up to about 40 ° C. (with Ca ²⁺ 1 mM), and the pH stability was about 4.5 to 9.0.

The effect of metal ions on the activity of this enzyme was examined in the presence of various metal salts at a concentration of 1 mM according to a method for measuring the activity. Table 3 shows the results.

[0052]

[Table 3]

As is clear from the results in Table 3, the activity of the present enzyme was significantly inhibited by Hg ²⁺ , Cu ²⁺ and EDTA, and inhibited by Ba ²⁺ and Sr ²⁺ . Ca ²⁺ , Mn
It was also found to be activated by ²⁺ .

The N-terminal amino acid sequence of this enzyme was analyzed using a protein sequencer model 473A (manufactured by Applied Biosystems), and the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, tyrosine-valine-serine-serine-leucine- It was found to have the N-terminal amino acid sequence of glycine-asparagine-leucine-isoleucine.

[0055]

[Experiment 5-2] <Properties of α-isomaltosyltransferase>
The purified α-isomaltosyltransferase preparation obtained by the method of Experiment 4-3 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration: 7.5 w / v%), and a molecular weight marker (Japan Bio-Rad. Laboratories, Inc.), and the molecular weight of the enzyme was measured to be about 112,000 ± 20,000 daltons.

The purified α-isomaltosyltransferase preparation was
w / v% ampholine (Amersham Pharmacia
Biotech) containing isoelectric focusing polyacrylamide gel electrophoresis, after electrophoresis, pH of protein band and gel
Was measured to determine the isoelectric point of the enzyme.
I was about 5.5 ± 0.5.

The effects of temperature and pH on the activity of this enzyme were examined according to the method for measuring the activity. The results are shown in FIG. 9 (effect of temperature) and FIG. 10 (effect of pH). The optimum temperature of the enzyme is pH 6.0, about 45 ° C for 30 minutes reaction,
Was about 6.0 in the reaction at 35 ° C. for 30 minutes. The temperature stability of this enzyme was measured using an enzyme solution (20 mM acetate buffer, pH
6.0) was maintained at each temperature for 60 minutes, cooled with water, and then measured for remaining enzyme activity. Also, pH
Stability was measured at 4 ° C for 2 hours in a 50 mM buffer at each pH.
After holding for 4 hours, the pH was adjusted to 6.0, and the remaining enzyme activity was measured. The respective results are shown in FIG. 11 (temperature stability) and FIG. 12 (pH stability). The temperature stability of the enzyme was up to about 40 ° C., and the pH stability was about 4.0 to 9.0.

The effect of metal ions on the activity of the enzyme was examined in the presence of various metal salts at a concentration of 1 mM according to the method for measuring the activity. Table 4 shows the results.

[0059]

[Table 4]

As is clear from the results in Table 4, the activity of the present enzyme was significantly inhibited by Hg ²⁺ and inhibited by Cu ²⁺ . Also, the fact that it is not activated by Ca ²⁺ indicates that EDTA
It was also found that it was not inhibited.

The N-terminal amino acid sequence of this enzyme was analyzed using “Protein Sequencer Model 473A” (manufactured by Applied Biosystems).
It was found to have the N-terminal amino acid sequence of aspartic acid-glycine-valine-tyrosine-histidine-alanine-proline-asparagine-glycine.

[0062]

[Experiment 6] <Production of α-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C11> Starch partially degraded product “Paindex # 4” 4.0 w / v%, yeast extract “Asahi Mist” 1.8 w / v v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate / 12 hydrate 0.06 w / v%, magnesium sulfate / 7 hydrate 0.05
100 ml of a liquid medium consisting of w / v% and water was placed in a 500 ml Erlenmeyer flask, sterilized in an autoclave at 121 ° C. for 20 minutes, cooled, and inoculated with Bacillus globisporus C11 (FERM BP-7144). A seed culture was obtained by culturing with rotation and shaking at 230 ° C. and 230 rpm for 48 hours.

A fermenter having a capacity of 30 liters is charged with about 20 liters of a medium having the same composition as that for seed culture, sterilized by heating, cooled to a temperature of 27 ° C., and inoculated with 1 v / v% of a seed culture solution.
While maintaining the temperature at 27 ° C. and the pH at 6.0 to 8.0, the cells were cultured with aeration and stirring for 48 hours. After culturing, the enzyme activity in the culture was about 0.55 units / ml, the α-isomaltosyltransferase activity was about 1.8 units / ml, and the cyclic tetrasaccharide-forming activity was about 1.1 units / ml. Yes, centrifugation (10,000 rpm
After measuring the enzyme activity of about 18 l of the collected supernatant, α-isomaltosylglucosaccharide-forming enzyme had an activity of about 0.51 unit / ml (total activity of about 9,180
Α-isomaltosyltransferase has an activity of about 1.7 units / ml (total activity of about 30,400 units) and a cyclic tetrasaccharide-forming activity of about 1.1 units / ml (total activity of about 19,40 units).
0 units).

[0064]

[Experiment 7] <Preparation of Bacillus globisporus C11-derived enzyme>

[Experiment 7-1] <Purification of Bacillus globisporus C11-derived enzyme> About 18 l of the culture supernatant obtained in Experiment 6 was diluted with 8
After salting out with 0% saturated ammonium sulfate solution and leaving at 4 ° C. for 24 hours,
The salted out precipitate is centrifuged (10,000 rpm, 30
Min) and collect 10 mM phosphate buffer (pH 7.5)
And then dialyzed against the same buffer to obtain about 416 crude enzyme solutions.
ml were obtained. This crude enzyme solution has 8,440 units of α-isomaltosylglucosidase activity, about 28,000 units of α-isomaltosyltransferase activity, and about 17,700 units of cyclic tetrasaccharide-forming activity. There was found. This crude enzyme solution was used as the “Sepabeads (Se)” described in Experiment 4-1.
pabeads) FP-DA13 "gel. All of α-isomaltosylglucosaccharide-forming enzyme activity, α-isomaltosyltransferase activity, and cyclic tetrasaccharide formation are described in “Sepabeads (Sepa beads)
beads) was eluted in the non-adsorbed fraction without adsorbing on the FP-DA13 "gel. This enzyme solution is 10m containing 1M ammonium sulfate.
M Phosphate buffer (pH 7.0), and the dialysate is centrifuged to remove insolubles. The product is manufactured by Amersham-Pharmacia Biotech Co., Ltd., "Sephacryl".
ryl) HR S-200 "gel was used for affinity chromatography (gel amount: 500 ml).
The enzymatic activity was determined by measuring "Sephacryl H".
Adsorbed on the RS-200 "gel, and a linear gradient of ammonium sulfate 1M to 0M, followed by maltotetraose 0
When eluted with a linear gradient from mM to 100 mM, α-isomaltosyltransferase and α-isomaltosylglucosaccharide-forming enzyme were separated and eluted, and the α-isomaltosyltransferase activity was determined by a linear gradient of ammonium sulfate. The enzyme was eluted at about 0.3 M, and the α-isomaltosylglucosaccharide-forming enzyme activity eluted at a concentration of about 30 mM in a linear gradient of maltotetraose. Therefore, α-isomaltosyltransferase active fraction and α
-Isomaltosyl glucosaccharide-forming enzyme fraction was separately collected and collected. As in the case of the C9 strain described in Experiment 4, it was found that no cyclic tetrasaccharide-forming activity was observed in any of the fractions of the present column chromatography, and that the obtained α-isomaltosyltransferase fraction was It was also found that the enzyme solution mixed with the α-isomaltosylglucosaccharide-forming enzyme fraction exhibited a cyclic tetrasaccharide-forming activity, and the activity of forming a cyclic tetrasaccharide from the partially degraded starch was α-isomaltosylglucosaccharide. It was found that the enzyme was exerted by the synergistic action of both enzyme activities of the enzyme and α-isomaltosyltransferase.

Hereinafter, a method for separately purifying α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase will be described.

[0066]

[Experiment 7-2] <Purification of α-isomaltosylglucosaccharide-forming enzyme> The α-isomaltosylglucosaccharide-forming enzyme fraction was purified from a 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate.
Dialysate, centrifuging the dialysate to remove insolubles, and removing the insolubles from Tosoh Corporation "Butyltoyopearl (Butyl-T)
(650 ml) and subjected to hydrophobic chromatography (gel amount: 350 ml). This enzyme is referred to as "Butyl-Toyopearl".
a1) 650M gel and eluted with a linear gradient of ammonium sulfate from 1M to 0M. The adsorbed enzyme was eluted at a concentration of about 0.3M ammonium sulfate, and fractions showing this enzyme activity were collected and collected. Again, this recovered solution was dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialysate was centrifuged to remove insolubles, and a “Sephacryl HRS-200” gel was used. It was purified using affinity chromatography. Table 5 shows the activity, specific activity, and yield of α-isomaltosylglucosaccharide-forming enzyme in each step of this purification.

[0067]

[Table 5]

The purity of the purified α-isomaltosylglucosaccharide-forming enzyme preparation was assayed by gel electrophoresis containing 7.5% w / v polyacrylamide. It was a high standard.

[0069]

[Experiment 7-3] <Purification of α-isomaltosyltransferase>
The α-isomaltosyltransferase fraction separated from the α-isomaltosylglucosaccharide-forming enzyme fraction by the affinity chromatography described in Experiment 7-1 was placed in a 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. Dialysis,
The dialysate was centrifuged to remove insolubles, and “Butyl-Toyopearl” manufactured by Tosoh Corporation was used.
arl) 650M "gel (350 ml of gel). This enzyme is referred to as "Butyl-Toyopearl 65".
The enzyme was adsorbed on a “0M” gel and eluted with a linear gradient of ammonium sulfate from 1M to 0M. The adsorbed enzyme was eluted at a concentration of about 0.3M ammonium sulfate, and fractions showing this enzyme activity were collected and collected. Again, this recovered solution was dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialysate was centrifuged to remove insolubles.
ryl) HR S-200 "gel was used for purification by affinity chromatography. The amount of α-isomaltosyltransferase activity in each step of this purification,
Table 6 shows the specific activity and yield.

[0070]

[Table 6]

[0071]

[Experiment 8] <Properties of α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase>

[Experiment 8-1] <Properties of α-isomaltosylglucosaccharide-forming enzyme> The purified α-isomaltosylglucosaccharide-forming enzyme sample obtained by the method of Experiment 7-2 was subjected to SDS-polyacrylamide gel electrophoresis. (Enzyme concentration 7.5 w / v%), and the molecular weight of the enzyme was measured in comparison with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.) that migrated simultaneously. The molecular weight was about 137,000 ± 2.
It was 000 Dalton.

The purified α-isomaltosylglucosaccharide-forming enzyme preparation was subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2% w / v ampholine (manufactured by Amersham-Pharmacia Biotech). And the pH of the gel was measured to determine the isoelectric point of the enzyme.
The isoelectric point was pI about 5.2 ± 0.5.

The effects of temperature and pH on the activity of the enzyme
The test was conducted according to the method of measuring the sex. Note that the effect of temperature
And Ca²⁺The measurement was performed in the absence and in the presence of 1 mM. This
The results are shown in FIG. 13 (effect of temperature) and FIG.
Hibiki). The optimal temperature of the enzyme is pH 6.0, 60 minutes
About 45 ° C (Ca²⁺Absent), about 50 ° C (Ca
²⁺1 mM), and the optimal pH is 35 ° C for 60 minutes.
Was about 6.0. The temperature stability of the enzyme
(20 mM acetate buffer, pH 6.0)^{2 +}Non-existence
Temperature or in the presence of 1 mM for 60 min.
And then determine the remaining enzyme activity
Was. The pH stability was determined by buffering the enzyme with 50 mM buffer at each pH.
After keeping the solution at 4 ° C for 24 hours, adjust the pH to 6.0
It was determined by measuring the remaining enzyme activity. So
The results are shown in FIG. 15 (temperature stability) and FIG.
Qualitative). Temperature stability of this enzyme up to about 40 ℃
(Ca ²⁺Absent), up to about 45 ° C. (Ca²⁺1 mM
PH stability was about 5.0 to 10.0.

The effect of metal ions on the activity of this enzyme was examined in the presence of various metal salts at a concentration of 1 mM according to the method for measuring the activity. Table 7 shows the results.

[0075]

[Table 7]

As is clear from the results in Table 7, the activity of the present enzyme was significantly inhibited by Hg ²⁺ , Cu ²⁺ and EDTA, and inhibited by Ba ²⁺ and Sr ²⁺ . Ca ²⁺ , Mn
It was also found to be activated by ²⁺ .

