JP2004211097A - Maltohexaose and maltoheptaose producing amylase and its preparation process and application - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new amylase capable of producing large amounts of maltohexaose and maltoheptaose from starch, and its preparation process and use. <P>SOLUTION: A microorganism-derived amylase mainly producing maltohexaose and maltoheptaose from starch without substantial decomposition of low molecular weight oligosaccharides smaller than maltohexaose. A microorganism producing the amylase, production of the amylase by the microorganism and sugars prepared by action of the amylase on carbohydrates and a composition containing the sugars, are provided. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a novel maltohexaose / maltoheptaose-forming amylase, a method for producing the same, and uses thereof, and more particularly, to a maltohexaose / maltoheptaose-forming amylase that mainly produces maltohexaose and maltoheptaose from starch. And a method for producing the same, a microorganism producing the same, and a maltohexaose and / or maltoheptaose-containing saccharide produced using the maltohexaose / maltoheptaose-forming amylase, or a hydrogenated product thereof. The present invention relates to the obtained maltohexaitol and / or maltoheptitol-containing saccharide, and further to a composition containing these.

  The method of specifically producing specific maltooligosaccharides such as maltose and maltotetraose by causing a specific amylase to act on starch has been carried out on an industrial scale, and is widely used in compositions such as foods and pharmaceuticals. Have been.

  In recent years, saccharides containing a large amount of maltooligosaccharides, such as maltohexaose and maltoheptaose, have been demanded because sugars having a relatively high molecular weight among maltooligosaccharides have low sweetness and are easily digested and absorbed.

  As an amylase that produces a large amount of maltohexaose from starch, several enzymes of microbial origin are known, but an amylase that specifically produces maltooligosaccharides higher than maltoheptaose is not known.

  Amylase, which mainly produces maltohexaose from starch, is roughly classified into two types based on its activity.One is exo-type maltohexaose, which cuts out maltohexaose sequentially from the non-reducing end of the starch sugar chain. Amylase, ie, maltohexahydrolase (EC 3.2.1.18), and the other is an endo-type maltohexaose-generating amylase that also acts on the inside of starch sugar chains to produce relatively large amounts of maltohexaose, so-called It is an endo-type α-amylase (EC 3.2.1.1).

  Regarding exo-type maltohexaose amylase, Non-Patent Document 1 reports that Aerobacter aerogenes is produced in the cells, but this enzyme has low optimum temperature and stable temperature, and is industrially low. Heat resistance is insufficient for use.

  Regarding the endo-type maltohexaose-forming amylase, Non-Patent Document 2 reports that Bacillus subtilis produces, and Non-patent Document 3 reports that Bacillus circulans G-6 produces. Non-Patent Document 4 reports that Bacillus circulans F-2 is produced, and Non-Patent Document 5 reports that Bacillus species H-167 is produced. Among these, the amylase derived from Bacillus circulans G-6, Bacillus circulans F-2, and Bacillus species H-167 is capable of converting maltohexaose from starch to a maximum of about 25 to 30% by mass (hereinafter, referred to as "the present specification"). Unless otherwise specified, mass% is abbreviated as%.) It produces no maltoheptaose, and decomposes the produced maltohexaose into maltose and maltotetraose as the reaction proceeds. It is known.

  On the other hand, Bacillus subtilis-derived amylase does not show decomposition of the produced maltohexaose, but the production rate of maltohexaose from starch is limited to about 25% at the maximum. Furthermore, enzymes derived from these microorganisms actually have a low maltohexaose content under starch saccharification conditions and a large amount of low-molecular oligosaccharides, and in addition, have a low production amount from microorganisms. Actually, no industrial production of maltohexaose-rich saccharides has been carried out using amylase produced by these microorganisms.

  On the other hand, Non-Patent Document 6 describes that purified α-amylase prepared from malt mainly produces maltohexaose and maltoheptaose in the early stage of the reaction when acting on starch. However, it has also been described that as the reaction proceeds, maltohexaose and maltoheptaose are further almost completely decomposed into low-molecular oligosaccharides such as maltose and maltotetraose. A saccharified product containing aose and maltoheptaose could not be produced.

Kainuma et al., "FEBS Letters", Vol. 26, pp. 281-285 (1972). "Starch", JF Kennedy et al., Vol. 31, pp. 235-241 (1979). Takasaki, "Agricultural and Biological Chemistry", Vol. 46, pp. 1539-1547 (1982) Taniguchi et al., Starch Science, Vol. 29, pp. 107-116 (1982) Hayashi et al., Agricultural and Biological Chemistry, 52, 443-448 (1988). "Agricultural and Biological Chemistry", Vol. 42, pp. 259-267 (1978)

  The present invention relates to an amylase that produces maltohexaose and maltoheptaose at a high content from starch, hardly decomposes the produced maltohexaose and maltoheptaose, and contains maltohexaose and / or maltoheptaose containing the amylase. An object of the present invention is to provide a method for producing a saccharide and its use.

  The present inventors have searched for a novel amylase that produces maltohexaose and maltoheptaose from starch in order to solve the above problems, and have searched extensively for microorganisms that produce the enzyme. As a result, a novel microorganism, Alcaligenes latus D2271, belonging to the genus Alcaligenes isolated from soil near Mt. Komagatake, Yamanashi Prefecture, produces a novel amylase that produces maltohexaose and maltoheptaose from starch in high content (this specification). In this regard, the present amylase is sometimes referred to as "maltohexaose / maltoheptaose-forming amylase".) It is found that maltohexaose and / or maltoheptaose can be produced by allowing the present enzyme to act on starch. The present invention has been completed by establishing a method for producing saccharides, and at the same time, establishing compositions such as foods, drinks, cosmetics, pharmaceuticals, and molded articles containing the saccharides.

  The maltohexaose / maltoheptaose-forming amylase of the present invention has an optimum temperature, a high temperature at a stable temperature, and an optimum pH, a wide pH range at a stable pH, and a high enzyme production amount from a producing microorganism. It has been found that it can be advantageously used for the production of maltohexaose and / or maltoheptaose-rich saccharides and maltohexaitol and / or maltoheptitol-rich saccharides from starch, and its industrial significance. Is extremely large.

  Hereinafter, the identification test results of the novel microorganism Alcaligenes latus D2271 of the present invention are shown. The identification test was performed in accordance with "Classification and Identification of Microorganism" (edited by Takeharu Hasegawa, Gakkai Shuppan Center, 1985).

<A cell morphology>
(1) Potato-dextrose agar culture, 27 ° C .: 0.7 to 1.3 × 1.3 to 2.4 μm bacilli. Single, rarely paired and linked cells are also observed. Motility due to periflagellate. No spores. Non acid resistant. Gram negative. It accumulates poly-β-hydroxybutyrate.
(2) Yeast extract / malt extract agar culture, 37 ° C .: The size of cells in one day of culture is 0.5 to 1.0 × 1.3 to 2.4 μm, and 0.5 to 1.0 in three days of culture. 0 × 1.0 to 2.5 μm. Alone.

