JPH09224665A - Beta-fructofuranosidase, production and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the above enzyme, produced by a specific microorganism, having the high fructosyl group transfer ability and useful for producing a saccharide having the transferred fructosyl groups therein. SOLUTION: This β-fructofuranosidase has the following physicochemical properties: (1) capable of hydrolyzing a β-fluctofuranoside bond in sucrose or raffinose, liberating fructose and further catalyzing a reaction for transferring the β-fructofuranosyl group from a glucide having the β-fructofuranoside bond to other gulcides, etc.; (2) having 49000±5000Dal molecular weight [measured by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)]; (3) isoelectric point: 4.6±0.5; (4) optimum pH: about 5.5-6.0 (at 40 deg.C for 10min) and (5) optimum temperature: about 45 deg.C (at pH6.0 for 10min and raised to about 50 deg.C when Ca ions are present). The enzyme is obtained by culturing Bacillus sp. V230 (FERM BP-5054).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel β-fructofuranosidase having a high fructosyl transfer ability, a method for producing the same and use thereof.

[0002]

2. Description of the Related Art A fructosyl-transferring sugar, such as xylosylfructoside and lactosucrose, which is produced by the transglycosylation activity of β-fructofuranosidase (also known as fructosyltransferase) from sucrose and other sugars It is an oligosaccharide that has attracted attention in the fields of foods and pharmaceuticals because it has properties such as caries resistance and bifidobacteria growth promoting property.

Β that produces these fructosyl transfer sugars
-As a fructofuranosidase, Arthrobacter species K-1 (Arthroba spp.
cter sp. K-1), and Bacillus megaterium I
The enzyme produced by FO 13498 is known.

Arthrobacter Species K-1
Β-fructofuranosidase is a product of “Agricultural and Biological Chemistry (Agri
cultural and Biological C
the optimum pH of the reaction according to "History", Vol. 54, No. 4, 913 to 919, (1990).
Is set to 6.5 to 6.8. In this pH range,
Under industrial temperature conditions of enzymatic reaction, sugar browning is likely to occur, and this tendency is particularly noticeable in the reaction that produces fructose, which may impose a great load on the decolorization operation in the purification process of the reaction sugar solution. To be done. In addition, β of Bacillus megaterium IFO 13498
-Fructofuranosidase is described in JP-A-4-200386.
As shown in the publication, the optimum pH of the reaction is 6.
It is 0, and the degree of browning of the enzyme reaction solution under that condition is considered to be low. However, the optimum temperature of this enzyme is as low as 40 to 45 ° C., and there is concern about microbial contamination in the enzyme reaction at temperatures around this temperature.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides β-fructofuranosidase, which has a high fructosyl transfer ability and is advantageous in industrial practice, a method for producing a fructosyl transfer sugar using the enzyme, and its use. It is a thing.

[0006]

In order to solve the above-mentioned problems, the present inventors have found that the optimum temperature of the reaction is high, the optimum pH is in the stable weak acidic region of sugar, and the sugar transfer ability is high. In search of a novel β-fructofuranosidase with high activity, microorganisms producing the enzyme were extensively searched. As a result, it was found that a novel microorganism V230 belonging to the genus Bacillus isolated from the soil of Okayama city produces the desired β-fructofuranosidase, and further, this enzyme was added to sucrose and other sugars. The present invention was completed by establishing a method for producing a saccharide containing a fructosyl-transferred saccharide by allowing it to act on a solution containing a saccharide, and also establishing a composition containing this saccharide.

The identification test results of the microorganism V230 belonging to the genus Bacillus of the novel microorganism of the present invention are shown below. The identification test was performed according to "Classification and Identification of Microorganisms" (Takeshi Hasegawa, Academic Society Publishing Center, 1985).

<A Cell Morphology> (1) Meat broth agar culture, 27 ° C., 1 day of culture: 1.0 to 1.5 × 3.0 to 7.5 μm bacilli. Single, rarely paired and linked cells are also observed. There is motility due to periflagellates. Non-acidic. Gram stain variable. Does not accumulate poly-β-hydroxybutyrate. Extremely rare endospores are observed after 4 days of culture. (2) potato extract / peptone / glucose agar medium,
27 ° C., 1 day of culture: 0.9 to 1.5 × 2.7 to 6.0
μm bacilli. Single, rarely paired and linked cells are also observed. There is mobility. Gram stain variable. Endospore formation.
The spore settlement position is at the end of the cell, with no swelling of the sporangium.
The spores are ellipses of 0.8 to 1.0 × 1.0 to 1.6 μm. Spore formation is also observed in culture using a soil extract medium.