The N-terminal amino acid sequence of this enzyme was analyzed using “Protein Sequencer Model 473A” (manufactured by Applied Biosystems).
It was found to have the N-terminal amino acid sequence of valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucine.

The result of the N-terminal amino acid sequence was determined in Experiment 5-1.
Was found to be the same as the N-terminal sequence of α-isomaltosylglucosaccharide-forming enzyme derived from Bacillus glovisiporus C9, and the common N-terminal amino acid sequence of α-isomaltosylglucosaccharide-forming enzyme was The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing was found to be the N-terminal amino acid sequence of tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucine.

[0079]

[Experiment 8-2] <Properties of α-isomaltosyltransferase>
The purified α-isomaltosyltransferase preparation obtained by the method of Experiment 7-3 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration: 7.5 w / v%), and a molecular weight marker (Japan Bio-Rad. Laboratories, Inc.), and the molecular weight of the enzyme was measured to be about 102,000 ± 20,000 daltons.

The purified α-isomaltosyltransferase preparation was
w / v% ampholine (Amersham Pharmacia
Biotech) containing isoelectric focusing polyacrylamide gel electrophoresis, after electrophoresis, pH of protein band and gel
Was measured to determine the isoelectric point of the enzyme.
I was about 5.6 ± 0.5.

The effect of temperature and pH on the activity of the present enzyme was examined according to the method for measuring the activity. The results are shown in FIG. 17 (effect of temperature) and FIG. 18 (effect of pH). The optimum temperature of the enzyme is pH 6.0, about 50 ° C for 30 minutes reaction,
Was about 5.5 to 6.0 at 35 ° C. for 30 minutes. The temperature stability of the present enzyme was determined by keeping the enzyme solution (20 mM acetate buffer, pH 6.0) at each temperature for 60 minutes, cooling with water, and measuring the remaining enzyme activity.
The pH stability was determined by maintaining the enzyme in each 50 mM buffer at 4 ° C. for 24 hours and then adjusting the pH to 6.0.
It was determined by measuring the remaining enzyme activity. The respective results are shown in FIG. 19 (temperature stability) and FIG. 20 (pH stability). The temperature stability of this enzyme is up to about 40 ° C,
pH stability was about 4.5-9.0.

The effect of metal ions on the activity of this enzyme was examined in the presence of various metal salts at a concentration of 1 mM according to the method for measuring the activity. Table 8 shows the results.

[0083]

[Table 8]

As is clear from the results in Table 8, the activity of the present enzyme was significantly inhibited by Hg ²⁺ and inhibited by Cu ²⁺ . Also, the fact that it is not activated by Ca ²⁺ indicates that EDTA
It was also found that it was not inhibited.

The N-terminal amino acid sequence of this enzyme was analyzed using “Protein Sequencer Model 473A” (manufactured by Applied Biosystems).
It was found to have the N-terminal amino acid sequence of aspartic acid-glycine-valine-tyrosine-histidine-alanine-proline-tyrosine-glycine. This N-terminal amino acid sequence result was compared with the N-terminal sequence of α-isomaltosyltransferase derived from Bacillus globisporus C9 in Experiment 5-2, and the common N-terminal amino acid sequence of α-isomaltosyltransferase was represented by SEQ ID NO: 4
Isoleucine-aspartic acid of the amino acid sequence shown in
Glycine-valine-tyrosine-histidine-alanine-
It was found to be the N-terminal amino acid sequence of proline.

[0086]

[Experiment 9] <Amino acid sequence of α-isomaltosylglucosaccharide-forming enzyme>

[Experiment 9-1] <Internal partial amino acid sequence of α-isomaltosylglucosaccharide-forming enzyme> A part of the purified α-isomaltosylglucosaccharide-forming enzyme preparation obtained by the method of Experiment 7-2 was used. After dialysis against 10 mM Tris-HCl buffer (pH 9.0), the mixture was diluted with the same buffer to a concentration of about 1 mg / ml. To this sample solution (1 ml) was added 10 μg of trypsin (available from Wako Pure Chemical Industries, Ltd.).
The peptide was formed by reacting for 22 hours. Reverse-phase HPLC was performed to isolate the resulting peptide.
Micro Bonder Pack C18 column (2.1mm diameter x
(Length: 150 mm, manufactured by Waters Co., Ltd.)
9% / min at room temperature from 0.1% trifluoroacetic acid-8% acetonitrile solution to 0.1% trifluoroacetic acid-40
The test was performed under the conditions of a linear gradient of a 120% acetonitrile solution for 120 minutes. The peptide eluted from the column
It was detected by measuring the absorbance at a wavelength of 210 nm. 3 peptides [P64 well separated from other peptides
(Retention time about 64 minutes), P88 (retention time about 88 minutes),
P99 (retention time: about 99 minutes)], and each was vacuum-dried and then dissolved in 200 μl of a 0.1% trifluoroacetic acid-50% acetonitrile solution. The peptide samples were subjected to a protein sequencer, and the amino acid sequences of up to eight residues were analyzed. As a result, the amino acid sequences shown in SEQ ID NOs: 5 to 7 in the sequence listing were obtained. Table 9 shows the obtained internal partial amino acid sequences.

[0087]

[Table 9]

[0088]

[Experiment 9-2] <Internal partial amino acid sequence of α-isomaltosyltransferase> Purified α-isomaltosyltransferase obtained by the method of Experiment 7-3
A portion of the isomaltosyltransferase preparation was added to 10 mM Tris-
After dialysis against a hydrochloric acid buffer (pH 9.0), the mixture was diluted with the same buffer to a concentration of about 1 mg / ml. 10 μg of lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.) was added to this sample solution (1 ml), and the mixture was reacted at 30 ° C. for 22 hours to form a peptide. Reverse-phase HPLC was performed to isolate the resulting peptide. Micro Bonder Pack C18 column (diameter 2.1 mm x length 1
Flow rate 0.9 ml using a 50 mm (Waters company)
/ Min at room temperature under a linear gradient condition of 0.1% trifluoroacetic acid-8% acetonitrile solution to 0.1% trifluoroacetic acid-40% acetonitrile solution for 120 minutes. The peptide eluted from the column was
It was detected by measuring the absorbance at 10 nm. Three peptides [P22 (retention time: about 22 minutes), P63 (retention time: about 63 minutes), and P71 (retention time: about 71 minutes)] that were well separated from other peptides were collected, dried in vacuo, and then 200 μl. Was dissolved in a 0.1% trifluoroacetic acid-50% acetonitrile solution. The peptide samples were subjected to a protein sequencer, and the amino acid sequence was analyzed for each of up to eight residues.
1 to 10 were obtained. Table 10 shows the obtained internal partial amino acid sequences.

[0089]

[Table 10]

[0090]

[Experiment 10] <Action on various carbohydrates>
It was tested whether it could be a substrate for α-isomaltosyl glucosaccharide-forming enzyme. Maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, isomaltose,
Isomalt triose, panose, isopanose, α,
A solution containing α-trehalose, kojibiose, nigerose, neotrehalose, cellobiose, gentibiose, maltitol, maltotriitol, lactose, sucrose, erulose, serginose, maltosylglucoside, and isomaltosylglucoside was prepared.

These solutions were added to a purified α-isomaltosylglucosaccharide-forming enzyme preparation derived from Bacillus globisporus C9 obtained by the method of Experiment 4-2, or from a Bacillus globisporus C11 obtained by the method of Experiment 7-2. Of purified α-isomaltosylglucosaccharide-forming enzyme is added in an amount of 2 units per gram of solid substrate, and the substrate concentration is adjusted to 2 w / v%.
Worked at 6.0 for 24 hours. The reaction solution before and after the enzymatic reaction was analyzed by the TLC method described in Experiment 1 to confirm the presence or absence of enzymatic action on each carbohydrate. Table 11 shows the results.

[0092]

[Table 11]

As is evident from the results in Table 11, α-isomaltosyl glucosaccharide-forming enzyme is one of the saccharides tested among those having a glucose polymerization degree of 3 or more and a maltose structure at the non-reducing end. It has been found to work well for quality. In the case of a saccharide having a glucose polymerization degree of 2, maltose, kojibiose, nigerose, neotrehalose,
It has been found that maltotriitol and elrose also slightly act.

[0094]

[Experiment 11] <Product from maltooligosaccharide>

[Experiment 11-1] <Preparation of product> Purified α-isomaltosylglucosugar obtained by the method of Experiment 7-2 in an aqueous solution of maltose, maltotriose, maltotetraose and maltopentaose having a concentration of 1%. 2 units (maltose and maltotriose), 0.2 units (maltotetraose), per gram of solid,
0.1 units (maltopentaose), 35 ° C, pH
The reaction was carried out at 6.0 for 8 hours, and the reaction was stopped at 100 ° C. for 10 minutes. The sugar composition of the enzyme reaction solution was analyzed by HPLC.
It was measured using the method. HPLC is based on “YMC Pack
ODS-AQ303 ”column (YM
Column temperature 40 ° C., flow rate 0.5 ml /
The detection was carried out under the condition of min water, and the detection was carried out using a differential refractometer “RI-80”.
12 "(manufactured by Tosoh Corporation). Table 12 shows the results.

[0095]

[Table 12]

[0096] As evident from the results in Table 12, the result of the action of the enzyme, from the substrate maltose, mainly glucose and α- iso maltosyl glucose (aka, 6 2 ^-O-alpha
- glucosyl maltose, panose) are generated from the substrate maltotriose, principally maltose and alpha-iso-maltosyl glucose (aka, 6 3 ^{-O-alpha-glucosyl} maltotriose) are generated, while a small amount glucose, maltotetraose, alpha-iso-maltosyl glucose (aka, 6 2 ^{-O-alpha-glucosyl} maltose, panose), that product X is produced was found. From substrate maltotetraose, mainly generated and product X and maltotriose, small amounts while maltose, maltopentaose, alpha-iso-maltosyl glucose (aka, 6 ³ -O
-Α-glucosylmaltotriose), and product Y was found to be produced. From the substrate maltopentaose,
It was found that mainly maltotetraose and product Y were produced, and maltotriose, maltohexaose, product X and product Z were produced in small amounts.

The product X, which is the main product from the substrate maltotetraose, and the product Y, which is the main product from the substrate maltopentaose, were isolated and purified. Preparative H
PLC column "YMC-Pack ODS-A R35
5-15S-15 12A "(manufactured by YMC Corporation) to obtain a product X standard having a purity of 99.9% or more from each of the above-mentioned reactants from maltotetraose and maltopentaose. The product was isolated with a solids yield of about 8.3% and a product Y having a purity of 99.9% or more with a solids yield of about 11.5%.

[0098]

[Experiment 11-2] <Structural analysis of product> Using the product X sample and the product Y sample obtained by the method of Experiment 11-1,
Methylation analysis and NMR analysis were performed according to a conventional method. The results of the methylation analysis are summarized in Table 13. Regarding the results of the NMR analysis, the ¹ H-NMR spectrum is shown in FIG. 21 (product X) and FIG. 22 (product Y), and the ¹³ C-NMR spectrum and assignment are shown in FIG. 23 (product X) and FIG. Compound Y) is summarized in Table 14.

[0099]

[Table 13]

From these results, it is found that the product X from maltotetraose by the α-isomaltosylglucosaccharide-forming enzyme is a pentasaccharide having a glucose group α-bonded to the 6-position hydroxyl group of non-reducing terminal glucose of maltotetraose. in, alpha-iso-maltosyl maltotriose (aka, 6 4 ^-O-α- glucosyl maltotetraose) represented by the structural formula 1 to be proved.

Structural formula 1: αD-Glcp (1 → 6) αD
-Glcp (1 → 4) αD-Glcp (1 → 4) αD-
Glcp (1 → 4) D-Glcp

Further, the product Y from maltopentaose
Is a 6 sugar glucosyl group is bound α to the 6-position hydroxyl group of the non-reducing end glucose of maltopentaose, alpha-iso-maltosyl maltotetraose (aka represented by structural formula 2, 6 5 ^{-O-alpha-glucosyl} Maltopentaose).

Structural formula 2: αD-Glcp (1 → 6) αD
-Glcp (1 → 4) αD-Glcp (1 → 4) αD-
Glcp (1 → 4) αD-Glcp (1 → 4) D-Gl
cp

[0104]

[Table 14]

Based on the above, the action of α-isomaltosylglucosaccharide-forming enzyme on maltooligosaccharides was determined as follows.