<B Cultural properties>
(1) Potato dextrose agar plate culture, 37 ° C
Shape: circular The size is about 1 mm per day. 3-4mm in 4 days.
Periphery: whole edge Uplift: umbilical luster: dull light Surface: wrinkle Color: opaque, white to ocher (2) Yeast extract / malt extract agar plate culture, 37 ° C
Shape: circular Size is about 3-5mm in 4 days
Periphery: whole edge Uplift: semi-lens shape
Gloss: Wet light Surface: Smooth Color: White to cream (3) Potato dextrose agar slant culture, 37 ° C
Growth degree: good Shape: filamentous (4) Yeast extract / malt extract gelatin puncture culture, 37 ° C
: Liquefaction

<C Physiological properties>
(1) Nitrate reducing: positive (succinic acid medium)
(2) Denitrification reaction: negative (3) Methyl red test: negative (4) VP test: negative (5) Indole formation: negative (6) Hydrogen sulfide formation: negative (7) Starch hydrolysis: positive ( 8) Use of citric acid: Negative (9) Use of inorganic nitrogen source: Both ammonium salt and nitrate can be used.
(10) Pigment formation: negative (11) Urease: positive (12) Oxidase: positive (13) Catalase: positive (14) Growth range: pH 5-8, temperature 10-41 ° C.
(15) Attitude to oxygen: aerobic (16) Use of carbon source and presence or absence of acid generation
Utilization Acid-generating ability D-glucose used negative D-galactose used negative D-mannose used negative D-fructose used negative L-arabinose used negative D-xylose used negative L-rhamnose used negative maltose used Negative Sucrose Use Negative Lactose Use Negative Trehalose Use Negative Raffinose Use Negative Mannitol Do not use Negative Dextrin Use Negative Dulchitol Not use Negative (17) Amino acid decarboxylation test: L-lysine, L-arginine, ornithine Also negative.
(18) DNase: negative (19) Production of 3-keto lactose: negative (20) GC content of DNA: 67%

  Based on the above mycological properties, reference to "Bergey's Manual of Systematic Bacteriology Volume 1" (Bergey's Manual of Systematic Bacteriology Volume 1), (1984) It was investigated. As a result, the bacterium was found to be a strain belonging to Alcaligenes latus.

  Based on these results, the present inventors named the microorganism microorganism Alcaligenes latus D2271, and dated February 23, 1994, the Ministry of International Trade and Industry, located at 1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture, Japan. Deposited at the National Institute of Bioscience and Biotechnology, the National Institute of Advanced Industrial Science and Technology, and the Patented Microorganisms Depositary Center, and deposited as “Accession No. FERM BP-4578”.

  In the present invention, not only the above-mentioned bacteria but also other strains belonging to Alcaligenes latus having maltohexaose-maltoheptaose-forming amylase-producing ability, mutants thereof, and the like can be used.

  The medium used for culturing the microorganism of the present invention may be any medium capable of growing the present microorganism and producing the enzyme of the present invention, and may be either a synthetic medium or a natural medium. Any carbon source can be used as long as it can be assimilated by the present strain, and for example, natural substances such as sugars such as maltose, dextrin, and starch, and sugar-containing substances such as molasses and yeast extract can be used. The concentration of these carbon sources in the medium is appropriately selected depending on the type of the carbon source. For example, the concentration of starch in the culture solution is desirably 20% by weight or less, and usually 5% or less from the viewpoint of the growth and proliferation of bacteria. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract, and meat extract. In addition, as the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt and the like are used as needed. Can be

  The cultivation is usually carried out aerobically at a temperature of 10 to 40 ° C, preferably 25 to 37 ° C, at a pH of 5 to 8, preferably at a pH of 6 to 7.5. The culturing time may be any time as long as the microorganism can proliferate, and is preferably 10 to 100 hours. The concentration of dissolved oxygen in the culture solution is not particularly limited, but is usually preferably 0.5 to 20 ppm. For this purpose, means such as adjusting the amount of ventilation, stirring, using oxygen, and increasing the pressure in the culture tank are employed. The culture method may be either batch culture or continuous culture.

  After culturing the microorganism in this way, the enzyme of the present invention is collected from the obtained culture. This enzyme activity is observed in the extracellular culture of the culture, and the extracellular culture may be collected as a crude enzyme, or the whole culture may be used as a crude enzyme. Conventional solid-liquid separation means is employed for separating the extracellular culture solution from the cells. For example, means for centrifuging the culture itself as it is, means for adding a filter aid to the culture, or means for filtration by pre-coating, means for membrane filtration using a flat membrane, hollow fiber membrane, etc. are applied. I can do it. Although the extracellular culture solution can be used as a crude enzyme solution as it is, it is preferably concentrated by ordinary means. For example, an ammonium sulfate salting-out method, an acetone and alcohol precipitation method, a membrane concentration method using a flat membrane, a hollow fiber membrane, or the like is employed.

  Further, the extracellular culture solution and the concentrate thereof can be immobilized by ordinary means. For example, a method of bonding to an ion exchanger, a method of covalent bonding and adsorption to a resin or a membrane, a method of entrapping using a polymer substance, and the like are employed.

  The crude enzyme may be used as it is, or it may be purified by ordinary means. As an example, the crude enzyme preparation obtained by salting out the extracellular culture solution with ammonium sulfate is dialyzed, then anion-exchange column chromatography using DEAE-Toyopearl resin, and subsequently, a hydrophobic column using butyltoyopearl resin. A single enzyme can be obtained electrophoretically by chromatography and hydrophobic column chromatography using phenyl superrose HR5 / 5 resin.

The maltohexaose / maltoheptaose-forming amylase of the present invention thus obtained has the following physicochemical properties.
(1) Action It acts on starch to mainly produce maltohexaose and maltoheptaose. It does not substantially decompose low molecular oligosaccharides of maltohexaose or less and hardly acts on maltoheptaose.
(2) Molecular weight: 43,000 ± 3,000 daltons by SDS-gel electrophoresis (3) Isoelectric point: 7.6 ± 0.5 by electrophoresis containing ampholine
(4) Optimum pH
About 5.0 (in the presence of calcium ion)
(5) Optimum temperature around 70 ° C (in the presence of calcium ions)
(6) pH stability
About 4.5 to 10.5 (in the presence of calcium ion)
(7) Temperature stability
Stable up to around 60 ° C (in the presence of calcium ions)
(8) Activity promotion and stabilization Activity is promoted and stabilized by calcium ions.
(9) Inhibition Inhibited by copper ion, lead ion, zinc ion, mercury ion or EDTA.

  The activity of the maltohexaose-maltoheptaose-forming amylase of the present invention is measured as follows. 0.2 ml of the enzyme solution was added to 5 ml of soluble starch 0.3 w / v% (containing 20 mM acetate buffer, pH 5.5, and 1 mM CaCl2) as a substrate, and reacted at 40 ° C. for 10 minutes. The reaction is stopped by adding to 15 ml of 0.02N sulfuric acid aqueous solution. 0.2 ml of a 0.1 N iodine solution is added to the reaction termination solution, stirred, left at 25 ° C. for 15 minutes, and the absorbance at 660 nm is measured. One unit of the enzyme activity is defined as the amount of the enzyme that completely eliminates the iodine coloration of 15 mg of soluble starch in a reaction at 40 ° C. for 10 minutes.