<B Culture Properties> (1) Meat broth agar plate culture, 27 ° C. Shape: circular, size 1.0 to 1. 5 mm. 1.5 to 3 mm in 3 days. Edge: All edges Raised: Trapezoidal Luster: Dull light Surface: Smooth to verrucous Transparency: Semi-transparent to opaque Color tone: Cream (2) Meat agar slope culture, 27 ° C Growth: Good shape: Filiform. Protuberance is thin, surface is smooth, wet light to dull light, translucent, cream color (3) Meat juice gelatin stab culture, 27 ° C: liquefaction

<C Physiological Properties> (1) Reducibility of nitrate: Positive (2) Denitrification reaction: Negative (3) Methyl red test: Negative (4) VP test: Negative (5) Indole formation: Negative ( 6) Production of hydrogen sulfide: Negative (7) Decomposition of starch: Positive (8) Decomposition of casein: Negative (9) Accumulation of poly-β-hydroxybutyrate: Negative (10) Degradation of protocatechuic acid: Negative (11) Use of citric acid: Negative (12) Use of inorganic nitrogen source: Both ammonium salt and nitrate can be used. (13) Dye formation: Negative (14) Fluorescence dye formation: Negative (15) Urease: Positive (16) Oxidase: Positive (17) Catalase: Positive (18) Growth range: pH 5 to 8, temperature 15 to 45 ° C. (19) Attitude toward oxygen: Aerobic (20) Utilization of carbon source and presence / absence of acid production Availability Acid production ability D-glucose utilization positive D-galactose utilization positive D-mannose utilization positive D-fructose utilization positive L-arabinose used Positive D-xylose used positive L-rhamnose used positive Maltose used positive Sucrose used positive Lactose used negative Trehalose used positive Raffinose used positive Sorbitol used positive Mannitol used positive Zlutitol not used negative Glycerol not used Negative dextrin used Positive (21) Amino acid decarboxylation test: Negative to any of L-lysine, L-arginine and ornithine. (22) Use of amino acid: L-glutamate sodium, L-aspartate sodium, L-arginine, L-tryptophan, L-histidine, L-valine, and D-alanine are all used. (23) DNase: Negative (24) 3-Keto-lactose production: Negative (25) G-C content of DNA: 41% (26) Cell wall diamino acid: meso-diaminopimelic acid

Based on the above-mentioned mycological properties, "Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Sy
structural Bacteriology Vol
ume 2) ”, Vol. 2, (1986), and examined the difference between the known bacteria. As a result, this bacterium was aerobic and formed endospores, although Gram's staining was indeterminate, and thus it was found to be a microorganism belonging to the genus Bacillus. Furthermore, this bacterium is a Bacillus megaterium (Bacillus megaterium) from the above-mentioned mycological properties.
However, the endospores are formed at the end of the cell, casein degradability is negative, citric acid availability is negative, and growth at 10 ° C is negative.
It turned out to be distinctly different from megaterium.

[0012] From these results, the present inventors have confirmed that the microorganism of the present invention is Bacillus species.
p. ) Named V230, deposited on March 24, 1995 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, Tsukuba City, Ibaraki Prefecture, with deposit number FERM BP-505.
Commissioned as 4.

In the present invention, not only the strain of the present invention but also mutants thereof can be used.

The medium used for culturing the microorganism of the present invention may be any one that can grow the microorganism and produce the enzyme of the present invention, and may be either a synthetic medium or a natural medium. Any carbon source may be used as long as it can be assimilated by the microorganism.
For example, sugars such as sucrose, lactose, maltose, dextrin and starch, sugar-containing substances such as molasses and yeast extract and the like 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 sucrose in the culture solution is preferably 20 w / v% or less,
From the viewpoint of growth and proliferation of bacteria, usually 5 w / v% or less is preferable. 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. Further, as the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate salt, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt, etc. are appropriately used as necessary. Used.

The culture is usually carried out at a temperature of 15 to 45 ° C., preferably 25 to 37 ° C., pH 5 to 8, preferably pH.
It is performed aerobically under the condition selected from 5.5 to 7.5. The culture time may be any time as long as the present microorganism can grow, and is preferably 5 to 100 hours. The dissolved oxygen concentration of the culture solution is not particularly limited, but usually 0.5
To 20 ppm is preferred. For that purpose, means such as adjusting the amount of aeration, stirring, using oxygen, and increasing the pressure in the culture tank are adopted. Further, the culture system may be either batch culture or continuous culture.

After culturing the microorganism in this manner, the enzyme of the present invention is recovered from the resulting culture. This enzyme activity is found in the extracellular culture solution of the culture, and the extracellular culture solution may be recovered as a crude enzyme, or the entire culture can be used as a crude enzyme. An ordinary solid-liquid separation means is adopted for separating the extracellular culture liquid from the bacterial cells. For example,
A means for centrifuging the culture itself as it is, a means for adding a filter aid to the culture, or a means for filtering and separating with a precoat filter precoated with a filter aid, a means for membrane filtration using a flat membrane, a hollow fiber membrane, etc. Are adopted. Although the extracellular culture solution can be used as a crude enzyme solution as it is, it is preferably concentrated before use. 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 and the like are adopted.

Further, the extracellular culture solution and its concentrate are
It can also be fixed. For example, a binding method to an ion exchanger, a covalent binding / adsorption method with a resin or a membrane, an encapsulation method using a polymer substance, etc. are adopted.

The crude enzyme may be used as it is, or may be purified before use. For example, after dialysis of a crude enzyme preparation obtained by salting out the extracellular culture solution with ammonium sulfate and concentrating it, anion exchange column chromatography using DEAE-Toyopearl resin, followed by hydrophobic column chromatography using butyl Toyopearl resin, By purifying again by anion exchange column chromatography using DEAE-Toyopearl resin, an electrophoretically single enzyme can be obtained.