(1) The present enzyme uses α-1,
An intermolecular molecule having an action of acting on maltooligosaccharides having a glucose polymerization degree of 2 or more of 2 or more and transferring the glucosyl residue at the non-reducing terminal to position 6 of the glucosyl residue at the non-reducing terminal of another molecule. Catalyzes the 6-glucosyl transfer of α-isomaltosylglucosaccharide (also known as 6-O-α-glucosylmaltooligo) having a 6-O-α-glucosyl group at the non-reducing end and having an increased degree of glucose polymerization by one. sugar)
And a maltooligosaccharide whose glucose polymerization degree has been reduced by one. (2) The enzyme also slightly catalyzes 4-glucosyl transfer, and slightly produces maltooligosaccharides having a glucose polymerization degree increased by 1 and maltooligosaccharides having a glucose polymerization degree decreased by 1 from maltooligosaccharides.

[0107]

[Experiment 12] <Reducing power generation test> The following test was performed to examine whether or not α-isomaltosylglucosaccharide-forming enzyme has a reducing power generating ability. Purified α-isomaltosylglucosaccharide-forming enzyme derived from Bacillus globisporus C9 obtained by the method of Experiment 4-2 in a 1% aqueous maltotetraose solution and purification from Bacillus globisporus C11 obtained by the method of Experiment 7-2 α-Isomaltosyl glucosaccharide-forming enzyme is added in an amount of 0.1 g / g substrate solids.
25 units were added, the mixture was allowed to act at 35 ° C. and pH 6.0, a part of the reaction solution was taken over time, the reaction was stopped at 100 ° C. for 10 minutes to stop the reaction, and the reducing power of the reaction solution was measured. That is, the amount of reducing sugars in the solution before and after the enzyme reaction was measured by the Somogi-Nelson method, and the total amount of sugars in the solution before and after the enzyme reaction was measured by the anthrone sulfate method.
Was calculated using the following formula.

[0108]

[Formula 1] Formula:

Table 15 shows the results.

[0110]

[Table 15]

As is evident from the results in Table 15, the α-isomaltosylglucosaccharide-forming enzyme does not substantially increase the reducing power of the reaction product when maltotetraose is used as a substrate. The α-isomaltosylglucosaccharide-forming enzyme was found to have no or no detectable hydrolysis.

[0112]

[Experiment 13] <Dextran production test> In order to examine whether the α-isomaltosylglucosaccharide-forming enzyme has a dextran-producing effect, “Bioscience Biotechnology and Biochemistry (Bios)
science Biotechnology andB
iochemistry), Vol. 56, 169 to 1
73 (1992). Experiment 4-2 on 1% maltotetraose aqueous solution
The purified α-isomaltosylglucosaccharide-forming enzyme derived from the C9 strain obtained by the method of the above and the purified α-isomaltosylglucosaccharide-forming enzyme derived from the Bacillus globisporus C11 strain obtained by the method of Experiment 7-2 were used as substrates. After adding 0.25 units per gram of the solid, the mixture was allowed to act at 35 ° C. and pH 6.0 for 4 hours and 8 hours, and then kept at 100 ° C. for 15 minutes to stop the reaction. 50 μl of the enzyme reaction solution was placed in a centrifuge tube, and a three-fold amount of ethanol was added thereto. After sufficiently stirring, the mixture was allowed to stand at 4 ° C. for 30 minutes. Then, centrifugation (1
5,000 rpm for 5 minutes), and after removing the supernatant, 1 ml of 75% w / w ethanol was added thereto, followed by stirring and washing. Again, the supernatant was removed by centrifugation, dried under vacuum, and 1 ml of deionized water was added and stirred sufficiently.
The total sugar amount (in terms of glucose) in the solution was measured by the phenol-sulfuric acid method, and was used as the total sugar amount in the test. Bacillus inactivated by heat treatment at 100 ° C for 10 minutes as a blank for reaction
The same procedure was carried out using purified α-isomaltosylglucosaccharide-forming enzyme derived from Globisporus C9 or Bacillus Globisporus C11 to obtain the total sugar amount of the blank. Dextran production was calculated by the following formula.

Formula: Dextran production amount (mg / ml) = [(total sugar content of test)-(total sugar content of blank)] × 20

Table 16 shows the results.

[0115]

[Table 16]

As is evident from the results in Table 16, dextran was not produced even when α-isomaltosylglucosaccharide-forming enzyme was allowed to act on maltotetraose, that is, the α-isomaltosylglucosaccharide-forming enzyme was not produced. It was found that the enzyme had substantially no dextran-producing action or its production was below the detection limit.

[0117]

[Experiment 14] <Transfer receptor specificity>
It was tested whether it could be a transfer receptor for this enzyme.
D-glucose, D-xylose, L-xylose, D
-Galactose, D-fructose, D-mannose,
D-arabinose, D-fucose, L-sorbose, L
-Rhamnose, methyl-α-glucoside, methyl-β-
Glucoside, N-acetyl-glucosamine, sorbitol, α, α-trehalose, isomaltose, isomalttriose, cellobiose, gentibiose, maltitol, lactose, sucrose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin A solution was prepared.

In these receptor solutions (concentration 1.6%),
Partially degraded starch [Paindex # 10] as a sugar donor
0 "(concentration: 4%), and purified Bacillus globisporus C9-derived purified α-isomaltosylglucosaccharide-forming enzyme preparation obtained by the method of Experiment 4-2 or Bacillus obtained by the method of Experiment 7-2. A purified α-isomaltosylglucosaccharide-forming enzyme preparation derived from Globisporus C11 was added in an amount of 1 unit per 1 g of solid saccharide donor, and the resulting mixture was added in an amount of 30 units.
The reaction was performed at pH 6.0 for 24 hours. The reaction solution after the enzyme reaction is analyzed by gas chromatography (hereinafter abbreviated as "GLC method") when the acceptor is a monosaccharide or disaccharide, and by HPLC when the acceptor is a trisaccharide or more. Then, it was confirmed whether each carbohydrate would be a transfer receptor for the present enzyme. In the GLC, the GLC device is “GC-16A”
(Manufactured by Shimadzu Corporation), a stainless steel column (3 mmφ × 2) filled with “2% silicon OV-17 / Chromosolve W” manufactured by GL Sciences, Inc.
m) As a carrier gas, nitrogen gas was heated at a flow rate of 40 ml / min from 160 ° C. to 320 ° C. at a rate of 7.5 ° C./min, and the detection was analyzed by a flame ion detector. In HPLC, the equipment of HPLC is "CCPD" manufactured by Tosoh Corporation,
The column was "ODS-AQ-303" (manufactured by YMC Co., Ltd.), the eluent was water at a flow rate of 0.5 ml / min, and the detection was analyzed by differential refraction. Table 17 shows the results.

[0119]

[Table 17]

As is clear from the results in Table 17, various carbohydrates can be used as α-isomaltosylglucosaccharide-forming enzyme as transfer receptors, and in particular, D- / L-xylose,
Methyl-α-glucoside, methyl-β-glucoside,
Transforms well to α, α-trehalose, isomaltose, isomalttriose, cellobiose, gentibiose, maltitol, lactose and sucrose, followed by D-glucose, D-fructose, D-fucose, L-sorbose and N -Acetylglucosamine, and further to D-arabinose.

Regarding the properties of the above-mentioned α-isomaltosylglucosaccharide-forming enzyme, an enzyme having a 6-glucosyltransferring activity previously reported, “Bioscience.
Biotechnology and Biochemistry (B
ioscience Biotechnology a
nd Biochemistry), Vol. 56, No. 1
Dextrin dextranase described in pages 69 to 173 (1992) and "Journal of the Japanese Society of Agricultural Chemistry", No. 3,
7, pp. 668-672 (1963). Table 18 summarizes the results.

[0122]

[Table 18]

As is clear from Table 18, it was found that α-isomaltosylglucosaccharide-forming enzyme has novel physicochemical properties completely different from known dextrin dextranase and transglucosidase.

[0124]

[Experiment 15] <Production of cyclic tetrasaccharide>
-A cyclic tetrasaccharide formation test was performed by the action of isomaltosyl glucosaccharide-forming enzyme and α-isomaltosyltransferase. Maltose, maltotriose, maltotetraose, maltopentaose, amylose, soluble starch, partially degraded starch (trade name “Paindex # 100”, manufactured by Matsutani Chemical Co., Ltd.) or glycogen (oyster-derived, Wako Pure Chemical Industries, Ltd.) Solution) was prepared.

To these aqueous solutions (concentration: 0.5%), Bacillus globisporus C11 obtained by the method of Experiment 7-2 was added.
The purified α-isomaltosylglucosaccharogenic enzyme preparation of 1 unit per gram of the solid substance was added to the purified α-isomaltosylglucosaccharide-forming enzyme preparation according to the method of Experiment 7-3.
Isomaltosyltransferase preparation was added to 10 g of solid
Units were added and these were allowed to act at 30 ° C., pH 6.0. The working conditions were performed in the following four systems.

(1) After the α-isomaltosylglucosaccharide-forming enzyme was allowed to act for 24 hours, the enzyme was inactivated by heat. Then, after the α-isomaltosyltransferase was allowed to act for 24 hours, the enzyme was heat-activated. Deactivated. (2) α-Isomaltosylglucosaccharide-forming enzyme and α-isomaltosylglucosaccharide-forming enzyme
After simultaneous action with isomaltosyltransferase for 24 hours, the enzyme was heat-inactivated. (3) After allowing only the α-isomaltosylglucosaccharide-forming enzyme to act for 24 hours, the enzyme was heat-inactivated. (4) After allowing only α-isomaltosyltransferase to act for 24 hours, the enzyme was heat-inactivated.

In order to examine the amount of cyclic tetrasaccharide produced in the heat-inactivated reaction solution, α-glucosidase / glucoamylase was treated in the same manner as in Experiment 1, the remaining reducing oligosaccharide was hydrolyzed, and HPLC was performed. The cyclic tetrasaccharide was quantified by the method. Table 19 shows the results.

[0128]

[Table 19]

As is evident from the results in Table 19, none of the carbohydrates tested showed any cyclic tetrasaccharides with only the action of α-isomaltosylglucosaccharide-forming enzyme and the action of α-isomaltosyltransferase alone. In contrast, α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase were simultaneously used to produce a cyclic tetrasaccharide. When the α-isomaltosyltransferase is allowed to act after the α-isomaltosylglucosaccharide-forming enzyme is acted upon, the amount of the produced is relatively low, about 11% or less, whereas the amount of α-isomaltosylglucosidase is relatively low. When maltosyl glucosaccharide-forming enzyme and α-isomaltosyltransferase are allowed to act simultaneously, the amount of cyclic tetrasaccharide produced by any of the carbohydrates is improved, and in particular, glycogen is increased to about 87%, and partially degraded starch It turned out to increase to about 64%.

The mechanism of cyclic tetrasaccharide formation in the simultaneous use of α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase is inferred from the reaction characteristics of both enzymes as follows.

(1) α-Isomaltosyl glucosaccharide-forming enzyme is used for α-isomaltosylglucosaccharide-forming enzymes such as glycogen and partially degraded starch.
Acts on the non-reducing terminal glucose group of the 1,4 glucan chain,
The glucose group is intermolecularly transferred to the hydroxyl group at the 6-position of the glucose group at the non-reducing terminal of another α-1,4 glucan chain, and an α-1,4 glucan chain having an α-isomaltosyl group at the non-reducing terminal is generated. (2) α-Isomaltosyltransferase acts on an α-1,4 glucan chain having an isomaltosyl group at a non-reducing terminal, and converts the isomaltosyl group into an α-1,4 glucan having another isomaltosyl group at another non-reducing terminal. The intermolecular transfer to the hydroxyl group at the 3-position of the non-reducing terminal glucose group of the chain results in the formation of an α-1,4 glucan chain having an isomaltosyl-1,3-isomaltosyl group at the non-reducing terminal. (3) Subsequently, α-isomaltosyltransferase acts on an α-1,4 glucan chain having an isomaltosyl-1,3-isomaltosyl group at its non-reducing end, and isomaltosyl-1,3-glucan is transferred by intramolecular transfer. The isomaltosyl group is cleaved from the α-1,4 glucan chain and cyclized to form a cyclic tetrasaccharide. (4) The separated α-1,4 glucan chain is again
By undergoing the reactions of (1) to (3),
A cyclic tetrasaccharide is produced. With simultaneous use of α-isomaltosyl glucosaccharide-forming enzyme and α-isomaltosyltransferase,
As described above, it was speculated that both enzymes repeatedly act to increase the amount of cyclic tetrasaccharide produced.