  When producing maltohexaose and / or maltoheptaose-containing saccharide using the maltohexaose / maltoheptaose-forming amylase of the present invention, industrially, starch, amylopectin, amylose, starch hydrolyzate, etc. Is preferably used as a substrate.

  In addition, if necessary, maltohexaose / maltoheptaose-forming amylase is allowed to act on the production of starch-containing foods and drinks to contain maltohexaose and / or maltoheptaose to prevent starch aging. In addition, the shelf life of food and drink can be extended.

  In general, the maltohexaose maltohepta of the present invention is added to a starchy solution of about 5 to 45%, preferably a solution in which a calcium salt such as calcium chloride is coexisted at a concentration of about 0.5 to 50 mM. The ausogenic amylase is added at a rate of about 0.5 to 20 units per gram of starch, and reacted at pH 3 to 8 at a reaction temperature of 40 to 90 ° C. for 1 to 100 hours.

  Further, in order to increase the maltohexaose and maltoheptaose contents in the reaction product as much as possible, the degree of liquefaction by starch acid or α-amylase should be as low as possible, and should be less than DE10, preferably less than DE5. It is preferable to cause maltohexaose-maltoheptaose-forming amylase to act.

  In the reaction, other starch-related enzymes such as cyclomaltodextrin glucanotransferase (EC 2.4.1.19) and α-amylase (EC 3.2.1) are used together with maltohexaose / maltoheptaose-forming amylase. .1), β-amylase (EC 3.2.1.2), glucoamylase (EC 3.2.1.3), α-glucosidase (EC 3.2.2.120), pullulanase (EC 3.2.1. 41), isoamylase (EC 3.2.1.68), maltotetraose-forming amylase (EC 3.2.1.60) and the like are used in combination to obtain the resulting maltohexaose and maltoheptaose-containing saccharides. It is optional to vary or to increase the maltohexaose and maltoheptaose content.

  In particular, it is possible to advantageously increase the yield of maltohexaose and maltoheptaose using starch in combination with maltohexaose / maltoheptaose-forming amylase and starch debranching enzymes such as pullulanase and isoamylase. Using maltohexaose and maltoheptaose-containing saccharide having a total content of maltohexaose and maltoheptaose of about 30 to 50% per solid as a raw material, contaminating saccharides, dextrin and the like are separated, It is also advantageous to collect maltohexaose and / or maltoheptaose.

  Examples of the separation method include use of a semipermeable membrane described in JP-A-48-4647, use of an organic precipitant described in JP-A-49-102854, and JP-A-59-148794. Use of column chromatography using a salt type strongly acidic cation exchange resin described in the publication is appropriately selected, and if necessary, a maltohexaose-rich saccharide or a maltoheptaose-rich saccharide having a purity of 90% or more is required. It is also possible to collect quality. In particular, the method of separating and removing contaminating saccharides by a method using a salt type strongly acidic cation exchange resin to collect a maltohexaose-rich solution and / or a maltoheptaose-rich solution is advantageous on an industrial scale. Can be implemented. At this time, any of the known fixed bed system, moving bed system, and simulated moving bed system may be used.

  The enzyme reaction solution or the solution from which contaminating carbohydrates are separated are filtered, centrifuged, etc. to remove insolubles, and then purified by activated carbon, desalting with H-type or OH-type ion exchange resin, etc. After the process, it is concentrated to a syrup-like product. If necessary, the product may be further dried to a powdery product by a method such as spray drying.

  The maltohexaose and / or maltoheptaose-containing saccharide of the present invention thus obtained usually contains maltohexaose and / or maltoheptaose in an amount of 30% or more, and more preferably 40% or more, based on solid matter. This powdered product, although slightly fluctuating depending on its content, is practically hard to absorb moisture, does not consolidate, has good fluidity and is easy to handle, and its packaging, transportation, storage, etc. The physical and human costs required for managing the system can be greatly reduced.

  In addition, this powdered product is substantially a hardly hygroscopic powder, has high heat resistance, and has good stability, so that powdered sweeteners, instant juices, instant soups, granules, tablets, etc., which have been considered extremely difficult in the past, can be used. As an excipient, bulking agent, powder base, etc., and further substituting flours such as flour, corn grits, starch for part or all, for example, pudding mix, hot cake mix, confectionery material, bread making material, cereal It can be advantageously used for materials. Furthermore, it is possible to advantageously carry out reduction of maltohexaose and / or maltoheptaose-containing saccharide to produce chemically more stable maltohexaitol and / or maltoheptaitol-containing saccharide.

  For example, a sugar containing maltohexaose and maltoheptaose is converted into an aqueous solution having a concentration of about 40 to 60%, placed in an autoclave, Raney nickel is added as a catalyst at about 8 to 10%, and the temperature is raised to 90 to 140 ° C. while stirring. After the hydrogenation was completed by raising the hydrogen pressure to 20 to 150 kg / cm2, Raney nickel was removed, and then decolorization and desalting were carried out in the same manner as in the production of maltohexaose and maltoheptaose-containing saccharides. It is purified and concentrated to a syrup-like product, or further dried by spray drying or the like to obtain a powdery product.

  The thus obtained maltohexaitol and maltoheptitol-containing saccharide usually contain maltohexaitol and maltoheptitol in an amount of 30% or more per solid. Using the thus obtained maltohexaitol and maltoheptitol-containing saccharide as a raw material, contaminating saccharides are separated by a fractionation method or the like in the same manner as in the case of maltohexaose and maltoheptaose, and further purified. It may also be advantageous to collect maltohexaitol and / or maltoheptitol with a purity.

  Maltohexaose and / or maltoheptaose-containing saccharide or maltohexaitol and / or maltoheptitol-containing saccharide produced by the method described above are used as low-potency sweeteners, Widely used as a viscosity modifier, moisturizer, illuminant, fragrance inhibitor, crystallization inhibitor, anti-sagging agent for candy, anti-aging agent for starch, etc., and also as a nutritional supplement, food, beverage, food, feed, cosmetics It is advantageously used in various compositions such as pharmaceuticals, molded products, etc., as well as daily necessities, agricultural, forestry and fishery products, reagents, and chemical industrial products.

  Maltohexaose and / or maltoheptaose-containing saccharide or maltohexaitol and / or maltoheptyitol-containing saccharide can be used as a seasoning for sweetening as it is while having a low sweetness. Then, for example, powdered candy, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L- Phenylalanine methyl ester, saccharin, glycine, alanine and the like, may be used in combination with one or more appropriate amounts of other sweeteners, and if necessary, mixed with dextrin, starch, lactose, etc. Can also be used.

  In addition, the taste of maltohexaose and / or maltoheptaose-containing saccharide or maltohexaitol and / or maltoheptyitol-containing saccharide may be other than acidity, salt to taste, astringency, umami, and bitterness. It is well harmonized with various substances having taste and has high acid resistance and heat resistance, so that it can be advantageously used for sweetening and taste improvement of general foods and drinks, quality improvement, and the like.