The β-fructofuranosidase of the present invention thus obtained has the following physicochemical properties. (1) Action Hydrolyzes β-fructofuranoside bonds of sucrose, raffinose and erulose to release fructose.
Further, the sugar having these β-fructofuranoside bonds is used as a sugar donor to catalyze the transfer of the β-fructofuranosyl group to an acceptor selected from other sugars, sugar alcohols and alcohols. (2) Molecular weight By SDS-gel electrophoresis, 49,000 ± 5,000.
Dalton (3) Isoelectric focusing Amphoraine-containing electrophoresis method 4.6 ± 0.5 (4) Optimum pH temperature 40 ° C, 10 minutes reaction, about 5.5 to 6.0 (5) Optimum Temperature pH 6.0 for 10 minutes, around 45 ° C in the absence of calcium ions, around 50 ° C in the presence of calcium ions (6) pH stability pH of about 5.0 at 24 ° C for 24 hours. Stable in the range of to 8.0 (7) Temperature stability pH 6.0, stable up to about 45 ° C under the condition of 1 hour holding (8) Inhibition 1 mM Cu ⁺⁺ , Pb ⁺⁺ , Fe ⁺⁺ , Fe ⁺ Inhibited by ⁺⁺ and Hg ⁺⁺ .

The activity of β-fructofuranosidase of the present invention is measured as follows. Sucrose 1.0 as a substrate
Aqueous solution containing w / v% (20 mM acetate buffer, pH 6.
(Containing 0.5 mM calcium chloride) to 0.5 ml of the enzyme solution.
After adding 2 ml and reacting at 40 ° C. for 10 minutes, a somoggy copper solution is added to stop the reaction, and the reducing power is measured by the Somogy Nelson method. As a control, 100
The same measurement is performed using the enzyme solution inactivated by heating at 0 ° C for 10 minutes. One unit of enzyme activity is defined as the amount of enzyme that increases the reducing power corresponding to 2 μmol glucose per minute by the above measurement method.

The β-fructofuranosidase of the present invention is
It acts on sucrose to produce glucose and fructose, but even if the sucrose concentration is increased, the transfer reaction between sucrose molecules is unlikely to occur, and the production of oligosaccharides having a larger molecular weight than sucrose is extremely small. However, when sucrose is used as a sugar donor and another suitable sugar, for example, a reducing sugar such as xylose, galactose, lactose, maltose, or isomaltose is used as an acceptor to act the enzyme, each reducing property is reduced. Transferring the fructosyl group to sugar, xylosylfructoside,
Galactosyl fructoside, lactosucrose (aka,
It produces lactosylfructoside), erulose (also known as maltosylfructoside), and isomaltosylfructoside. In addition, when the present enzyme is similarly acted by using a non-reducing disaccharide composed of glucose such as trehalose or neotrehalose as another sugar as an acceptor, fructosyl trehalose and fructosyl neotrehalose are produced. Not only monosaccharides and disaccharides, but also oligosaccharides having a higher degree of polymerization such as xylooligosaccharides, galactooligosaccharides, maltooligosaccharides and isomaltooligosaccharides, and sugar alcohols such as sorbitol and maltitol can be acceptors. It is also advantageous to use two or more types of receptors simultaneously. In addition to sucrose, oligosaccharides having a β-fructofuranoside bond such as raffinose and erulose can also be used as the donor.

The substrate concentration in the fructosyl-transferring sugar production reaction using the β-fructofuranosidase of the present invention is
Although not particularly limited, industrially 10 w / w% (hereinafter,
In the present specification, w / w% is abbreviated as% unless otherwise specified. ) Or more, and preferably 20 to 60%. The reaction temperature may be a temperature at which the enzyme is not inactivated in the presence of the substrate, that is, up to about 60 ° C., preferably about 50 to 55 ° C. The reaction pH may be usually adjusted to a range of about 3.5 to 8.0, preferably pH of about 4.5 to 6.5. The reaction time may be appropriately selected depending on the progress of the enzymatic reaction, and is usually about 0.1 to 100 hours at a usage amount of about 0.1 to 50 units per gram of the solid substrate.

The production rate of fructosyl-transferred sugar varies depending on the substrate concentration of the enzyme reaction solution, the type of acceptor used, the reaction conditions and the like. For example, when 20% sucrose and 20% lactose are used as substrates, the production rate of lactosucrose is about 40% at maximum.

The reaction solution is filtered, centrifuged, etc. to remove insoluble materials by a conventional method, and then decolorized with activated carbon, H type,
After a purification process such as desalting with an OH type ion exchange resin, the product is concentrated to give a syrup-like product. If desired, further drying into a powdered product is optional.

Further, in order to increase the content of transferase, it is also possible to separate and purify the target saccharide from the enzyme reaction solution to obtain a high fructosyl transferase content. Examples of the method include a method of removing monosaccharides by a fermentation method using an invertase-deficient yeast (yeast fermentation method), and a method of decomposing reducing sugars by heat treatment with an alkaline solution (alkaline treatment method). A method for separating and removing contaminant saccharides by a membrane filtration method, column chromatography, etc. can be appropriately adopted. Above all, JP-A-58-2379
No. 9 and JP-A No. 58-72598 disclose column chromatography using a salt-type strongly acidic cation exchange resin to remove contaminating saccharides and collect a desired fraction containing a high fructosyl transfer sugar. The method can be advantageously carried out on an industrial scale. At this time, any of the known fixed bed method, moving bed method, and pseudo moving bed method may be adopted.