[0132]

[Experiment 16] <Effect of degree of starch liquefaction> Corn starch was used as a 15% starch milk, and calcium carbonate was added to the milk at a concentration of 0.1%.
The pH was adjusted to 6.0 by adding 1%, and 0.2 to 2.0% of α-amylase (trade name “Tamamir 60L” (manufactured by Novo)) was added per gram of starch, and reacted at 95 ° C. for 10 minutes. Then autoclaved at 120 ° C for 20 minutes,
After quenching to about 35 ° C., a liquefied solution of DE 3.2 to 20.5 was obtained, and the purified α-isomaltosyl glucosaccharide-forming enzyme derived from Bacillus globisporus C11 obtained by the method of Experiment 7-2 was added thereto. 2 units per gram of the solid, and 20 units per gram of the purified α-isomaltosyltransferase derived from Bacillus globisporus C11 obtained by the method of Experiment 7-3, and the mixture was added at 35 ° C. for 24 hours. Reacted. The enzyme was inactivated by heat treatment at 100 ° C. for 15 minutes. Subsequently, the mixture was treated with α-glucosidase and glucoamylase in the same manner as in Experiment 1, the remaining reducing oligosaccharide was hydrolyzed, and the cyclic tetrasaccharide was quantified by the HPLC method. Table 20 shows the results.

[0133]

[Table 20]

As is evident from the results in Table 20, the formation of cyclic tetrasaccharide by α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase is affected by the degree of starch liquefaction. The lower the degree, that is, the lower the value of DE, the higher the rate of formation of the cyclic tetrasaccharide from the starch, and conversely, the higher the degree of liquefaction, that is, the higher the value of DE, the higher the cyclic rate from the starch. It was found that the production rate of tetrasaccharide was low. In order to increase the rate of formation of a cyclic tetrasaccharide from starch, it has been found that the degree of partial decomposition of starch is suitably about 20 or less, preferably about 12 or less, more preferably about 5 or less. .

[0135]

[Experiment 17] <Effect of concentration of partially degraded starch> An aqueous solution of partially degraded starch “Paindex # 100” (DE about 2 to 5) having a final concentration of 0.5 to 40% was prepared. The purified α-isomaltosylglucosaccharide-forming enzyme preparation derived from Bacillus globisporus C11 obtained by the method of Example-2 was subjected to 1 unit per gram of the solid material, and the amount of the sample was measured in Experiment 7-3.
A purified α-isomaltosyltransferase sample derived from Bacillus globisporus C11 obtained by the method of 1) was added in an amount of 10 units per gram of solid material, and both enzymes were used simultaneously at 30 ° C. and pH
After acting at 6.0 for 48 hours, the enzyme was deactivated by heat treatment at 100 ° C. for 15 minutes. Then, as in Experiment 1, α
-Treated with glucosidase and glucoamylase, hydrolyzed the remaining reducing oligosaccharides, and quantified the cyclic tetrasaccharide by HPLC. Table 18 shows the results.

[0136]

[Table 21]

As is clear from the results shown in Table 21, when the concentration of the partially degraded starch was as low as 0.5%, the amount of cyclic tetrasaccharide produced was about 64%, whereas the amount of the cyclic tetrasaccharide was as high as 40%. In terms of the concentration, the amount of cyclic tetrasaccharide produced was about 40%, indicating that the amount of cyclic tetrasaccharide produced varies depending on the concentration of the partial starch hydrolyzate as a substrate. From these results, the amount of cyclic tetrasaccharide produced was
It was found that the lower the concentration of partially decomposed starch, the higher the tendency.

[0138]

[Experiment 18] <Effect of addition of cyclodextrin glucanotransferase> Starch partially decomposed product “Paindex #
100 "aqueous solution (concentration 15%) was prepared, and the purified α-isomaltosylglucosaccharide-forming enzyme preparation derived from Bacillus globisporus C11 obtained by the method of Experiment 7-2 was 1 unit per gram of solid matter. C1 obtained by the method of 7-3
1 unit of purified α-isomaltosyltransferase from one strain is added to 10 units per gram of solid, and 0 to 0.5 units of Bacillus stearothermophilus-derived cyclodextrin glucanotransferase (CGTase) is added to 1 gram of solid. , 30 ° C, pH by simultaneous use of both enzymes
After acting at 6.0 for 48 hours, the enzyme was deactivated by heat treatment at 100 ° C. for 15 minutes. Then, as in Experiment 1, α
-Treated with glucosidase and glucoamylase, hydrolyzed the remaining reducing oligosaccharides, and quantified the cyclic tetrasaccharide by HPLC. Table 22 shows the results.

[0139]

[Table 22]

As is clear from the results in Table 22, CGT
It was found that the addition of ase increased the amount of cyclic tetrasaccharide produced.

[0141]

[Experiment 19] <Preparation of cyclic tetrasaccharide> An aqueous solution (about 100 l) of corn-derived phytoglycogen (manufactured by Kewpie Co., Ltd.) was used at a concentration of 4 w / v%, pH 6.0, and a temperature of 30 ° C.
After the purification, the purified α-isomaltosylglucosidase-producing enzyme preparation obtained by the method of Experiment 7-2 was added to the purified α-isomaltosyl obtained by the method of Experiment 7-3 at 1 unit per gram of solid. A transferase preparation was added at 10 units per gram of solid, allowed to act for 48 hours, and then heat-treated at 100 ° C. for 10 minutes to inactivate the enzyme. Take a part of this reaction solution, HP
When the amount of cyclic tetrasaccharide produced was examined by LC, the sugar composition was about 84%. This reaction solution was adjusted to pH 5.0 at a temperature of 45 ° C.
After treatment with α-glucosidase and glucoamylase in the same manner as in Experiment 1, the remaining reducing oligosaccharides and the like are hydrolyzed, the pH is adjusted to 5.8 with sodium hydroxide, and the temperature is adjusted to 90. C. for 1 hour to inactivate the enzyme, and insolubles were removed by filtration. After concentrating the filtrate to about 16% solid content using a reverse osmosis membrane,
After decoloring, desalting, filtering and concentrating according to a conventional method, about 6.2 kg of a sugar solution containing about 3,700 g of a solid content was obtained.

The obtained sugar solution was applied to a column (about 225 liters of gel) filled with an ion exchange resin “Amberlite CR-1310 (Na type)” manufactured by Organo, and the column temperature was changed to 6 ° C.
Chromatographic separation was performed at 0 ° C. at a flow rate of about 45 l / h. The sugar composition of the eluate was monitored by the HPLC method described in Experiment 1, and a fraction having a purity of the cyclic tetrasaccharide of 98% or more was collected.
After desalting, decoloring, filtering and concentrating according to a conventional method, about 7.5 kg of a sugar solution containing about 2,500 g of solids was obtained. When the sugar composition of the obtained sugar solution was measured by an HPLC method, the purity of the cyclic tetrasaccharide was about 99.5%.

[0143]

[Experiment 20] <Crystallization of cyclic tetrasaccharide from aqueous solution> Experiment 1
The cyclic tetrasaccharide fraction having a purity of about 98% or more obtained by the method of Example 9 is concentrated to a solid concentration of about 50% by an evaporator, and then about 5 kg of the concentrated sugar solution is put into a cylindrical plastic container and gently rotated. About 65 hours in about 20 hours
When the solution was crystallized by lowering the temperature to 20 ° C., a white crystalline powder was obtained. 1 of the micrograph
An example is shown in FIG. Subsequently, the mixture was separated using a centrifugal filter, and 1,360 g of the crystalline material was recovered as a wet weight. Further, after drying at 60 ° C. for 3 hours, the cyclic tetrasaccharide crystalline powder was
170 g were obtained. When the sugar composition of the obtained crystal powder was measured by HPLC method, the purity of the cyclic tetrasaccharide was extremely high at 99.9% or more.

When the crystalline powder of this cyclic tetrasaccharide was analyzed by powder X-ray diffraction method, as shown in FIG. 26, the main diffraction angles (2θ) were 10.1 °, 15.2 °, and 20.3 °. A diffraction spectrum characterized by ° and 25.5 ° was obtained. The water content of the crystalline powder was measured by the Karl Fischer method and found to be 13.0%, indicating that the crystalline powder contained 5 to 6 molecules of water per 1 molecule of the cyclic tetrasaccharide. did.

Further, the thermogravimetric measurement of this cyclic tetrasaccharide crystal powder gave a thermogravimetric curve shown in FIG. 27. From the relationship between the change in weight and the temperature, 4 to 5 at a temperature increase to 150 ° C. Weight loss corresponding to molecular water is observed,
Subsequently, a weight loss corresponding to one molecule of water was observed at a temperature of about 250 ° C., and a weight loss considered to be due to thermal decomposition of the cyclic tetrasaccharide itself was observed at a temperature of about 280 ° C. From these, the cyclic tetrasaccharide 5 to 6 hydrous crystal of the present invention contains 1 molecule of water by removing 4 to 5 molecules of water per crystal molecule by raising the temperature to 150 ° C. at normal pressure. It becomes crystalline, and becomes 1
It has been found that molecular water is released from the crystals to form anhydrous crystals.

[0146]

[Experiment 21] <Conversion to cyclic tetrasaccharide 1 hydrous crystal> Experiment 20
The cyclic tetrasaccharide 5-6 hydrous crystal powder obtained by the above method was placed in a glass container, and the glass container was held in an oil bath kept at 140 ° C. in advance for 30 minutes. When the obtained cyclic tetrasaccharide powder was analyzed by powder X-ray diffraction method, it was completely different from powder X-ray diffraction of 5 to 6 hydrous crystals before heat treatment, and as shown in FIG. 28, the main diffraction angle (2θ) 8.3
Diffraction spectra were obtained which were characterized by °, 16.6 °, 17.0 ° and 18.2 °. The water content of the crystalline powder was measured by the Karl Fischer method and found to be about 2.7%, indicating that one molecule of cyclic tetrasaccharide contained one molecule of water. Further, the thermogravimetric measurement of this crystal powder yielded a thermogravimetric curve shown in FIG. 29. From the relationship between the weight change and the temperature,
At around 70 ° C., a weight loss corresponding to one molecule of water was observed, and at a temperature around 290 ° C., a weight loss considered to be due to thermal decomposition of cyclic tetrasaccharide itself was observed. From these, it was found that the cyclic tetrasaccharide crystalline material of the present invention was a monohydrate crystal.

[0147]

[Experiment 22] <Conversion to anhydrous crystals> Cyclic tetrasaccharide 5 to 6 hydrous crystal powder obtained by the method of Experiment 20 was vacuum dried at a temperature of 40 ° C to 120 ° C for 16 hours. When the water content of the obtained cyclic tetrasaccharide powder was measured by the Karl Fischer method, the water content was about 4.2% when the vacuum drying temperature was 40 ° C.
When the vacuum drying temperature is 120 ° C., the water content is about 0.2
% Was found to be substantially anhydrous. When the vacuum-dried cyclic tetrasaccharide powder was analyzed by powder X-ray diffraction method, it was completely different from powder X-ray diffraction of 5 to 6 hydrous crystals before vacuum drying and that of 1 hydrous crystal. 40 ° C.), as shown in FIG. 31 (vacuum drying temperature 120 ° C.), the main diffraction angles (2θ) are 10.8 °, 1θ,
Diffraction spectra characterized by 4.7 °, 15.0 °, 15.7 ° and 21.5 ° were obtained. Although peak intensity was observed between the two diffraction spectra, the diffraction angle of the peak was basically the same, and it was presumed that they were the same anhydrous crystal in crystallography. In addition, the baseline of the diffraction spectrum has a mountain-like shape, and the degree of crystallinity is lower than that of the crystalline tetrasaccharide having 5 to 6 hydrous crystals and that of the monohydrated crystal before vacuum drying. Was found to be present. From this, vacuum drying 40 ° C
In this case, the cyclic tetrasaccharide powder containing about 4.2% of water was presumed to be a powder in which the cyclic tetrasaccharide amorphous containing the water and the cyclic tetrasaccharide anhydrous crystal were mixed. From the above, it is possible to obtain 5
It has been found that the water-containing crystals of Nos. 6 to 6 lose water molecules and are converted into amorphous and amorphous crystals in an amorphous state. The thermogravimetric analysis of the anhydrous cyclic tetrasaccharide powder having a water content of 0.2% was performed in the same manner as in Experiment 20, and as shown in FIG.
From around 70 ° C., only the weight loss considered to be due to thermal decomposition of the cyclic tetrasaccharide was observed.