  For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, Hishio, sprinkle, mayonnaise, dressing, vinegar, three tablespoons vinegar, powdered sushi vinegar, Chinese ingredients, Tentsuyu, noodle soup, sauce, ketchup, takuan pickles, Chinese cabbage pickles It can be advantageously used as a variety of seasonings such as nonomoto, yakiniku sauce, curry roux, stew nosouce, soup soup, dash nosouce, compound seasoning, mirin, new mirin, table sugar, coffee sugar and the like. In addition, for example, various types of Japanese confectionery such as rice crackers, hail, rice bran, rice cakes, steamed buns, sea cucumber, sea cucumber, sheep shrimp, mizu-yori, nishikidama, jelly, castella, candy, bread, biscuit, crackers, cookies, pies , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy and other sweets, ice cream, sherbet, etc., ice confectionery, fruit syrup pickles, ice honey and other syrups, Pastes such as flower paste, peanut paste, fruit paste, spreads, fruits such as jam, marmalade, syrup pickles, sugar fruits, processed foods of vegetables, pickles such as fukugami pickles, betta pickles, senmai pickles, pickled pickles, hams, sausages, etc. Meat products, Manufactured from fish meat products such as meat ham, fish sausage, kamaboko, chikuwa, and tempura, various delicacies such as sea urchin, salted squid, vinegar konbu, sakisume, fugumirin dried, seaweed, wild vegetables, sardines, small fish, shellfish, etc. Prepared foods such as boiled tsukudani, boiled beans, potato salad, konbu-maki, dairy products, fish meat, animal meat, fruits, vegetable bottles, canned foods, sake, synthetic liquors, liqueurs, liquors such as Western sake, tea, coffee, Soft drinks such as cocoa, juice, carbonated drinks, lactic acid drinks, and lactic acid drinks, pudding mixes, hot cake mixes, instant meals, instant soups, and other instant foods, as well as various foods such as baby foods, therapeutic foods, and drinks It can be advantageously used for sweetening, improving taste, and improving physical properties.

  Further, it can be used for raising the preference of feed, feed and the like for domestic animals such as livestock, poultry and fish. In addition, various solids such as tobacco, toothpaste, lipstick, lip balm, oral liquid, tablets, troches, liver oil drops, mouth fresheners, mouth flavors, gargles, etc. It can be advantageously used as a sweetener for various compositions, as a taste improver, a flavor enhancer, and as a quality improver.

  As a quality improving agent and a stabilizer, it can be advantageously applied to various physiologically active substances which easily lose their active ingredients and activities, or health foods and pharmaceuticals containing the same. For example, lymphokine-containing liquids such as interferon-α, interferon-β, interferon-γ, Tsumore necrosis factor-α, Tsumore necrosis factor-β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, interleukin 2 , Hormone-containing solutions such as insulin, growth hormone, prolactin, erythropoietin, oocyte stimulating hormone, placental hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, pox seedling, tetanus toxoid, hub antitoxin, human immunoglobulin, etc. Liquids containing biologicals, liquids containing antibiotics such as penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin, kanamycin sulfate, thiamine, riboflavin , L-ascorbic acid, liver oil, carotenoids, ergosterol, tocopherol, etc., vitamin-containing solutions, lipase, esterase, urokinase, protease, β-amylase, isoamylase, glucanase, lactase, etc. enzyme-containing solutions, ginseng extract, terrapin Extracts, chlorella extract, aloe extract, propolis extract, and other extracts, viruses, lactic acid bacteria, live bacteria such as yeast, and various physiologically active substances such as royal jelly are also stable and high-quality without losing their active ingredients and activities. Health foods and pharmaceuticals can be easily manufactured.

  As described above, the composition according to the present invention includes not only foods and drinks, cosmetics, and pharmaceuticals used orally or parenterally, but also, for example, daily necessities, agricultural, forestry and fishery products, reagents, and chemicals. It has a wide range of uses such as industrial goods.

  In addition, the method of incorporating maltohexaose and / or maltoheptaose-containing saccharide or maltohexaitol and / or maltoheptitol-containing saccharide of the present invention into these compositions is carried out until the product is completed. Any known method such as mixing, dissolving, dipping, penetrating, spraying, coating, spraying, pouring, or solidifying may be appropriately selected. The amount to be contained varies depending on the composition, but is generally 0.1% or more, desirably 0.5% or more as maltohexaose, maltoheptaose, maltohexaitol or maltoheptitol. Is preferred. Next, the present invention will be described more specifically by experiments.

<Experiment 1: Production of enzyme>
Soluble starch 2.0 w / v%, peptone 0.5 w / v%, yeast extract 0.1 w / v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate dihydrate 0.06 w / v %, Magnesium sulfate heptahydrate 0.05% w / v%, calcium carbonate 0.5% w / v%, and water, 100 ml each in a 500 ml Erlenmeyer flask, and sterilized in an autoclave at 121 ° C. for 15 minutes. The mixture was cooled, inoculated with Alcaligenes latus D2271 (FERM BP-4578), and subjected to rotational shaking culture at 37 ° C. and 200 rpm for 20 hours to obtain a seed culture.

  About 20 l of a medium having the same composition as in the case of seed culture was placed in a fermenter having a capacity of 30 l, heat sterilized, cooled to a temperature of 37 ° C., and inoculated with 1 v / v% of a seed culture solution. The cells were cultured with aeration and stirring for about 24 hours. About 19 l of the culture was centrifuged to obtain about 18 l of the culture supernatant. The activity of maltohexaose / maltoheptaose-forming amylase in the culture supernatant was 58 units / ml.

<Experiment 2: Purification of enzyme>
The culture supernatant obtained in Experiment 1 was concentrated on a UF membrane to recover about 800 ml of a concentrated enzyme solution containing about 1,200 units of maltohexaose / maltoheptaose-forming amylase per ml. 300 ml of the obtained concentrated enzyme solution was dialyzed against 10 mM Tris-HCl buffer (pH 8.0) containing 5 mM calcium chloride for 24 hours, and centrifuged to remove insolubles. The dialysate supernatant (400 ml) was subjected to ion exchange column chromatography (gel amount: 100 ml) using DEAE-Toyopearl gel 650 gel (manufactured by Tosoh Corporation).

  The maltohexaose / maltoheptaose-forming amylase of the present invention did not adsorb to the DEAE-Toyopearl gel, and its activity was detected in the flow-through fraction. After collecting the enzymatically active fraction, it was dialyzed against the same buffer containing 0.5 M ammonium sulfate, and the dialysate was centrifuged to remove insolubles. Then, butyl toyopearl 650 gel (manufactured by Tosoh Corporation) ) Was used for the hydrophobic column chromatography (gel amount: 100 ml). The adsorbed maltohexaose / maltoheptaose-forming amylase was eluted from the column with a linear gradient of ammonium sulfate from 0.5M to 0M, and an enzyme active fraction was collected. Subsequently, hydrophobic column chromatography (gel amount: 10 ml) using Phenyl Super Rose HR5 / 5 (Pharmacia LKB, Sweden) was performed, and the eluted enzyme-active fraction was collected.