The liquid from which the contaminant sugars have been separated is subjected to a purification process such as decolorization with activated carbon and desalting with an H-type or OH-type ion exchange resin after removing insolubles by a conventional method such as filtration and centrifugation. And concentrate to give a syrup-like product. If necessary, it is optionally further dried by a method such as spray drying to give a powdered product.

The thus-obtained β-fructofuranosidase-containing sugar of the present invention containing fructosyl-transferring sugars usually contains 5% or more, preferably 10% or more, of fructosyl-transferring sugar per solid matter. .

The fructosyl-transferred sugar-containing sugar produced by the above-mentioned method has a good taste and sweetness, and also has an osmotic pressure adjusting property, a moisturizing property, a light-imparting property, a crystallization preventing property and a starch aging preventing property. In addition, it also has functions such as caries resistance, bifidobacteria growth promotion, mineral absorption promotion, etc.,
Widely used in various compositions such as food and drink, favorite foods, feeds, feeds, cosmetics, pharmaceuticals, molded products, and also household products, agricultural, forestry and fishery products, reagents, chemical industrial products and the like.

The fructosyl-transferred sugar-containing sugar can be used as it is as a seasoning for sweetening, but if necessary, for example, starch syrup, glucose, maltose, trehalose, sucrose, isomerized sugar, honey, Maple sugar, sorbitol, maltitol, lactitol,
Used in admixture with one or more appropriate amounts of other sweeteners such as dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, etc. It may be used, and if necessary, it can be used by mixing with a bulking agent such as dextrin, starch, lactose and the like.

The taste of the sugar containing fructosyl-transferred sugar is in good harmony with various substances having other tastes such as sourness, salt to taste, astringency, umami and bitterness, so that the sweetness of general foods and drinks It can be advantageously used for attaching, improving taste, and improving quality.

For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hisoio, sprinkle, mayonnaise, dressing, vinegar, three tablespoons vinegar, powdered sushi vinegar, Chinese cabbage, tempura sauce,
Noodle soup, sauce, ketchup, pickled tuna, pickled cabbage, sauce of roasted meat, curry roux, stew, soup, soup stock, dashi stock, mixed seasonings, mirin, new mirin, table sugar, coffee sugar, etc. , Can be advantageously used as various seasonings.

Further, for example, various Japanese sweets such as rice crackers, hail, rice cakes, rice cakes, steamed buns, uiro, bean paste, yokan, water yoyo, nishikidama, jelly, castella, candy, etc.,
Bread, biscuits, crackers, cookies, pies, puddings, butter cream, custard cream, cream puffs, waffles, sponge cakes, donuts, chocolate, chewing gum, caramel, candy and other Western confectionery, ice cream, sherbet, and other frozen desserts,
Fruit syrup pickles, syrups such as ice honey, flower paste, peanut paste, fruit paste, pastes such as spreads, jam, marmalade, syrup pickles, fruits such as sugar fruit, processed foods of vegetables, Fukugami pickles,
Pickles such as Beta-ra-zuke, Sen-mai-zuke, and Rakkyo-zuke, meat products such as ham and sausage, fish ham, fish-meat sausage, fish products such as kamaboko, chikuwa, and tempura, sea urchin, salted squid, vinegared kombu, sakinume, and fugu mirin. Tsutsudani cooked with various delicacies such as dried vegetables, seaweed, edible wild plants, sardines, small fish, and shellfish, boiled beans, potato salad, prepared foods such as kelp rolls, dairy products, fish meat, meat, fruits, vegetables bottled , Canned goods, sake, synthetic liquor, liqueur, liquor such as Western liquor, tea, coffee, cocoa, juice, carbonated drink, lactic acid drink, soft drink such as lactic acid bacteria drink, pudding mix, hot cake mix, instant sirko, instant soup Improving foods, such as baby foods, therapeutic foods, sweetening to various foods and drinks such as drinks, improving taste,
It can be advantageously used for improving physical properties.

Further, it can also be used for domestic animals such as livestock, poultry and fish for the purpose of improving the palatability of feed, feed and the like, or improving the fattening. Other, cigarettes,
It can be used for various solid substances such as toothpaste, lipstick, lip balm, oral solution, tablets, troches, liver oil drops, mouth refreshing agents, mouth flavoring agents, mouthwashes, etc. , It can be advantageously used as a sweetener for various compositions such as pharmaceuticals, or as a taste improver, a corrigent, and a quality improver.

As the quality improver and stabilizer, active ingredients,
It can be advantageously applied to various physiologically active substances that easily lose activity, or health foods, pharmaceuticals, etc. containing the same. For example,
Interferon-α, Interferon-β, Interferon-γ, Tumor necrosis factor-
α, Tumor necrosis factor-β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, lymphokine-containing solution such as interleukin-2, insulin, growth hormone, prolactin, erythropoietin, egg cell stimulating hormone, placental hormone, etc. Liquid, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, smallpox seedling, tetanus toxoid, hub antitoxin, liquid containing biologics such as human immunoglobulin, penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin, kanamycin sulfate. Antibiotic-containing liquid such as, thiamine, riboflavin, L-ascorbic acid, liver oil, carotenoid, ergosterol, tocopherol-containing vitamin-containing liquid, lipase , Elastase, urokinase,
Enzyme-containing liquids such as protease, β-amylase, isoamylase, glucanase and lactase, ginseng extract, terrapin extract, chlorella extract, aloe extract,
Extracts such as propolis extract, viruses, lactic acid bacteria,
Various bioactive substances such as live bacteria such as yeast and royal jelly can also be improved in quality and / or stabilized without losing their active ingredients and activities by using the fructosyl transfer sugar-containing saccharide of the present invention, Stable and high-quality health foods and pharmaceuticals can be easily manufactured.