[0148]

[Experiment 23] <Saturation concentration of cyclic tetrasaccharide in water> Temperature 1
In order to examine the saturated concentration of the cyclic tetrasaccharide in water at 0 to 90 ° C., 10 ml of water was placed in a sealed glass container, and the cyclic tetrasaccharide 5 to 6 hydrous crystals obtained by the method of Experiment 20 were added at each temperature. After adding more than the amount that completely dissolves,
The glass container is sealed and kept at a temperature of 10-9 until saturation is reached.
The mixture was stirred for 2 days while keeping the temperature at 0 ° C. After the cyclic tetrasaccharide saturated solution at each temperature was subjected to microfiltration to remove the undissolved cyclic tetrasaccharide, the water content of the filtrate was examined by a dry weight loss method, and the saturated concentration at each temperature was determined. The results are shown in Table 23.

[0149]

[Table 23]

[0150]

[Experiment 24] <Thermal stability> Cyclic tetrasaccharide 5-6 hydrous crystals obtained by the method of Experiment 20 were dissolved in water, and the concentration was 10 w / v%.
Was prepared in a glass test tube, sealed, and heated at 120 ° C. for 30 to 90 minutes. After allowing to cool, measure the degree of coloring of these solutions,
Purity was measured by the PLC method. Coloring degree is 480n
It was evaluated by the absorbance in a 1 cm cell at m. The results are shown in Table 24.

[0151]

[Table 24]

As is clear from the results in Table 24, the cyclic tetrasaccharide aqueous solution was not colored even at a high temperature of 120 ° C., the purity of the saccharide composition did not decrease, and the cyclic tetrasaccharide was a heat-stable saccharide. It turned out to be.

[0153]

[Experiment 25] <pH stability> The cyclic tetrasaccharide 5-6 hydrous crystals obtained by the method of Experiment 20 were mixed with various buffer solutions (20 mM).
And a cyclic tetrasaccharide at a concentration of 4 w / v% and a pH of 2 to 1
A cyclic tetrasaccharide solution adjusted to 0 was prepared. 8 ml of each solution is placed in a glass test tube, sealed, and then heated to 100 ° C.
For 24 hours. After cooling, the solutions were measured for the degree of coloring and the purity was measured by the HPLC method. The coloring degree is
Evaluated by absorbance at 480 nm in a 1 cm cell. The results are shown in Table 25.

[0154]

[Table 25]

As is evident from the results in Table 25, the aqueous solution of cyclic tetrasaccharide was heated at a high temperature of 120 ° C. for 24 hours,
There is no coloring in a wide range of 2 to 10, and although the purity of the saccharide composition slightly decreases at pH 2, the purity of the saccharide composition does not decrease at all in the range of pH 3 to 10, and the cyclic tetrasaccharide has a wide p-type.
It has been found that the saccharide is extremely stable even when boiled in the H range, in other words, the acidic side at pH 3 to 5, the neutral side at pH 6 to 8, and the alkaline side at pH 9 to 10.

[0156]

[Experiment 26] <Aminocarbonyl reaction> The cyclic tetrasaccharide 5 to 6 hydrous crystals obtained by the method of Experiment 20 were dissolved in water, and then a commercially available reagent of special grade glycine and a phosphate buffer were added thereto. 1 adjusted to pH 7.0 with liquid
A 10 w / v% cyclic tetrasaccharide solution containing w / v% glycine was prepared. 4 ml of the solution was placed in a glass test tube, sealed, and heated at 100 ° C. for 30 to 90 minutes. After cooling in a room, their coloring degree was measured to examine aminocarbonyl reactivity. The degree of coloring was evaluated by the absorbance at 480 nm in a 1 cm cell. The results are shown in Table 26.

[0157]

[Table 26]

As is clear from the results shown in Table 26, the cyclic tetrasaccharide was not colored even when heated in the presence of glycine, and did not cause browning with glycine. In other words, the aminocarbonyl reaction (also called the Maillard reaction) ) Is found to be a stable carbohydrate that is unlikely to occur.

[0159]

[Experiment 27] <Aminocarbonyl reaction> Cyclic tetrasaccharide 5-6 hydrous crystals obtained by the method of Experiment 20 and commercially available polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) were dissolved in deionized water, and 5 w / v%
A 10% w / v cyclic tetrasaccharide solution containing polypeptone was prepared. 4 ml of the solution was placed in a glass test tube, sealed, and heated at 120 ° C. for 30 to 90 minutes. After cooling in a room, their coloring degree was measured to examine aminocarbonyl reactivity. At the same time, the solution containing only polypeptone was heated similarly as a blank. The degree of coloring was evaluated based on the value obtained by subtracting the absorbance of the blank from the absorbance at 480 nm in a 1 cm cell. The results are shown in Table 27.

[0160]

[Table 27]

As is evident from the results in Table 27, the cyclic tetrasaccharide is a stable saccharide which does not cause browning with polypeptone when heated in the presence of polypeptone, in other words, is a stable saccharide which is unlikely to cause an aminocarbonyl reaction. It turned out to be.

[0162]

[Experiment 28] <Inclusion function> The hydrous crystals of cyclic tetrasaccharide 5 or 6 obtained by the method of Experiment 20 were dissolved in deionized water, and 20%
An aqueous solution was prepared. For 100 g of the aqueous solution, 2 g of methanol, 3 g of ethanol, and 4.6 g of acetic acid were added for inclusion. Thereafter, each was filtered and the filtrate was freeze-dried to remove unclathrates. As a control, the same procedure was performed using a branched cyclodextrin (trade name “Iso-Elite P”, marketed by Maruha Co., Ltd.) known to have an inclusion ability.

In order to measure the amount of clathrate in the lyophilized powder, 1 g of each lyophilized powder was dissolved in 5 ml of water, and 5 ml of diethyl ether was added thereto for extraction, and the extraction was repeated again. The extract in diethyl ether was quantified by gas chromatography. The results are shown in Table 28.

[0164]

[Table 28]

As is clear from the results in Table 28, the cyclic tetrasaccharide has an inclusion ability, and the inclusion ability was found to be about twice as high as that of the branched cyclodextrin per weight. .

[0166]

[Experiment 29] <Sweetness> The hydrated crystals of cyclic tetrasaccharide 5 to 6 obtained by the method of Experiment 20 are dissolved in deionized water to prepare an aqueous solution having a solid concentration of 10%. The concentration of sucrose (a commercially available granulated sugar) was changed, and a sensory test was conducted by five panelists. As a result, the degree of sweetness of the cyclic tetrasaccharide was about 20% of that of sucrose.

[0167]

[Experiment 30] <Digestibility test> Using the hydrous crystals of cyclic tetrasaccharide 5 or 6 obtained by the method of Experiment 20, "Journal of the Japanese Society of Nutrition and Food", Vol. 43, pp. 23-29 (1990) According to the method of Okada et al. Described above, the digestibility of cyclic tetrasaccharide by salivary amylase, synthetic gastric juice, pancreatic amylase, and small intestinal mucosal enzyme in a test tube was examined. As a control, maltitol, which is known as an indigestible carbohydrate, was used. The results are shown in Table 29.

[0168]

[Table 29]

As is clear from the results in Table 29, the cyclic tetrasaccharide was not digested at all by salivary amylase, synthetic gastric juice, or pancreatic amylase, but was slightly digested by small intestinal mucosal enzyme, but the degree was 0.74%. The value was 1/5 or less as compared with the value of the indigestible carbohydrate maltitol as a control, and it was found that the cyclic tetrasaccharide was a carbohydrate extremely difficult to digest.

[0170]

[Experiment 31] <Fermentability test> Using the cyclic tetrasaccharide 5 to hydrous crystal obtained by the method of Experiment 20, "Journal of Nutrition Science and Vitaminology (Journal of Nutritiona)"
l Science and Vitaminlog
y) ", Vol. 37, pp. 529-544 (1991)
), The fermentability of cyclic tetrasaccharides with rat cecal contents was examined. The cecal contents used were male Wistar rats that had been sacrificed under ether anesthesia, anaerobically collected, and suspended in a four-fold amount of a 0.1 M aqueous sodium hydrogen carbonate solution. About 7% of the cyclic tetrasaccharide was added based on the weight of the cecal contents, and the amount of the remaining cyclic tetrasaccharide immediately after the addition and 12 hours later was determined by gas chromatography.
As a result, the cyclic tetrasaccharide concentration immediately after the addition was 68.0 mg per g of cecal content, and the cyclic tetrasaccharide concentration after 12 hours was 63.0 mg per g of cecal content, with 93% remaining without fermentation. It was found that the cyclic tetrasaccharide was a carbohydrate that was extremely difficult to ferment.

[0171]

[Experiment 32] <Assimilation test> Using the hydrous crystals of cyclic tetrasaccharide 5 to 6 obtained by the method of Experiment 20, "Intestinal Flora and Food Factors", edited by Tomohiko Mitsuoka, Gakkai Shuppan Center (1984)
), The assimilation of various intestinal bacteria was examined. That is, about 10 ⁷ CFU of freshly cultured bacteria was added to 5 ml of PYF medium supplemented with 0.5% of cyclic tetrasaccharide.
The cells were inoculated and cultured under anaerobic conditions at 37 ° C. for 4 days. As a control, saccharide glucose was used, which was easily assimilated. The assimilation was determined by determining that the pH of the culture solution after the cultivation was 6.0.
In the above case, it was regarded as not assimilated (-), and when it was less than 6.0, as assimilated (+). Furthermore, the amount of carbohydrate remaining in the culture solution was measured by the anthrone method to determine the amount of carbohydrate reduction,
The determination of assimilation was confirmed. The results are shown in Table 30.

[0172]

[Table 30]

As can be seen from the results in Table 30, the cyclic tetrasaccharide was not assimilated by any of the intestinal bacterial strains tested,
It was found that the control glucose was assimilated into any of them, and that the cyclic tetrasaccharide was a carbohydrate that was extremely difficult to assimilate by intestinal bacteria.

[0174]

[Experiment 33] <Acute toxicity test> Using a mouse, an acute toxicity test was carried out by orally administering the hydrous crystals of cyclic tetrasaccharide 5 to 6 obtained by the method of Experiment 20. As a result, the cyclic tetrasaccharide was a low-toxic substance, and no deaths were observed even at the maximum administrable dose. Therefore, although it cannot be said that it is an accurate value, its LD50 value was 50 g / kg mouse body weight or more.

From the results of the above Experiments 30 to 33, the cyclic tetrasaccharide is hardly digested and absorbed even when taken orally, and is a dietary sweetener or a high-sweetness sweetener as a calorie-free or low-calorie edible material. It can be expected to be used as an excipient, a dietary food and drink thickener, a bulking agent, an excipient, and further as a dietary fiber, a fat substitute food material and the like.

Hereinafter, a method for producing a saccharide mixture containing a sugar alcohol together with the cyclic tetrasaccharide of the present invention will be described in Example A.
In Example B, a composition containing a carbohydrate mixture containing a sugar alcohol together with a cyclic tetrasaccharide is shown.

[0177]

Example A-1 Bacillus globisporus C9 (F
ERM BP-7143) was cultured in a fermenter for 48 hours according to the method of Experiment 3. After the cultivation, the bacteria were filtered off using an SF membrane, and about 18 l of the culture filtrate was collected.
The filtrate was concentrated on a UF membrane, and about 1 liter of a concentrated enzyme solution containing 8.8 units / ml of α-isomaltosylglucosaccharide-forming enzyme and 26.7 units / ml of α-isomaltosyltransferase.
Was recovered.