  The enzyme activity recovery rate of the purified enzyme preparation obtained by the above purification means was about 25% of that of the culture supernatant. The specific activity of the purified enzyme preparation was 1,860 units per mg of protein. The protein was quantified according to the Lowry method using bovine serum albumin as a standard. When the purity of the purified enzyme sample was assayed by gel electrophoresis containing 7.5 w / v% polyacrylamide, the protein band was a single, highly pure sample.

<Experiment 3: Properties of enzyme>
The maltohexaose / maltoheptaose-forming amylase standard obtained by the method of Experiment 2 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration: 10 w / v%), and simultaneously molecular weight markers (Nihon Bio-Rad Laboratories Co., Ltd.) The molecular weight of the enzyme was 43,000 ± 3,000 daltons as compared to that of the enzyme (manufactured by the company). The purified maltohexaose / maltoheptaose-forming amylase was subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2 w / v% ampholine (manufactured by Pharmacia LKB, Sweden). When the isoelectric point of the enzyme was determined by measurement, the isoelectric point was 7.6 ± 0.5.

  The effects of temperature and pH on the activity of the present enzyme were examined in the presence of 5 mM calcium chloride according to the activity measurement method. The results are shown in FIG. 1 (effect of temperature) and FIG. 2 (effect of pH). The optimum temperature of the enzyme was around 70 ° C., and the optimum pH was about 5.0. The thermostability of this enzyme was determined by keeping the enzyme solution (20 mM acetate buffer containing 5 mM calcium chloride, pH 6.0) at each temperature for 1 hour, cooling with water, and measuring the remaining enzyme activity. The pH stability was determined by maintaining the enzyme in a 20 mM buffer solution of each pH containing 5 mM calcium chloride at 40 ° C. for 1 hour, adjusting the pH to 6, and measuring the remaining enzyme activity. . The respective results are shown in FIG. 3 (thermal stability) and FIG. 4 (pH stability). The thermostability of this enzyme was around 60 ° C., and the pH stability was about 4.5 to 10.5. In addition, under the conditions in which calcium ions were removed from the enzyme solution and the substrate solution, the amylase showed almost no starch-decomposing activity and had a remarkably high dependence on calcium ions. The enzyme activity was inhibited by 1 mM copper ion, lead ion, zinc ion, mercury ion, or 10 mM EDTA.

<Experiment 4: Action on starch>
The purified maltohexaose / maltoheptaose-forming amylase obtained by the method of Experiment 2 was added to a soluble starch (20 mM acetate buffer containing 5 mM calcium chloride, pH 5.5) at a final concentration of 2 w / v% at a temperature of 40 ° C. The reaction was performed by adding 50 units per gram of starch, and the reaction solution was collected over time and heated at 100 ° C. for 10 minutes to inactivate the enzyme. Using this enzyme reaction solution, the hydrolysis rate, blue value, and sugar composition were analyzed.

  The hydrolysis rate was determined as the ratio of the amount of reducing sugar to the total amount of sugar by quantifying the total amount of sugar in the reaction solution by the anthrone sulfate method and the amount of reducing sugar by glucose conversion by the Somogy-Nelson method. The blue value is determined by adding 0.1 ml of the reaction solution to 15 ml of a 0.05 N aqueous sulfuric acid solution, stirring the mixture, and adding 0.2 ml of a 0.1 N iodine solution to develop a color. The absorbance at 660 nm is measured as iodine coloration. It was determined as a percentage of the iodine color degree at each time with respect to the iodine color degree.

  The sugar composition was analyzed by high performance liquid chromatography (hereinafter abbreviated as HPLC) after desalting the reaction solution. The HPLC apparatus is a CCPD system (manufactured by Tosoh Corporation), the analytical column is MCI GEL CK04SS (10 mmφ × 200 mm, manufactured by Mitsubishi Kasei Corporation), the eluent is water at a flow rate of 0.2 ml / min, and the detection is a differential refractometer. went. The results are shown in Tables 1 and 2.

  As shown in the results of Table 1, the decrease in iodine coloration was large compared to the increase in the hydrolysis rate of starch, and a small amount of low molecular weight such as maltose and maltotriose was observed from the beginning of the reaction. The oligosaccharide was confirmed to be an endo-amylase, and further, the anomeric form of the reaction product was examined by optical rotation. Α-amylase.

  Also, as is clear from the results in Table 2, maltohexaose and maltoheptaose are generated more than other oligosaccharides from the beginning of the reaction, and these components increase with the reaction, and even when the reaction time is prolonged. Since the contents of maltohexaose and maltoheptaose were not reduced, the amylolytic enzyme of the present invention is a novel maltohexaose and maltoheptaose that essentially produces maltohexaose and maltoheptaose from starch. Α-amylase of this type. The production rates of maltohexaose and maltoheptaose from starch were about 45% in total.

<Experiment 5: Substrate specificity>
Amylose, amylopectin, glycogen, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, pullulan, dextran, glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexa at a final concentration of 2 w / v% The purified maltohexaose / maltoheptaose-forming amylase obtained by the method of Experiment 2 was added to aose or maltoheptaose at 50 units per gram of solid substrate at 40 ° C. and pH 5.5 in the presence of 5 mM calcium chloride. Time acted. The reaction solution before and after the enzymatic reaction is subjected to thin-layer chromatography (hereinafter abbreviated as TLC) using Kieselgel 60 (manufactured by Merck; aluminum plate, 20 × 20 cm) to confirm the presence or absence of enzymatic action on each carbohydrate. did. TLC was developed once at room temperature using 1-butanol: pyridine: water = 6: 4: 1 (volume ratio) as a developing solvent. Color development was performed by spraying a 20 v / v% sulfuric acid-methanol solution and heating at 110 ° C. for about 10 minutes.

  As a result, it acts on amylose, amylopectin, and glycogen to produce mainly maltohexaose and maltoheptaose as in the case of using soluble starch as a substrate, and produces α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin Does not act on pullulan, dextran, glucose, maltose, maltotriose, maltotetraose, maltopentaose, or maltohexaose, and slightly produces glucose and maltohexaose as decomposition products from maltoheptaose. However, it turned out to be extremely difficult to work.

<Experiment 6: Acute toxicity test>
Using a mouse, a powdery maltohexaose-rich saccharide produced by the method of Example 7, a powdery maltoheptaose-rich saccharide, and a syrup-like maltohexaitol-rich saccharide produced by the method of Example 8 An acute toxicity test was performed by orally administering the saccharide and the syrup-like maltoheptitol-rich saccharide to each. As a result, maltohexaose, maltoheptaose, maltohexaitol, and maltoheptyitol were all low-toxic substances, and no deaths were observed even at the maximum administrable dose. Therefore, although the exact value cannot be said, the LD50 values were all 50 g / kg or more.