As described above, the composition of the present invention is not limited to foods and drinks or medicines used orally or parenterally, but other than that, for example, cosmetics, daily necessities,
It has a wide range of applications such as agricultural, forestry and fishery products, reagents, and chemical industry products.

The method of incorporating the fructosyl transfer sugar-containing saccharide of the present invention into these compositions may be such that it is contained in the steps until the product is completed. For example, mixing, dissolving, dipping, permeating, Known methods such as spraying, coating, spraying, pouring, and solidifying are appropriately selected. The amount to be contained varies depending on the composition, but it is generally 0.1% or more, preferably 0.5% as the fructosyl transfer sugar.
The above amount is suitable.

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

<Experiment 1 Production of Enzyme> Sucrose 1.0 w / v
%, Polypeptone 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 0.3 w /
Liquid medium consisting of v% and water was placed in a 500 ml Erlenmeyer flask, 100 ml each, and the mixture was placed in an autoclave for 121
Sterilize at ℃ for 15 minutes, cool, inoculate with Bacillus species V230 (FERM BP-5054),
What was subjected to rotary shaking culture at 30 ° C. and 200 rpm for 20 hours was used as a seed culture.

About 7 liters of a medium having the same composition as in the case of seed culture was placed in a fermenter having a volume of 10 liters, and the mixture was sterilized by heating and cooled to a temperature of 30 ° C., and 1 v / v% of a seed culture was inoculated at a temperature of 30 The culture was performed at 20 ° C. for about 20 hours with aeration and stirring.

About 6.5 l of the culture solution was centrifuged (15,000).
(0 G, 20 minutes) to obtain about 6.3 l of culture supernatant.
The β-fructofuranosidase activity of this culture supernatant was 3.6 units / ml.

<Experiment 2 Purification of Enzyme> Ammonium sulphate was added to the culture supernatant obtained in Experiment 1 to dissolve it at a saturation level of 0.8, allowed to stand at 4 ° C. overnight, and then centrifuged. The ammonium sulfate salted out product was recovered.

The obtained ammonium sulfate salt precipitate was dissolved in 10 mM acetate buffer (pH 6.0) containing 5 mM calcium chloride, dialyzed against the same buffer for 24 hours and centrifuged (15,000 G, 20). The insoluble matter was removed for 1 minute). The dialysate supernatant (210 ml) was subjected to ion exchange column chromatography (gel amount 380 ml) using DEAE-Toyopearl gel 650 gel (manufactured by Tosoh Corporation).

The β-fructofuranosidase of the present invention is
It is adsorbed to the DEAE-Toyopearl gel and collected in a fraction eluted with 0.1 M sodium chloride. The collected fraction was dialyzed against 10 mM acetate buffer (pH 6.0) containing 1 M ammonium sulfate, and the dialysate was centrifuged (15,000 G, 20 minutes) to remove insoluble matter, and then butyl toyopearl. 65
Hydrophobic column chromatography using 0 gel (manufactured by Tosoh Corporation) (gel amount 100 ml) was performed. The adsorbed β-fructofuranosidase was eluted from the column by a linear gradient from ammonium sulfate 1M to 0M, and the enzyme active fraction was collected.

Then, DEAE-Toyopearl gel 65
Ion exchange column chromatography using 0 gel (gel amount 10 ml) was performed, and the eluted enzyme active fraction was collected.

The recovery rate of enzyme activity in the purified enzyme preparation obtained by the above purification means was about 13% with respect to that of the culture supernatant. The specific activity of the purified enzyme preparation was 205 units per mg of protein. The protein was quantified according to the Lowry method using bovine serum albumin as a standard.

The purified β-fructofuranosidase preparation was assayed for the purity of the enzyme preparation by gel electrophoresis containing 7.5 w / v% concentration polyacrylamide, and it was confirmed that the protein band was single and the purity was high. It was a product.

<Experiment 3 Properties of enzyme> The purified β-fructofuranosidase preparation obtained by the method of Experiment 2 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration 10 w / v).
%), And compared with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.) that was electrophoresed at the same time,
When the molecular weight of this enzyme was measured, the molecular weight was 49.00
It was 0 ± 5,000 Daltons. Also, TSKgel
G3000SW column (φ7.8mm × 300mm,
As a result of measuring the molecular weight by a gel filtration method using Tosoh Corporation, the result was 37,000 ± 3,000 daltons.

The same purified β-fructofuranosidase preparation as described above was subjected to isoelectric point polyacrylamide gel electrophoresis containing 2 w / v% ampholine (Pharmacia LKB, Sweden), and after electrophoresis, protein staining was performed. The pH of the gel was measured and the isoelectric point of this enzyme was determined to be 4.6 ± 0.5.