The potato starch milk was made into a starch milk having a concentration of about 2%, calcium chloride was added thereto to a final concentration of 1 mM, the pH was adjusted to 6.0, and the mixture was heated at 95 ° C. for about 20 minutes to perform gelatinization. Then, the mixture was cooled to about 35 ° C., and the α-isomaltosylglucosaccharide-forming enzyme prepared by the above method and α
A concentrated enzyme solution containing -isomaltosyltransferase was added at a ratio of 0.25 ml per gram of solid starch, and reacted at pH 6.0 at a temperature of 35 ° C for 48 hours. The reaction was heated to 95 ° C. and held for 10 minutes, then cooled,
After filtration, purify according to the usual method (decolorization, desalting)
Then, the resultant was concentrated to obtain a syrup having a concentration of 50% of cyclic tetrasaccharide in a yield of about 90% based on the raw starch solids. Put the syrup in an autoclave, add Raney nickel 6%, raise the temperature to 90 to 120 ° C with stirring, and increase the hydrogen pressure to 2 ° C.
After completing the hydrogenation by raising the pressure to 0 to 120 kg / cm ² , the Raney nickel was removed, and then decolorization, desalting, purification and concentration were carried out according to a conventional method, and the syrup having a concentration of 70% was converted into a raw starch solid. Obtained in about 80% yield per product.

The present syrup is a saccharide mixture containing a sugar alcohol together with a cyclic tetrasaccharide and having a DE of less than 0.5, comprising 62.1% of cyclic tetrasaccharide, 0.7% of sorbitol,
Contains 1.4% isomaltitol, 11.1% maltitol and 24.7% other sugar alcohols,
It has mild sweetness, moderate viscosity, moisturizing properties, inclusion properties, low-calorie sweeteners, taste improvers, quality improvers, water separation inhibitors,
As a stabilizer, a discoloration inhibitor, an excipient, a clathrate, a powdered base material, etc., it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

[0180]

Example A-2 Potato starch was made into about 6% starch milk,
Calcium carbonate was added thereto to a concentration of 0.1% to adjust the pH to 6.0, and α-amylase (trade name “Tamamir 60L”, manufactured by Novo Co.) was added in an amount of 0.2% per gram of starch solids. And reacted at 95 ° C. for 10 minutes, then autoclaved at 120 ° C. for 20 minutes, and rapidly cooled to about 35 ° C. to obtain a liquefied solution of about 4 DE.
To this was added a concentrated solution containing α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferase prepared by the method of Example A-1 at a rate of 0.25 ml per gram of starch solids. , PH 6.0, 48 at 35 ° C
Allowed to react for hours. The reaction solution was heated to 95 ° C. and maintained for 10 minutes, then cooled and filtered to obtain a cyclic tetrasaccharide-containing syrup at a yield of about 90% based on the raw starch solids. This syrup was hydrogenated, purified and concentrated according to the method of Example A-1, and then purified (decolorized and desalted) according to a conventional method.
Concentration gave a syrup with a concentration of 70% in a yield of about 80% per raw starch solids. This syrup is a carbohydrate mixture containing a sugar alcohol together with a cyclic tetrasaccharide and having a DE of less than 0.5. Per solid, 60.1% cyclic tetrasaccharide, 0.9% sorbitol, 1.5% isomaltitol, 1.5% maltitol Thor 1
Contains 1.3% and 26.2% of other sugar alcohols, has mild sweetness, moderate viscosity, moisturizing properties, and inclusion properties, and has a low calorie sweetener, taste improver, and quality improver. It can be advantageously used for various compositions such as various foods and drinks, cosmetics and pharmaceuticals, as a water separation preventing agent, a stabilizer, a discoloration preventing agent, an excipient and an inclusion agent.

[0181]

Example A-3 Bacillus globisporus C11
(FERM BP-7144) was cultured in a fermenter for 48 hours according to the method of Experiment 6. After culture, SF
The membrane was sterilized and filtered, and about 18 l of the culture filtrate was collected.
Further, the filtrate is concentrated on a UF membrane, and about 1 liter of a concentrated enzyme solution containing 9.0 units / ml of α-isomaltosylglucosaccharidase and 30.2 units / ml of α-isomaltosyltransferase is recovered. did. Tapioca starch is used as a starch milk having a concentration of about 25%, and α-amylase (trade name “Neospitase”, manufactured by Nagase Seikagaku Co., Ltd.) is added at 0.2% per gram of the starch solid. After autoclaving at 120 ° C. for 20 minutes, the mixture was rapidly cooled to about 35 ° C. to obtain a liquefied solution having a DE of about 4, into which α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosylglucosaccharide-forming enzyme were prepared. -Concentrated enzyme solution containing isomaltosyltransferase was added at a rate of 0.25 ml per gram of solid starch, and cyclomaltodextrin glucanotransferase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.)
Is added to give 10 units per gram of starch solids,
The reaction was carried out at pH 6.0 and a temperature of 35 ° C. for 48 hours. After keeping the reaction solution at 95 ° C. for 30 minutes,
After the temperature was adjusted to 0 ° C., an α-glucosidase agent (trade name “transglucosidase L“ Amano ””, manufactured by Amano Pharmaceutical Co., Ltd.) was added at 300 units per gram of solid, and 24
After reacting for a time, a glucoamylase agent (trade name “Glucoteam”, manufactured by Nagase Seikagaku Corporation) is added to solid 1
After adding 30 units per gram and reacting for 17 hours, the reaction solution was heated to 95 ° C. and maintained for 30 minutes, cooled, filtered, and thereafter purified (decolorized, desalted) and concentrated according to a conventional method. A syrup containing 50% cyclic tetrasaccharide was obtained at a yield of about 90% based on the raw starch solids. Example A of this syrup
According to the method of -1, hydrogenation, purification, concentration,
A syrup having a concentration of 77% was placed in an auxiliary crystal can, and 5 to 6 hydrous crystals of cyclic tetrasaccharide and hydrated sorbitol were added as seed crystals in an amount of about 2% each, followed by slow cooling to obtain a mass kit. Spraying was performed from a nozzle at a high pressure of 150 kg / cm ² . At the same time, hot air of 85 ° C is blown from the top of the drying tower, and the crystal powder is collected on a transfer wire mesh conveyor provided at the bottom, and the powder is dried while sending hot air of 45 ° C from under the conveyor. Moved slowly out of the cabin and removed. The crystal powder was filled in a maturation tower, aged for 10 hours while sending warm air, and crystallization and drying were completed to obtain a powder containing hydrous crystals of cyclic tetrasaccharides 5 to 6 and sorbitol. This product is a saccharide mixture containing a sugar alcohol together with a cyclic tetrasaccharide and having a DE of less than 0.5. Per solid (in terms of anhydride), 58.1% of cyclic tetrasaccharide, 38.4% of sorbitol and other sugar alcohols Contains 3.5%, has a mild sweetness, moisturizing property, and inclusion property, and has a low calorie sweetener, a taste improving agent, a quality improving agent, a water separation preventing agent, a stabilizer, a discoloration preventing agent, It can be advantageously used for various compositions such as various foods and drinks, cosmetics, and pharmaceuticals as a form, a clathrate and the like.

[0182]

Example A-4 Corn starch was used as starch milk having a concentration of about 20%, and 0.1% of calcium carbonate was added thereto.
The pH was adjusted to 6.5, and α-amylase (trade name “Tamamir 60L”, manufactured by Novo Co.) was added at 0.
Add 3%, react at 95 ° C for 15 minutes, and then
The mixture was autoclaved for 20 minutes and further quenched to about 35 ° C. to obtain a liquefied solution having a DE of about 4, into which the α-isomaltosylglucosaccharide-forming enzyme obtained by the method of Example A-3 and α-isomaltosyltransferase were transferred. A concentrated enzyme solution containing an enzyme and a starch solid 1
0.25 ml per gram, and cyclomaltodextrin glucanotransferase (manufactured by Hayashibara Biochemical Laboratories, Inc.) was added to give 10 units per gram of starch solids. The reaction was carried out at 35 for 48 hours. The reaction solution is heated at 95 ° C for 30 minutes.
After keeping the pH for 5.0 minutes and adjusting the temperature to 50 ° C,
α-glucosidase agent (trade name “transglucosidase L“ Amano ”, manufactured by Amano Pharmaceutical Co., Ltd.)
Add 300 units per gram and allow to react for 24 hours, then add 30 units per gram of solid substance with glucoamylase agent (trade name "Glucozyme", manufactured by Nagase Seikagaku Corporation), react for 17 hours, and react the reaction solution. After heating to 95 ° C. and maintaining for 30 minutes, the mixture was cooled, filtered, and then purified (decolorized and desalted) according to a conventional method, and concentrated to obtain a syrup containing a cyclic tetrasaccharide having a concentration of 50% based on the raw starch solids. Yield about 9
Obtained at 0%. This syrup was hydrogenated, purified and concentrated according to the method of Example A-1 to obtain a syrup having a concentration of 85%.
About 6% of water-containing crystals was added thereto, and the mixture was gradually cooled while stirring to obtain an auxiliary crystal.
It was left for a day and crystallized and aged to prepare a block. Next, the block is pulverized by a cutting machine to form a cyclic tetrasaccharide 5 to 6.
A powder containing hydrous crystals was obtained. This product is a carbohydrate mixture containing starch sugar alcohol together with cyclic tetrasaccharide and having a DE of less than 0.5.
Contains 2.7%, 34.2% sorbitol and 3.1% other sugar alcohols, mild sweetness, moisturizing,
Various inclusions such as low-calorie sweeteners, taste improvers, quality improvers, anti-synthesis agents, stabilizers, discoloration inhibitors, excipients, clathrates, etc. It can be advantageously used for various compositions.

[0183]

Example B-1 <Sweetener> One part by weight of powder containing anhydrous sorbitol crystals together with 5 to 6 hydrous crystals of cyclic tetrasaccharide obtained by the method of Example A-3 was mixed with α-glycosyl stevioside and Toyo Seikagyo Co., Ltd. Company sales “αG Suite” 0.01
Parts by weight and L-aspartyl-L-phenylalanine methyl ester, sold by Ajinomoto Co., Inc. "Aspartame"
0.01 parts by weight are mixed uniformly, and put into a granule molding machine.
A granulated sweetener was obtained. It has excellent sweetness, about twice the sweetness of sucrose, and is substantially low in calories. This sweetener does not decompose the high-intensity sweetener blended with it and has excellent stability.As a low-calorie sweetener, low-calorie food and drink for obese and diabetics who have restricted caloric intake It is suitable for sweetening products and the like. In addition, the present sweetener is suitable for sweetening foods and the like that suppress tooth decay, because it produces less acid by caries-inducing bacteria and produces less insoluble glucan.

[0184]

Example B-2 <Hard candy> 30 parts by weight of a syrup containing a sugar alcohol together with the cyclic tetrasaccharide obtained by the method of Example A-1 were mixed with 100 parts by weight of a 55% strength sucrose solution, and then the pressure was reduced. The solution was concentrated under heating until the water content became less than 2%, and 1 part by weight of citric acid and an appropriate amount of a lemon flavor and a coloring agent were mixed with each other, followed by molding 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 or deformation. The product is also suitable as a low calorie, low caries hard candy.

[0185]

Example B-3 <Chocolate> Cocoa paste 40
20 parts by weight of a powder containing anhydrous sorbitol crystals together with 5 to 6 parts by weight of the cyclic tetrasaccharide obtained by the method of Example A-4, 10 parts by weight of cocoa butter, 30 parts by weight of sucrose, and 5 to 6 parts of the hydrous crystals of cyclic tetrasaccharide obtained by the method of Example A-4. After reducing the particle size, the mixture was placed in a conch and kneaded at 50 ° C. for 2 days and nights. During this time, 0.5 part by weight of lecithin was added and thoroughly mixed and dispersed. Next, the temperature was adjusted to 31 ° C. with a temperature controller, poured into a mold just before the butter was hardened, the mill was evacuated with a vibrator, and solidified by passing through a 10 ° C. cooling tunnel for 20 minutes. This was die-cut and packaged to obtain a product. This product is not hygroscopic,
Good color and gloss, good internal structure, melts smoothly in the mouth, and has elegant sweetness and mellow flavor.

[0186]

<Example B-4><Chewinggum> 3 parts by weight of a gum base were heated and melted to such an extent that they were softened, and 4 parts by weight of sucrose and 5 or 6 cyclic tetrasaccharide obtained by the method of Example A-3 were added to sorbitol. 3 parts by weight of a powder containing anhydrous crystals were added, an appropriate amount of a fragrance and a coloring agent were further mixed, kneaded by a roll, molded and packaged according to a conventional method to obtain a product. This product is a chewing gum with good texture and flavor. The product is also suitable as a low calorie, low cariogenic chewing gum.