  Hereinafter, examples of the present invention will be described. In Examples 1 to 9, maltohexaose / maltoheptaose-forming amylase and maltohexaose and / or maltoheptaose-containing saccharide using the same, or maltohexaitol and / or maltoheptaitol-containing saccharide The production method is described, and compositions containing the saccharide are described in Examples 10 to 21.

  Alkalinegenes Latus D2271 (FERM BP-4578) was cultured in a fermenter for about 30 hours with a fermenter according to the method of Experiment 1 with the same medium composition as in Experiment 1 except that the culture temperature was 30 ° C. The activity of maltohexaose / maltoheptaose-forming amylase in the culture supernatant was 66 units per ml of culture. The culture solution was subjected to MF membrane filtration, and the permeate was concentrated with a UF membrane, and the maltohexaose / maltoheptaose-forming amylase was recovered at a yield of about 82% with respect to the activity of the original culture solution. A concentrated enzyme solution having 1,300 units was obtained.

  5% potato starch milk (pH 5.5) was heated and gelatinized, cooled to 70 ° C. and added with calcium chloride to a concentration of 0.02%, and maltohexaose / maltohepta prepared in Example 1 was added. Aose-forming amylase was added at a ratio of 2 units per gram of starch, and liquefaction and saccharification were performed for 20 hours. The reaction solution was heated in an autoclave (120 ° C.) for 20 minutes to inactivate the enzyme, cooled, decolorized and filtered with activated carbon according to a conventional method, and desalted with H-type and OH-type ion exchange resins. Purification and further concentration yielded a syrup with a concentration of about 75% in a yield of about 95% based on the raw solids. The product contained 24.2% maltohexaose and 17.7% maltoheptaose per solid, and its sweetness was about 15% that of sugar. This product is widely used as a sweetener and as a body-imparting agent, viscosity modifier, humectant, sunshine-imparting agent, adhesive, fragrance-preventing agent, crystallization inhibitor, candy sagging inhibitor, etc. It can be advantageously used for various compositions such as molded articles.

  10% potato starch milk (pH 4.5) is gelatinized by heating, then cooled to a temperature of 50 ° C., and isoamylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) is added thereto so that the ratio becomes 1000 units per gram of starch. In addition, the reaction was carried out for 20 hours. The reaction solution was adjusted to pH 5.2, autoclaved (120 ° C.) for 10 minutes, cooled to 70 ° C., and added with calcium chloride to a concentration of 0.02%. The prepared maltohexaose / maltoheptaose-forming amylase was added at a ratio of 5 units per gram of starch, and liquefaction and saccharification were carried out for 8 hours. The reaction solution was heated in an autoclave (120 ° C.) for 20 minutes, cooled, and purified and concentrated in the same manner as in Example 2 to obtain a syrup having a concentration of 75% with a yield of about 93% based on the solid material. The sugar composition of this product is 40.3% of oligosaccharides having a glucose polymerization degree of 5 or less, 27.3% of maltohexaose, 19.2% of maltoheptaose, and 13.2% of dextrin having a glucose polymerization degree of 8 or more. Was. This product can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as in the case of Example 2 as a low-sweetness, low-sugar sweetener and the like.

  After adding calcium carbonate to 33% corn starch milk to a final concentration of 0.1% by weight, the pH was adjusted to 6.0, and α-amylase (Novo Co., trade name: Tamamir 60 L) was added to the gram starch. The reaction was carried out at 95 ° C. for 15 minutes. The reaction solution was subjected to autoclave (120 ° C.) for 30 minutes, and then cooled to 50 ° C., to which isoamylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was added. Hexaose / maltoheptaose-forming amylase was added at a ratio of 1.8 units per gram of starch and allowed to react for 40 hours. This reaction solution was heated and cooled in the same manner as in Example 2, purified, concentrated and spray-dried to obtain powdered maltohexaose and maltoheptaose-containing saccharides having a water content of less than 2%, and the yield per solid material was about 90. The sugar composition of this product is 38.8% of oligosaccharides having a degree of glucose polymerization of 5 or less, 26.7% of maltohexaose, 15.7% of maltoheptaose, and 18.8% of dextrin having a degree of glucose polymerization of 8 or more. Was. This product can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as in the case of Example 2 as a low-sweetness, low-sugar sweetener and the like.

Maltohexaose and maltoheptaose-containing saccharide obtained by the method of Example 3 were fractionated by column chromatography using a strongly acidic cation exchange resin to increase the contents of maltohexaose and maltoheptaose. Maltohexaose and maltoheptaose-containing saccharide produced by the method of Example 3 were adjusted to a concentration of 55% to obtain a raw sugar solution. The resin used is an alkali metal type strongly acidic cation exchange resin (manufactured by Mitsubishi Kasei Kogyo Co., Ltd., trade name: Diaion SK 104K ⁺ type, degree of cross-linking: 4%), and four jacketed stainless steel columns with an inner diameter of 5.4 cm. To make the total length of the resin layer 20 m. While maintaining the temperature in the column at 60 ° C., the raw sugar solution was added to the resin at 7 v / v%, and hot water at 60 ° C. was flowed at a flow rate of SV 0.12 to fractionate the mixture, and maltohexaose 35% or A maltohexaose having a maltoheptaose content of 25% or more and a maltoheptaose-rich fraction were collected, purified and concentrated in the same manner as in Example 2 to give a 75% syrup-like maltohexaose and maltoheptaose-containing sugar. The quality was obtained in about 50% yield per raw sugar liquor solids. The product contained about 45% maltohexaose and about 31% maltoheptaose per solid, and its sweetness was less than 10% that of sugar. This product can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as in the case of Example 2 as a low-sweetness, low-sugar sweetener and the like.

  The syrup-like maltohexaose and maltoheptaose-rich saccharide obtained by the method of Example 5 are further spray-dried, and the powdery maltohexaose and maltoheptaose-rich material having a water content of less than about 2% are used as raw material syrup solids. Obtained in about 98% yield per product. This product can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as in the case of Example 2 as a low-sweetness, low-sugar sweetener and the like.

Maltohexaose and maltoheptaose-rich saccharides produced by the method of Example 5 were adjusted to a concentration of 50% to prepare a raw sugar solution. As the resin, an alkali metal-type strongly acidic cation exchange resin (manufactured by Mitsubishi Kasei Kogyo Co., Ltd., trade name: Diaion SK 104 K ⁺ type, degree of crosslinking: 4%) was used, and packed in a column in the same manner as in Example 5. While maintaining the temperature in the column at 60 ° C., the raw sugar solution was added to the resin at 5 v / v%, and hot water at 60 ° C. was flowed at a flow rate of SV 0.10 to fractionate, and maltohexaose was 80% or more. And a high-content fraction of maltoheptaose of 70% or more were collected, and then purified, concentrated and spray-dried in the same manner as in Example 2 to obtain a powdery malt having a water content of less than about 2%. Hexaose-rich saccharides and powdered maltoheptaose-rich saccharides were obtained at a yield of about 30% and 20%, respectively, based on the raw sugar liquid solids. The purity of the powdery maltohexaose-rich saccharide and the powdery maltoheptaose-rich saccharide thus obtained are about 89% and about 81%, respectively, and their sweetness is less than 10% of that of sugar. Met. This product can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as in the case of Example 2 as a low-sweetness, low-sugar sweetener and the like.