The effect of pH and temperature on the β-fructofuranosidase activity of the present invention was examined in the presence or absence of 5 mM calcium chloride according to the method for measuring activity.
The results are shown in FIG. 1 (effect of pH) and FIG. 2 (effect of temperature). The optimum pH of this enzyme is about 5.5 to 6.0 in the presence or absence of calcium ion at a temperature of 40 ° C. for 10 minutes, and the optimum temperature of this enzyme is
After reacting at pH 6.0 for 10 minutes, the temperature was around 50 ° C in the presence of calcium ions and around 45 ° C in the absence of calcium ions. The pH stability of this enzyme is 5
After keeping in 0 mM buffer at 4 ° C. for 24 hours, the pH was adjusted to 6 and the residual enzyme activity was measured. The thermal stability was determined by keeping the enzyme solution (20 mM acetate buffer, pH 6.0) at each temperature for 1 hour, cooling with water, and measuring the remaining enzyme activity. The results are shown in FIG. 3 (pH stability) and FIG. 4 (thermal stability). The enzyme has a pH range of about 5.0 to 8.0 and 4
It was stable up to around 5 ° C. The pH stability and heat stability of this enzyme were completely the same in the presence of calcium ions as in the absence thereof. Moreover, this enzyme activity was inhibited by 1 mM of Cu ⁺⁺ , Pb ⁺⁺ , Fe ⁺⁺ , Fe ⁺⁺⁺ and Hg ⁺⁺ .

<Experiment 4 Substrate specificity> Final concentration 2 w / v
% Sucrose, raffinose, erulose, stachyose,
For lactosucrose, xylosylfructoside, maltose, cellobiose, lactose, inulin or levan,
Purified β-fructofuranosidase obtained by the method of Experiment 2 was added in 2 units per gram of substrate solid, and the mixture was added at 40 ° C. and p
It was operated at H5.5 for 24 hours. Kieselgel 60 (manufactured by Merck & Co .; aluminum plate, 2)
Thin layer chromatography (0 × 20 cm) (hereinafter, abbreviated as “TLC”) was performed to confirm the presence or absence of an enzymatic action on each sugar. The TLC was developed once at room temperature using 1-butanol: pyridine: water = 7: 3: 1 (volume ratio) as a developing solvent. For the color development, in the case of a substrate containing fructose as a constituent sugar, 0.2 w / v% naphthresorcinol-0.5N phosphoric acid solution was sprayed to
Heating is performed at 10 ° C. for about 5 minutes, and for other substrates, 20 w / v% sulfuric acid-methanol solution is sprayed.
The heating was performed at 0 ° C. for about 10 minutes.

As a result, the β-fructofuranosidase of the present invention acts on sucrose, raffinose, erulose, stachyose, lactosucrose and xylosylfructoside to specifically release fructose, maltose,
It was found to have no effect on cellobiose, lactose, inulin, and levan.

<Experiment 5 Receptor Specificity> Various monosaccharides, oligosaccharides and alcohols shown in Table 1 as receptors,
To the sugar solution having a final concentration of 10%, which was obtained by mixing equal amounts of sucrose as a donor in a weight ratio, the purified β-fructofuranosidase obtained by the method of Experiment 2 was added in 2 units per 1 gram of sucrose, and 40 ° C. It was allowed to act at pH 5.5 for 24 hours. The reaction solution before and after the enzyme reaction was subjected to TLC in the same manner as in Experiment 4 to give 0.
A 2 w / v% naphthresorcinol-0.5N phosphoric acid solution was sprayed and heated at 110 ° C. for about 5 minutes to cause color development, and it was determined whether or not a transfer sugar was produced. The results are shown in Table 1.

[0053]

[Table 1]

As shown in Table 1, the β-fructofuranosidase of the present invention uses D-xylose, D-galactose, maltose, isomaltose, cellobiose, maltotriose, panose, lactose with sucrose as a sugar donor. , It was found that the fructosyl group was transferred well to reducing sugars such as melibiose and non-reducing sugars such as trehalose. In addition, it was found that it is well transferred to sugar alcohols such as D-xylitol, D-sorbitol and maltitol and alcohols such as glycerol and ethylene glycol to produce fructosyl transfer sugar.

<Experiment 6 Fructosyl-transferring sugar from sucrose and reducing sugar> A confirmatory test was carried out to identify a fructosyl-transferring sugar produced in the transglycosylation reaction by the β-fructofuranosidase of the present invention. A part of the enzyme reaction solution using sucrose and lactose as the substrates obtained in Experiment 5 was diluted with 20 mM acetate buffer (pH 4.5) to a sugar concentration of 2%, and 0.5 ml of this was used for invertase (biochemical 0.1 unit (manufactured by Kogyo Co., Ltd.) or 0.1 unit of β-galactosidase (manufactured by Seikagaku Co., Ltd.) was added and reacted at 40 ° C. for 20 hours. The reaction liquid with the β-fructofuranosidase of the present invention, the invertase-treated liquid and the sugar component of the β-galactosidase-treated liquid were analyzed by gas chromatography (hereinafter, abbreviated as “GLC”). A part of the enzyme reaction solution was dried, dissolved in pyridine, and then trimethylsilylated to obtain a GLC analysis sample. The GLC column is a stainless steel column (3 mmφ × 2 m) packed with 2% silicon OV-17 / Chromozorb W (manufactured by GL Science Co., Ltd.), and the carrier gas is nitrogen gas at a flow rate of 40 ml / min and the column oven temperature. Is 16
The analysis was performed from 0 ° C. to 320 ° C. at a heating rate of 7.5 ° C./min. A flame ionization detector was used for detection.