[0187]

Example B-5 <Sugared condensed milk> In 100 parts by weight of raw milk, 3 parts by weight of a syrup containing sugar alcohol and 1 part by weight of sucrose were dissolved together with the cyclic tetrasaccharide obtained by the method of Example A-2.
The product was sterilized by heating with a plate heater, then concentrated to a concentration of 70%, and canned under aseptic conditions to obtain a product. This product has a mild sweetness and good flavor, and can be advantageously used for seasoning infant foods, fruits, coffee, cocoa, black tea and the like.

[0188]

<Example B-6><Lactic acid bacteria beverage> 175 parts by weight of skim milk powder, 80 parts by weight of powder containing anhydrous sorbitol crystals together with 5 to 6 cyclic tetrasaccharide hydrous crystals obtained by the method of Example A-3, and 50 parts by weight of the lactosucrose-rich powder disclosed in
0 parts by weight, sterilized at 65 ° C. for 30 minutes, cooled to 40 ° C., inoculated with 30 parts by weight of a lactic acid bacterium starter, and cultured at 37 ° C. for 8 hours to obtain a lactic acid bacterium beverage according to a conventional method. Obtained. This product is a low-calorie lactic acid bacterium beverage with good flavor. In addition, the product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacterium growth promoting effect.

[0189]

Example B-7 <Powder juice> Example A-33 was used to prepare 33 parts by weight of orange juice powder produced by spray drying.
50 parts by weight of powder containing anhydrous crystals of sorbitol together with 5 to 6 hydrous crystals of cyclic tetrasaccharide obtained by the method of 4
Parts by weight, anhydrous citric acid 0.65 parts by weight, malic acid 0.1
Parts by weight, 0.1 parts by weight of L-ascorbic acid, 0.1 parts by weight of sodium citrate, 0.5 parts by weight of pullulan, an appropriate amount of powdered fragrance, well mixed and stirred, pulverized to a fine powder, and then fluidized bed granulated. The syrup containing sugar alcohol was sprayed as a binder together with the cyclic tetrasaccharide obtained by the method of Example A-2, and granulated, weighed, and packaged for 30 minutes. Got the product. This product is a low calorie powdered juice with a fruit content of about 30%. Also,
This product had no off-flavor and off-flavor and was stable for a long time.

[0190]

Example B-8 <Custard cream> 100 parts by weight of corn starch, 100 parts by weight of a syrup containing a sugar alcohol together with the cyclic tetrasaccharide obtained by the method of Example A-1,
80 parts by weight of maltose, 20 parts by weight of sucrose and 1 part by weight of salt are mixed well, and 280 parts by weight of chicken egg are added and stirred,
1,000 parts by weight of boiled milk is gradually added to the mixture, and the mixture is further heated. The mixture is heated and stirred. When the corn starch is completely gelatinized and the whole becomes translucent, the fire is stopped, and the mixture is cooled to an appropriate amount. Was added, weighed, filled and packaged to obtain a product. This product has a smooth luster and is mildly sweet and delicious.

[0191]

Example B-9 <Uiro no Moto> 90 parts by weight of rice flour
20 parts by weight of corn starch, 40 parts by weight of sucrose, Example A
80 parts by weight of a powder containing anhydrous sorbitol crystals and 4 parts by weight of pullulan were uniformly mixed with the hydrous crystals of cyclic tetrasaccharide 5 to 6 obtained by the method described in the above-mentioned method 3 to produce uiro wax. Uiro no element, an appropriate amount of matcha and water were kneaded, and this was put in a container and steamed for 60 minutes to produce matcha uiro. This product has good shine, mouthfeel, good flavor, and low calories. In addition, aging of the starch is suppressed, and the shelf life is good.

[0192]

Example B-10 <Anno> To 10 parts by weight of raw material Azuki,
According to a conventional method, water was added and the mixture was boiled, astringently cut off, and crushed to remove water-soluble impurities, thereby obtaining about 21 parts by weight of azuki vine. To this raw bean paste, 14 parts by weight of sucrose, Example A
-5 parts by weight of a syrup containing sugar alcohol and 4 parts by weight of water together with the cyclic tetrasaccharide obtained by the method of -2, and boiling,
To this was added a small amount of salad oil and kneaded so as not to break the bean paste, to obtain about 35 parts by weight of the product bean paste. This product has no color burn, has a good texture, and has a good flavor, and is suitable as an ingredient for red bean bread, steamed bun, dumpling, monaka, frozen dessert and the like.

[0193]

Example B-11 <Bread> Contains 100 parts by weight of flour, 2 parts by weight of yeast, 5 parts by weight of sugar, and anhydrous sorbitol crystals together with 5 to 6 cyclic tetrasaccharide hydrous crystals obtained by the method of Example A-4. 1 part by weight of powder and 0.1 part by weight of inorganic food are kneaded with water according to a conventional method, and
Fermented for hours, then aged for 30 minutes and baked. This product is a high-quality bread with good hue and color, moderate elasticity and mild sweetness.

[0194]

Example B-12 <Ham> To 1,000 parts by weight of pork thigh, 15 parts by weight of salt and 3 parts by weight of potassium nitrate are uniformly rubbed, and piled up in a cold room all day and night. This is composed of 500 parts by weight of water, 100 parts by weight of sodium chloride, 3 parts by weight of potassium nitrate, 40 parts by weight of powder containing anhydrous sorbitol crystals together with 5 to 6 cyclic tetrasaccharide hydrous crystals obtained by the method of Example A-3, and spices. Soak in the salted solution for 7 days in a cold room,
Wash with cold water, wrap with string, smoke,
The product was cooked, cooled and packaged to obtain a product. This product is a high quality ham with good color and good flavor.

[0195]

Example B-13 <Powdered Peptide> Cyclic tetrasaccharide 5 obtained by the method of Example A-4 was added to 1 part by weight of “Hynew S” manufactured by Fuji Oil Co., Ltd.
2 parts by weight of a powder containing anhydrous sorbitol crystals together with 1 to 6 hydrous crystals were mixed, placed in a plastic vat, dried at 50 ° C. under reduced pressure, and pulverized to obtain a powdered peptide. This product has a good flavor and can be advantageously used not only as a confectionary material such as premix and frozen dessert, but also as a baby food such as an oral liquid food and a tube food liquid, a therapeutic nutrient and the like.

[0196]

Example B-14 <Cosmetic cream> 2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glyceryl monostearate, 5 to 6 cyclic tetrasaccharides obtained by the method of Example A-3 2 parts by weight of powder containing anhydrous crystals of sorbitol together with hydrated crystals, 1 part by weight of α-glycosylrutin, 1 part by weight of liquid paraffin, 10 parts by weight of glyceryl trioctanoate and an appropriate amount of a preservative are heated and dissolved according to a conventional method. 2 parts by weight of L-lactic acid, 1,3-
5 parts by weight of butylene glycol and 66 parts by weight of purified water were added, and the mixture was emulsified with a homogenizer, and an appropriate amount of a flavor was further added, followed by stirring and mixing to produce a cream. This product has antioxidant properties, high stability, high quality sunscreen, skin tonic,
It can be advantageously used as a whitening agent.

[0197]

[Example B-15] <Solid preparation> A human natural interferon-α sample (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.)
According to a conventional method, the antibody is applied to an immobilized anti-human interferon-α antibody column, human natural interferon-α contained in the sample is adsorbed, bovine serum albumin as a stabilizer is removed by passing through, and then the pH is changed. Let me
Human natural interferon-α was eluted using a physiological saline solution containing 5% of a powder containing anhydrous sorbitol crystals together with 5 to 6 hydrous cyclic tetrasaccharide crystals obtained by the method of Example A-4. This liquid was subjected to precision filtration, added to about 20 times the amount of anhydrous crystalline maltose powder sold by Hayashibara Shoji Co., Ltd. “Finetose”, dehydrated and powdered, and then compressed with a tableting machine to form one tablet (about 200 mg). A tablet containing about 150 units of human natural interferon-α per unit was obtained. The product is orally administered as a sublingual tablet, for example, about 1 to 10 tablets per day per adult, and can be advantageously used for the treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., in which the number of patients has rapidly increased in recent years.
In this product, both the non-reducing saccharide of the present invention and anhydrous crystalline maltose act as stabilizers, and their activities are maintained well for a long period of time even at room temperature.

[0198]

Example B-16 <Sugar-coated tablet> A 150 mg uncoated tablet was used as a core, and the cyclic tetrasaccharide 5 obtained by the method of Example A-3 was added thereto.
6 to 6 parts by weight of powder containing anhydrous crystals of sorbitol together with hydrous crystals, 2 parts by weight of pullulan (average molecular weight 200,000), 30 parts by weight of water, 25 parts by weight of talc, and 3 parts by weight of titanium oxide. Tablet weight is about 230
mg, and then the same cyclic tetrasaccharide 5-6
65 parts by weight of hydrous crystal powder, 1 part by weight of pullulan and 34 parts of water
A sugar-coated tablet having an excellent glossy appearance was obtained by sugar-coating using the overhanging solution consisting of parts by weight and further glazing with a wax solution.
The product has excellent impact resistance and maintains high quality for a long time.

[0199]

Example B-17 <Toothpaste> Dibasic calcium phosphate 4
5.0 parts by weight, pullulan 2.95 parts by weight, sodium lauryl sulfate 1.5 parts by weight, glycerin 20.0 parts by weight,
0.5 parts by weight of polyoxyethylene sorbitan laurate, 0.05 parts by weight of preservative, 12.0 parts by weight of powder containing anhydrous crystals of sorbitol together with 5 to 6 cyclic tetrasaccharide hydrous crystals obtained by the method of Example A-3 Part, maltitol 5.0
1 part by weight of water and 13.0 parts by weight of water were mixed in a conventional manner to obtain a toothpaste. This product has a moderate sweetness,
It is particularly suitable as a toothpaste for children.

[0200]

Example B-18 <Solid liquid food preparation> Example A-4
100 parts by weight of powder containing anhydrous crystals of sorbitol together with 5 to 6 hydrated crystals of cyclic tetrasaccharide produced by the method described above, 200 parts by weight of hydrated maltose 1 crystal, 200 parts by weight of hydrated α, α-trehalose 2 crystal, 270 parts by weight of egg yolk 209 parts by weight of skim milk powder, 4.4 parts by weight of sodium chloride, 1.8 parts by weight of potassium chloride, 4 parts by weight of magnesium sulfate, 0.01 part by weight of thiamine, 0.1 part by weight of sodium ascorbate.
A formulation consisting of 1 part by weight, 0.6 part by weight of vitamin E acetate and 0.04 part by weight of nicotinamide was prepared,
Each 25 grams of this compound was filled into a moisture-proof laminate pouch and heat-sealed to obtain a product. This product is obtained by dissolving one bag in about 150 to 300 ml of water to make a liquid food, and is used orally or by tube application to the nasal cavity, stomach, intestine, etc., and is advantageous for nutritional supplementation to living organisms. Available to

[0201]

<Example B-19><Treatment for wound treatment> 200 parts by weight of a powder containing anhydrous sorbitol crystals together with 5 to 6 hydrous crystals of cyclic tetrasaccharide prepared by the method of Example A-3 and 300 parts by weight of maltose were added to iodine. 50 parts by weight of methanol in which 3 parts by weight are dissolved are added and mixed, and further, 200 parts by weight of a 10 w / v% pullulan aqueous solution is added and mixed, and moderately extended.
A plaster for wound treatment showing adhesiveness was obtained. This product shortens the healing period due to the bactericidal action of iodine, and heals the wound surface neatly.

[0202]

As is evident from the above, the saccharide mixture comprising a starch saccharide alcohol together with the cyclic tetrasaccharide of the present invention is substantially non-reducing and excellent in stability, and has a good and elegant sweetness. Have. Therefore, the saccharide mixture of the present invention can be advantageously used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, and the like in various compositions such as various foods and drinks, cosmetics, and pharmaceuticals. In addition, as the reducing saccharide for a raw material used in the production of the saccharide mixture of the present invention, α-isomaltosyltransferase is allowed to act on a solution containing α-isomaltosylglucosaccharide such as panose, or starch is liquefied. Α-Isomaltosyltransferase and α-isomaltosylglucosaccharide-forming enzyme together with a starch debranching enzyme and / or cyclomaltodextrin-glucanotransferase are allowed to act on the resulting solution to form a reducing saccharide together with a cyclic tetrasaccharide. By obtaining the containing saccharide, hydrogenating it, converting the reducing saccharide to the corresponding saccharide alcohol to reduce the reducing property without isolating the cyclic tetrasaccharide, and then purifying, Low molecular weight, low viscosity and easy to handle DE
A substantially non-reducing carbohydrate mixture of less than 0.5 is obtained. The saccharide mixture of the present invention that can be obtained in this manner is, for example, a low-calorie sweetener, a taste improver, a quality improver, a water separation inhibitor, a stabilizer, a discoloration inhibitor, an excipient, and a clathrate. It can be used for a wide range of applications as a powdered base material.