The syrup-like maltohexaose and maltoheptaose-containing saccharide produced by the method of Example 3 were adjusted to a concentration of 50%, placed in an autoclave, and Raney nickel 10% was added as a catalyst, and the temperature was raised to 90 to 125 ° C with stirring. The hydrogenation was completed by raising the hydrogen pressure to 20-100 kg / cm ² . Then, after removing Raney nickel, purification and concentration were carried out in the same manner as in Example 2, and maltohexaitol and maltoheptitol-containing saccharide having a water content of 25% were obtained at a yield of about 90% based on the raw material syrup solids. Obtained. The product contained about 27% maltohexaitol and about 19% maltoheptitol per solid, and its sweetness was about 20% of that of sugar. This product is widely used as a low-sweetness, non-reducing sweetener, and as a body-imparting agent, viscosity modifier, humectant, sunshine-imparting agent, adhesive, fragrance-preventing agent, anti-crystallizing agent, anti-sagging agent for candy, etc. Used for food and drink.

  The maltohexaose and maltoheptaose-rich saccharides produced by the method of Example 5 were hydrogenated, purified and concentrated by the method of Example 8 to obtain maltohexaitol and maltoheptitol-rich saccharides. The raw sugar solution was prepared at a concentration of 50%. This sugar solution was subjected to column chromatography using a strongly acidic cation exchange resin according to the method of Example 7 to obtain a high-content fraction of maltohexaitol of 80% or more and a high-content fraction of maltoheptitol of 70% or more. And then purified and concentrated in the same manner as in Example 2 to obtain a syrup-like maltohexaitol-rich saccharide and a syrup-like maltoheptitol-rich saccharide having a concentration of about 75%. Yields of about 30% and 20% per product, respectively. The syrup-like maltohexaitol-rich saccharide and the syrup-like maltoheptitol-rich saccharide thus obtained have a purity of about 90% and about 82%, respectively, and their sweetness is 10% that of sugar. %. This product can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as in the case of Example 2 as a low-sweetness, low-sugar sweetener and the like.

<Sweetener>
One part by mass of the syrup-like maltohexaose and maltoheptaose-containing saccharide obtained by the method of Example 2 was uniformly mixed with 0.02 parts by mass of α-glycosyl stevioside (trade name α-G suite, manufactured by Toyo Seika Co., Ltd.). The syrupy sweetener obtained by mixing has excellent sweetness, has about twice the sweetness of sugar, and has a calorie of about one half that of sugar per degree of sweetness. Therefore, the present sweetener is suitable as a low-calorie sweetener for sweetening low-calorie foods and drinks for those who restrict caloric intake, for example, obese, diabetic, and the like. In addition, the present sweetener is also suitable for sweetening foods and the like that suppress tooth decay by producing less acid by caries-inducing bacteria and producing less insoluble glucan.

<Custard cream>
500 parts by mass of cornstarch, 400 parts by mass of a powdery maltohexaose and maltoheptaose-rich saccharide obtained by the method of Example 6, 500 parts by mass of maltose, and 5 parts by mass of sodium chloride were thoroughly mixed through a sieve, and chicken egg 1 , 400 parts by mass and stirred, 5,000 parts by mass of boiled milk were gradually added thereto, and the mixture was further heated over a low heat. The stirring was continued, and the corn starch was completely gelatinized and the whole became translucent. The custard cream was prepared by turning off the heat, cooling and adding a small amount of vanilla flavor. This product has a smooth and glossy taste and is not too sweet.

<Uiro>
90 parts by mass of rice flour, 20 parts by mass of corn starch, 20 parts by mass of sugar, 1 part by mass of matcha powder, 90 parts by mass of syrup-like maltohexaitol and maltoheptitol-containing saccharide obtained by the method of Example 8, and an appropriate amount of water Was added and kneaded, and the mixture was put in a container and steamed for 60 minutes to produce matcha uiro. This product had a good shine, a good mouthfeel, and a good flavor. In addition, aging of the starch was suppressed, and the starch was stable for a long time.

<Hard candy>
90 parts by mass of sugar are mixed and dissolved in 70 parts by mass of the syrup-like maltohexaose and maltoheptaose-containing saccharide obtained by the method of Example 3, and the mixture is heated and concentrated under reduced pressure until the water content becomes less than about 2%. 0.15 parts by mass of citric acid and a small amount of a lemon flavor and a coloring agent were mixed with the mixture, and then molded according to a conventional method to produce a hard candy. This product is a crisp hard candy with less hygroscopicity and less tendency to sag.

<Betta pickles>
Syrup-like maltohexaose and maltoheptaose-containing saccharide obtained by the method of Example 2 1 part by mass, maltose 3 parts by mass, licorice preparation 0.05 parts by mass, malic acid 0.008 parts by mass, sodium glutamate 0.07 If the mixture was homogeneously mixed with 0.03 parts by mass of potassium sorbate and 0.2 parts by mass of pullulan, the mixture was mixed to produce a pickled base. 30 kg of radish was soaked with salt in a conventional manner, and then medium-soaked with sugar, and then immersed in a seasoning solution prepared with 4 kg of honbetara-zuke to prepare betara-zuke. This product was good in both color and luster and aroma, had moderate sweetness, and was crisp.

<Tube nutrition>
Powdered maltohexaose and maltoheptaose-containing saccharides obtained by the method of Example 4 20 parts by mass, glycine 1.1 parts by mass, sodium glutamate 1 part by mass, calcium lactate 0.4 part by mass, magnesium carbonate 0.1 A formulation consisting of parts by weight, 0.01 part by weight of thiamine and 0.01 part by weight of riboflavin is prepared. Each 24 g of the compound was filled in a laminated aluminum package and heat-sealed to obtain a product. This product is used by dissolving one bag in about 33 to 500 ml of water to make a nutritional supplement and administrating it into the nasal cavity and gastrointestinal tract by the tube method. The product can be advantageously used as a parenteral nutritional supplement for humans as well as livestock.

<Tube nutrition>
80 parts by mass of a powdery maltohexaose and maltoheptaose-rich saccharide obtained by the method of Example 6, 190 parts by mass of dried egg yolk, 209 parts by mass of skim milk powder, 4.4 parts by mass of sodium chloride 1.85 parts by mass of potassium chloride A mixture comprising 4 parts by weight of magnesium sulfate, 0.01 part by weight of thiamine, 0.1 part by weight of sodium ascorbate, 0.6 part by weight of vitamin E acetate and 0.04 part by weight of nicotinamide is prepared. Each 25 g of the mixture was filled in a laminated aluminum package and heat-sealed to obtain a product. This product is used as a nutritional supplement by dissolving one bag in about 150-300 ml of water, and then used by administration to the nasal cavity, esophagus, stomach, etc. by the tube method.