As a result, the retention time of the peak of the fructosyl-transferred sugar from sucrose and lactose by the β-fructofuranosidase of the present invention was in agreement with that of the known sugar lactosucrose (lactosylfructoside), and the invertase treatment, Alternatively, it was found that the peaks disappeared by β-galactosidase treatment and they were decomposed into fructose and lactose, and galactose and sucrose, respectively. Considering the reaction characteristics of invertase and β-galactosidase,
The fructosyl transfer product from sucrose and lactose by the β-fructofuranosidase of the present invention is considered to be lactosucrose.

In the case of a fructosyl-transferring saccharide produced by using xylose, maltose or isomaltose as an acceptor,
As a result of GLC analysis of the reaction solution before and after the invertase treatment,
From the results of comparison with GLC analysis of known sugars, it was found that they were xylosylfructoside, erulose (maltosylfructoside) and isomaltosylfructoside, respectively. In the transglycosylation reaction by the β-fructofuranosidase of the present invention, when these reducing sugars are used as acceptors, the fructosyl-transferring sugars have β-fructo at the 1-position carbon atom in the reducing sugar residue as the acceptor. It is considered to be a furanoside bond.

<Experiment 7 Fructosyltrehalose> On the other hand, in order to investigate the structure of the saccharide transferred to trehalose, fructosyltrehalose was isolated and its structure was investigated. A sugar solution containing 20% each of sucrose and trehalose was adjusted to pH 6.0, and 4 units of the purified β-fructofuranosidase obtained by the method of Experiment 2 was added thereto per 1 gram of sucrose, and the mixture was reacted at 55 ° C. for 20 hours, Then, the enzyme was inactivated by heating at 100 ° C. for 10 minutes. This solution contained about 23% fructosyl trehalose. This solution is decolorized with activated carbon, filtered, desalted with H-type and OH-type ion exchange resins for purification, and concentrated to a concentration of about 50%,
Column chromatography was performed to collect a fraction containing a large amount of fructosyltrehalose.

As the fractionation resin, an alkali metal type strongly acidic cation exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name "XT-1016", Na type, crosslinking degree 4%) is used.
Two stainless steel columns with a jacket having an inner diameter of 3 cm and a length of 1 m were packed in a water suspension form and connected in series so that the total length of the resin layer was about 2 m. While maintaining the temperature in the column at 40 ° C., a sugar solution was added at 5 v / v% to the resin, and hot water at 40 ° C. was flowed at a flow rate of SV 0.15 to fractionate,
Fractions high in fructosyl trehalose were collected.

Fractions high in fructosyltrehalose were desalted, purified, concentrated to a concentration of about 40%, and chromatographed using a column packed with octadecyl silica gel (YMC, trade name "YMC-Pack ODS"). The fraction was enriched in fructosyl trehalose and collected. The fructosyl trehalose-rich liquid collected by repeating this method was desalted, purified, concentrated and vacuum dried to obtain a fructosyl trehalose-rich powder at a solid yield of about 10%. This powder preparation contained fructosyl trehalose in an amount of about 98% based on the solid content.

The structure was confirmed by an enzymatic method and GLC analysis of partial methylhexitol acetate using a powdered preparation containing a large amount of fructosyltrehalose. As a result, fructosyltrehalose produced by the action of β-fructofuranosidase of the present invention was decomposed into fructose and trehalose at a molar ratio of 1: 1 by invertase treatment, and the sugar was methylated. After partial hydrolysis with acid, and subsequent reduction and acetylation, partial methylhexitol acetate obtained was analyzed by GLC to find that it was 1,5,6-tri-O-acetyl-2,3,4-tri-. Since O-methylglucitol and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol were detected at an equimolar ratio, trehalose contained β-fructosyl groups. It was found to be a trisaccharide linked by -2,6.

<Experiment 8 Acute Toxicity> A lactosucrose-rich powder prepared by the method of Example A-4 described below was orally administered to a 7-week-old dd mouse to perform an acute toxicity test. No fatal cases were observed up to 15 g per 1 kg of body weight, and further administration was difficult. Therefore, the toxicity of this carbohydrate is extremely low. Similarly, using 7-week-old dd strain mice, Example A described later, respectively.
Powder containing a high amount of erulose obtained by the method of Example -6, Example A-8
Xylopylfructoside-rich syrup obtained by the method of
An acute toxicity test was conducted by oral administration using the fructosyltrehalose-rich powder obtained by the method of Example A-11 and the isomaltosylfructoside-containing syrup obtained by the method of Example A-12. , Weight 1kg
No deaths were seen up to 15 g, and further administration was difficult. Therefore, the toxicity of these carbohydrates is extremely low.

The β-fructofuranosidase of the present invention and a method for producing a fructosyl-transferring sugar-containing sugar using the same are described in Example A, and a composition containing the fructosyl-transferring sugar-containing sugar is described in Example. Shown as B.

Example A-1 Bacillus species V230 (FERM BP-5054) was used with a sucrose concentration of 2 w / v% in the medium composition and a medium volume of about 20 l using a fermenter with a volume of 30 l. Except for the above, the culture was performed with aeration and stirring for about 24 hours in the same manner as in Experiment 1. The β-fructofuranosidase activity of this culture supernatant was 7.3 units per 1 ml of the culture medium. The culture solution was subjected to MF membrane filtration, and the permeate was subjected to UF membrane concentration to obtain an enzyme solution having about 130 units of β-fructofuranosidase activity per ml of about 80% of the total activity of the original culture solution. Obtained in yield.