The establishment of a saccharide mixture containing a sugar alcohol together with the cyclic tetrasaccharide of the present invention, a method for producing the same, and the use of the mixture are provided by using starch, which is an inexpensive and unlimited resource, as a raw material. Thus, a completely new route that can be industrially provided in a large amount and at a low cost from a saccharide containing a reducing saccharide together with a cyclic tetrasaccharide that could not be easily obtained is pioneered, and the industrial practice of the present invention becomes extremely easy. . Therefore, the effects of the present invention are not limited to the food, cosmetics, and pharmaceutical industries, but also to the agriculture, fisheries and livestock industries, and the chemical industry, and the industrial significance of these industries is immense.

[0204]

[Sequence List] SEQUENCE LISTING <110> Kabushiki Kaisha Hayashibara Seibutsu Kagaku
Kenkyujo <120> Saccharide Composition, Preparation And Uses
Thereof <130> 10088801 <160> 10 <210> 1 <211> 9 <212> PRT <213> Bacillus globisporus <400> 1 Tyr Val Ser Ser Leu Gly Asn Leu Ile 1 5 <210> 2 <211> 10 <212> PRT <213> Bacillus globisporus <400> 2 Ile Asp Gly Val Tyr His Ala Pro Asn Gly 1 5 10 <210> 3 <211> 10 <212> PRT <213> Bacillus globisporus <400> 3 Ile Asp Gly Val Tyr His Ala Pro Tyr Gly 1 5 10 <210> 4 <211> 8 <212> PRT <213> Bacillus globisporus <400> 4 Ile Asp Gly Val Tyr His Ala Pro 1 5 <210> 5 <211> 8 <212 > PRT <213> Bacillus globisporus <400> 5 Asp Ala Ser Ala Asn Val Thr Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Bacillus globisporus <400> 6 Trp Ser Leu Gly Phe Met Asn Phe 1 5 <210> 7 <211> 8 <212> PRT <213> Bacillus globisporus <400> 7 Asn Tyr Thr Asp Ala Trp Met Phe 1 5 <210> 8 <211> 8 <212> PRT <213> Bacillus globisporus <400> 8 Gly Asn Glu Met Arg Asn Gln Tyr 1 5 <210> 9 <211> 8 <212> PRT <213> Bacillus globisporus <400> 9 Ile Thr Thr Trp Pro Ile Glu Ser 1 5 <210> 10 <211> 8 <212> PRT <213> Bacillus globisporus <400> 10 Trp A la Phe Gly Leu Trp Met Ser 1 5

[0205]

[Brief description of the drawings]

FIG. 1 is a view showing an elution pattern when a saccharide obtained by an α-isomaltosyltransferase reaction is subjected to high performance liquid chromatography.

FIG. 2 is a diagram showing a nuclear magnetic resonance spectrum ( ¹ H-NMR spectrum) of a cyclic tetrasaccharide obtained by an α-isomaltosyltransferase reaction.

FIG. 3 is a view showing a nuclear magnetic resonance spectrum ( ¹³ C-NMR spectrum) of a cyclic tetrasaccharide obtained by an α-isomaltosyltransferase reaction.

FIG. 4. The structure of the cyclic tetrasaccharide is cyclo ｛→ 6) -α-D.
-Glucopyranosyl- (1 → 3) -α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1
FIG. 3 shows that (3) -α-D-glucopyranosyl- (1 →｝).

FIG. 5 is a graph showing the effect of temperature on the enzymatic activity of α-isomaltosylglucosaccharide-forming enzyme derived from Bacillus globisporus C9.

FIG. 6 shows the effect of pH on the enzymatic activity of α-isomaltosylglucosaccharide-forming enzyme derived from Bacillus globisporus C9.
FIG.

FIG. 7 is a graph showing the temperature stability of α-isomaltosylglucosaccharide-forming enzyme derived from Bacillus globisporus C9.

FIG. 8 is a diagram showing the pH stability of α-isomaltosylglucosaccharide-forming enzyme derived from Bacillus globisporus C9.

FIG. 9 is a graph showing the effect of temperature on the enzymatic activity of α-isomaltosyltransferase derived from Bacillus globisporus C9.

FIG. 10 is a graph showing the effect of pH on the enzymatic activity of α-isomaltosyltransferase derived from Bacillus globisporus C9.

FIG. 11 shows the temperature stability of α-isomaltosyltransferase derived from Bacillus globisporus C9.

FIG. 12 is a diagram showing the pH stability of α-isomaltosyltransferase derived from Bacillus globisporus C9.

FIG. 13: α-derived from Bacillus globisporus C11
It is a figure which shows the influence of the temperature which has on the enzyme activity of isomaltosyl glucosaccharide formation enzyme.

FIG. 14: α-derived from Bacillus globisporus C11
It is a figure which shows the influence of pH which has on the enzyme activity of isomaltosyl glucosaccharide formation enzyme.

FIG. 15: α-derived from Bacillus globisporus C11
It is a figure which shows the temperature stability of an isomaltosyl glucosaccharide formation enzyme.

FIG. 16: α-derived from Bacillus globisporus C11
It is a figure which shows the pH stability of an isomaltosyl glucosaccharide formation enzyme.

FIG. 17: α-derived from Bacillus globisporus C11
It is a figure which shows the influence of the temperature which has on the enzyme activity of isomaltosyltransferase.

FIG. 18: α-derived from Bacillus globisporus C11
It is a figure which shows the influence of pH which has on the enzyme activity of isomaltosyltransferase.

FIG. 19: α-derived from Bacillus globisporus C11
It is a figure which shows the temperature stability of isomaltosyltransferase.

FIG. 20 shows α-derived from Bacillus globisporus C11.
FIG. 3 is a diagram showing the pH stability of isomaltosyltransferase.

FIG. 21 shows the relationship between α-isomaltosylmaltotriose obtained by α-isomaltosylglucosaccharide-forming enzyme reaction.
It is a figure which shows a < ¹ > H-NMR spectrum.

FIG. 22 is a diagram showing a ¹ H-NMR spectrum of α-isomaltosylmaltotetraose obtained by an α-isomaltosylglucosaccharide-forming enzyme reaction.

FIG. 23 shows the relationship between α-isomaltosylmaltotriose obtained by the α-isomaltosylglucosaccharide-forming enzyme reaction.
It is a figure which shows a < ¹³ > C-NMR spectrum.

FIG. 24 is a view showing a ¹³ C-NMR spectrum of α-isomaltosylmaltotetraose obtained by the α-isomaltosylglucosaccharide-forming enzyme reaction.

FIG. 25 is a diagram showing a halftone image in which a microscopic photograph of a 5 to 6 hydrous crystalline cyclic tetrasaccharide is displayed on a display.

FIG. 26 is a diagram showing a diffraction spectrum obtained by analyzing 5 to 6 hydrous crystalline cyclic tetrasaccharide-like powders by a powder X-ray diffraction method.

FIG. 27 is a view showing a thermogravimetric curve when thermogravimetric measurement is performed on 5 to 6 hydrous crystalline cyclic tetrasaccharide powders.

FIG. 28 is a view showing a diffraction spectrum when analyzing one hydrous crystalline cyclic tetrasaccharide powder of the present invention by a powder X-ray diffraction method.

FIG. 29 is a diagram showing a thermogravimetric curve when thermogravimetric measurement is performed on 1 hydrous crystalline cyclic tetrasaccharide powder of the present invention.

FIG. 30 is a diagram showing a diffraction spectrum when an anhydrous crystalline cyclic tetrasaccharide powder obtained by vacuum-drying 5 to 6 hydrous crystalline cyclic tetrasaccharide powder at 40 ° C. is analyzed by a powder X-ray diffraction method.

FIG. 31 is a view showing a diffraction spectrum when an anhydrous crystalline cyclic tetrasaccharide powder obtained by vacuum-drying 5 to 6 hydrous crystalline cyclic tetrasaccharide powder at 120 ° C. is analyzed by a powder X-ray diffraction method.

FIG. 32 is a graph showing a thermogravimetric curve obtained by thermogravimetric measurement of the anhydrous crystalline cyclic tetrasaccharide powder of the present invention.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A23L 1/236 A23L 1/236 B 4C057 1/30 1/30 Z 4C076 1/307 1/307 4C083 2/52 A61K 7 / 00 F 2/00 N A61K 7/00 7/16 47/10 7/16 47/26 47/10 A61P 3/00 47/26 C12N 9/10 ZNA // A61P 3/00 A23L 2/00 F C12N 9/10 ZNA G (72) Inventor Keion Fukuda 1-2-3 Shimoishii, Okayama City, Okayama Prefecture Inside the Hayashibara Biochemical Laboratory Co., Ltd. (72) Inventor Toshio Miyake 1-2-3 Shimoishii, Okayama City, Okayama Prefecture No. Co., Ltd. Hayashibara Biochemical Laboratory F-term (reference) 4B017 LC04 LG02 LK11 LK12 LL02 4B018 LE03 MD31 MD32 ME01 ME14 MF10 MF12 4B041 LC01 LC10 LE01 LK11 LK12 4B047 LB09 LE06 LG15 LG21 LG25 LG32 4B050 CC01 DD02 FF04EFFFFEFFFFE 4C057 AA04 BB01 BB04 4C076 AA36 AA43 BB02 DD27 DD29 DD38 DD67T EE30 EE30T FF36 4C083 AB282 AC122 AC132 AC441 AC442 AC782 AD211 AD212 CC41 DD22 EE32

Claims (10)

[Claims] 1. Cyclo II → 6) -α-D-glucopyranosyl- (1 → 3) -α-D-glucopyranosyl- (1
→ 6) -α-D-glucopyranosyl- (1 → 3) -α-
A saccharide mixture comprising a saccharide having a structure of D-glucopyranosyl- (1 →｝) and a sugar alcohol. 2. The saccharide mixture according to claim 1, wherein the sugar alcohol is one or more sugar alcohols selected from sorbitol, maltitol, isomaltitol and panitol. 3. Cyclo ｛→ 6) -α-D-glucopyranosyl- (1 → 3) -α-D-glucopyranosyl- (1)
→ 6) -α-D-glucopyranosyl- (1 → 3) -α-
The carbohydrate according to claim 1 or 2, which is obtained by hydrogenating a carbohydrate containing a reducing carbohydrate together with a carbohydrate having a structure of D-glucopyranosyl- (1 →｝) to reduce reductivity, and then purifying the carbohydrate. mixture. 4. Cyclo ｛→ 6) -α-D-glucopyranosyl- (1 → 3) -α-D-glucopyranosyl- (1)
→ 6) -α-D-glucopyranosyl- (1 → 3) -α-
A carbohydrate containing a reducing carbohydrate together with a carbohydrate having a structure of D-glucopyranosyl- (1 →｝) is reacted with α-isomaltosylglucosaccharide with α-isomaltosyltransferase, or starch having a DE of 20 or less. The saccharide mixture according to claim 3, which is a saccharide obtained by allowing an α-isomaltosylglucosaccharide-forming enzyme and an α-isomaltosyltransferase to act on the partially degraded product. 5. The method according to claim 3, wherein the reducing saccharide is one or more reducing saccharides selected from glucose, maltose, isomaltose and panose.
Or the saccharide mixture according to 4. 6. The saccharide mixture according to claim 1, wherein the saccharide mixture has a DE of less than 0.5 and is in the form of a syrup or a crystal-containing powder. 7. The cyclo ｛→ 6) -α-D-glucopyranosyl- (1 → 3)-according to any one of claims 3 to 5.
Carbohydrate having a reducing carbohydrate together with a carbohydrate having a structure of α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 3) -α-D-glucopyranosyl- (1 →｝) The method for producing a saccharide mixture according to any one of claims 1 to 6, comprising a step of reducing the reducibility by hydrogenating the saccharide, and a step of purifying and collecting the saccharide. 8. A composition containing the saccharide mixture according to claim 1. 9. The composition according to claim 8, wherein the composition is a food, drink, cosmetic or pharmaceutical. 10. The composition according to claim 9, wherein the food is a low calorie food or a low cariogenic food.