<Tablets>
50 parts by mass of aspirin, 10 parts by mass of maltose, 4 parts by mass of corn starch, and 4 parts by mass of a powdered maltohexaose and maltoheptaose-rich saccharide obtained by the method of Example 6 were uniformly mixed, and then a diameter of 12 mm and 20R Each tablet was tableted with a punch at 680 mg per tablet, tablet thickness 5.25 mm, and hardness 8 kg ± 1 kg. The product is an easy-to-drink tablet with moderate sweetness.

<Emulsion>
0.5 parts by mass of polyoxyethylene behenyl ether, 1 part by mass of polyoxyethylene sorbitol tetraoleate, 1 part by mass of lipophilic glyceryl monostearate, 0.5 part by mass of behenyl alcohol, 1 part by mass of avocado oil, of Example 9 3.5 parts by mass of the syrup-like maltohexaitol-rich saccharide obtained by the above method, 1 part by mass of α-glycosylrutin, an appropriate amount of vitamin E and an antiseptic were dissolved by heating according to a conventional method. 5 parts by mass of butylene glycol, 0.1 parts by mass of carboxyvinyl polymer and 85.3 parts by mass of purified water were added, and the mixture was emulsified with a homogenizer to produce an emulsion. This product is a moisturizing emulsion and can be advantageously used as a sunscreen, a fairing agent and the like.

<Skin cream>
2 parts by mass of polyoxyethylene glycol monostearate, 5 parts by mass of self-emulsifying glyceryl monostearate, 2 parts by mass of α-glycosylrutin, 1 part by mass of liquid paraffin, 10 parts by mass of glyceryl trioctanoate, obtained by the method of Example 7 4 parts by mass of the powdered maltohexaose-rich saccharide and an appropriate amount of a preservative were dissolved by heating according to a conventional method, and 5 parts by mass of 1,3-butylene glycol and 66 parts by mass of purified water were added thereto. And emulsified, and then an appropriate amount of a flavor was added, followed by stirring and mixing to produce a cream. This product is a cream with good elongation, and can be advantageously used as a sunscreen, a skin beautifying agent, a fairing agent and the like.

<Toothpaste>
45 parts by mass of dibasic calcium phosphate, 1.5 parts by mass of sodium lauryl sulfate, 25 parts by mass of glycerin, 0.5 parts by mass of polyoxyethylene sorbitan laurate, syrupy maltohexaose and maltoheptaose obtained by the method of Example 5 15 parts by mass of the high sugar content, 0.02 parts by mass of saccharin and 0.05 parts by mass of preservative were mixed with 13 parts by mass of water to obtain a toothpaste. This product has good gloss and detergency, and is suitable as toothpaste.

<Fertilizer pile>
Compound fertilizer (N = 14%, P2O5 = 8%, K2O = 12%), pullulan, maltohexaose and maltoheptaose-containing saccharide obtained by the method of Example 3, proportions of calcium sulfate and water by weight After sufficient mixing as 70, 5, 5, 15, and 5, the mixture was heated to 80 ° C. with an extruder (L / D = 20, compression ratio = 1.8, die diameter = 30 mm) to produce a fertilizer pile. This product does not require a fertilizer container, is easy to handle, has a strength suitable for full-layer fertilization, and can adjust the dissolution rate of fertilizer components by changing the mixing ratio. If necessary, it is easy to mix a plant hormone, an agricultural chemical, a soil conditioner and the like into the fertilizer pile.

  As is clear from the above, the maltohexaose / maltoheptaose-forming amylase of the present invention has an optimum temperature and a high temperature at a stable temperature, and has an optimum pH and a wide pH range at a stable pH. Has a high enzyme production amount and can be advantageously used for the production of maltohexaose and / or maltoheptaose-rich saccharides and maltohexaitol and / or maltoheptitol-rich saccharides from starch. As it turns out, its industrial significance is enormous.

  Furthermore, the maltohexaose and / or maltoheptaose-rich saccharide produced in this manner and the maltohexaitol and / or maltoheptoitol-rich saccharide obtained by hydrogenating the maltohexaose and / or maltoheptaitol-rich saccharide are low. It is widely used as a sweetening agent for sweetness, as a body-imparting agent, viscosity modifier, humectant, sunshine-imparting agent, and as a nutritional supplement, and is widely used in various compositions such as food and drink, cosmetics, pharmaceuticals, and molded products. It is also a great feature to be able to sing.

It is a figure which shows the influence of the temperature which has on the enzyme activity of maltohexaose maltoheptaose production | generation amylase of this invention. It is a figure which shows the influence of pH which has on the enzyme activity of maltohexaose maltoheptaose production | generation amylase of this invention. It is a figure which shows the influence of the temperature which gives the stability of the maltohexaose maltoheptaose production | generation amylase of this invention. It is a figure which shows the influence of pH on the stability of the maltohexaose maltoheptaose producing amylase of the present invention.

Claims (9)

  It produces maltohexaose and maltoheptaose mainly from starch, and does not substantially decompose low molecular weight oligosaccharides lower than maltohexaose. A maltohexaose and / or maltoheptaose-containing saccharide obtained by allowing amylase to act or by acting a starch debranching enzyme together with the maltohexaose / maltoheptaose-forming amylase.   The maltohexaose and / or maltoheptaose is maltohexaose and / or maltoheptaose-rich saccharide obtained by separating contaminating saccharides. // Maltoheptaose-containing saccharide.   The maltohexaose and / or maltoheptaose-containing saccharide according to claim 1 or 2, which contains 20% by mass or more of maltohexaose and a mass ratio of maltoheptaose to maltohexaose of 0.5 or more. .   Maltohexaose / maltoheptaose-forming amylase which has an action of mainly producing maltohexaose and maltoheptaose from starch and has substantially no decomposing action on low-molecular oligosaccharides of maltohexaose or less is a gene belonging to the genus Alcaligenes The maltohexaose and / or malto according to any one of claims 1 to 3, wherein the maltohexaose and / or malto has a molecular weight of 43,000 ± 3,000 daltons as determined by SDS-gel electrophoresis, which is derived from a microorganism. Heptaose-containing carbohydrate.   The maltohexaose and / or maltoheptaose-containing saccharide according to claim 4, wherein the microorganism of the genus Alcaligenes is Alcaligenes latus D2271 (National Institute of Bioscience and Human Technology, Accession No. FERM BP-4578). .   A maltohexaitol and / or maltoheptitol-containing saccharide obtained by further hydrogenating the maltohexaose and / or maltoheptaose-containing saccharide according to any one of claims 1 to 5.   The maltohexaitol and / or maltoheptitol is a maltohexaitol and / or maltoheptitol-rich saccharide obtained by separating contaminating saccharides. Maltohexaitol and / or maltoheptitol-containing saccharide.   A maltohexaose and / or maltoheptaose-containing saccharide according to any one of claims 1 to 5, or a maltohexaitol and / or maltoheptitol-containing saccharide according to claim 6 or 7. Composition. 9. The composition according to claim 8, wherein the composition is a food, a drink, a cosmetic, a medicine, or a molded product.

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