<Example A-2> An aqueous solution containing 20% each of sucrose and lactose was adjusted to pH 5.5, and β-fructofuranosidase prepared by the method of Example A-1 was added thereto per gram of sucrose. It was added so that the ratio was 1 unit and reacted at 55 ° C. for 16 hours. 90 ° C
After heating for 30 minutes at 37 ° C to inactivate the enzyme, it is cooled, decolorized with activated carbon according to a conventional method, filtered, desalted with H-type and OH-type ion exchange resins for purification, and further concentrated to a concentration of about 75% syrup was obtained with a solids yield of about 95%.

This product contains about 37% lactosucrose per solid matter, has a good taste, sweetness, moderate viscosity, and
Has moisturizing properties, sweeteners, taste improvers, stabilizers, excipients,
It can be advantageously used in various compositions such as food and drink, cosmetics and pharmaceuticals as a bifidobacteria growth promoter, mineral absorption promoter and the like.

Example A-3 An aqueous solution containing 22% sucrose and 18% lactose was adjusted to pH 6.0, and the β-fructofuranosidase prepared in Example A-1 was added thereto per gram of sucrose. 1 unit, and invertase-deficient yeast were added so as to be 5% by wet weight per solid weight, and 1
The reaction solution was adjusted to pH 6 with a specified sodium hydroxide solution.
The reaction was carried out at 35 ° C. for 20 hours while controlling to 7 to 7.
The reaction solution was heated at 90 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized with activated carbon according to a conventional method, filtered, and
And OH type ion exchange resin for desalination and purification, and further concentration to obtain a syrup having a concentration of about 75% in a solid yield of about 70.
%.

This product contains about 65% lactosucrose per solid matter, and has a good taste, sweetness, moderate viscosity, and
It has a moisturizing property and can be advantageously used in various compositions such as food and drink, cosmetics and pharmaceuticals as a sweetener, a taste improver, a stabilizer, a bifidobacteria growth promoter, a mineral absorption promoter and the like.

Example A-4 A solution containing lactosucrose of about 37% based on the solid substance reacted and purified by the method of Example A-2 was used as a raw sugar solution, which was concentrated to a concentration of about 45%.
I made it. To increase the lactosucrose content of this sugar solution,
Alkali metal strong acid cation exchange resin (Tokyo Organic Chemical Industry Co., Ltd., trade name "XT-1016", Na type,
Column chromatography was performed using a crosslinking degree of 4%). Resin was packed in four stainless steel columns with an inner diameter of 5.4 cm and which were connected in series to form a resin layer with a total length of 20
m. While maintaining the temperature in the column at 40 ° C, 5v / v% of sugar solution was added to the resin, and warm water at 40 ° C was added to the solution.
Fractionation was carried out at a flow rate of V0.2, and a lactosucrose-rich fraction was collected. Further purification, concentration, vacuum drying,
Grinding gave a lactosucrose-rich powder with a solids yield of about 30%.

This product contains about 90% lactosucrose per solid and has low reducing property, good sweetness, sweetness, moisturizing property and low caries property, and is a sweetener and taste improver. , Stabilizers, excipients, bifidobacteria growth promoters, mineral absorption promoters, etc. can be advantageously used in various compositions such as food and drink, cosmetics and pharmaceuticals.

Example A-5 A sugar solution containing 20% each of sucrose and maltose was adjusted to pH 5.5, and β-fructofuranosidase prepared by the method of Example A-1 was added to 1 g of sucrose. 1 unit per unit, and reacted at 50 ° C. for 24 hours. 90 ° C
For 30 minutes to deactivate the enzyme, then cooled, decolorized with activated carbon according to a conventional method, filtered, purified by desalting with H-type and OH-type ion exchange resins, and further concentrated to a concentration of about 7%.
5% syrup was obtained with a solids yield of about 95%.

This product contains about 28% erulose per solid.
%, It has a good taste, sweetness, moderate viscosity, and moisturizing property, and can be advantageously used for various compositions such as food and drink, cosmetics, and pharmaceuticals.

Example A-6 A solution containing about 28% erulose per solid matter reacted and purified by the method of Example A-5 was used as a raw sugar solution and concentrated to a concentration of about 45%. In order to increase the ellulose content of this sugar solution, as a fractionation resin, an alkali metal type strongly acidic cation exchange resin (sold by Dow Chemical Co., trade name “Dowex 50W × 4”,
Column chromatography was carried out according to the method of Example A-4 except that Ca type) was used, and a fraction containing high erulose was collected. Further purification, concentration, vacuum drying and crushing gave a powder with a high erulose content in a solids yield of about 25%.

This product contains about 84% erulose per solid matter and has a low reducing property, a good taste sweetness, a moisturizing property and a low caries property, and can be used for various foods, cosmetics, pharmaceuticals and the like. It can be advantageously used in a composition.

Example A-7 A sugar solution containing 30% sucrose and 15% xylose was adjusted to pH 5.5, and β-fructofuranosidase prepared by the method of Example A-1 was added to the sucrose solution. It was added at a rate of 0.5 unit per gram and reacted at 50 ° C. for 40 hours. 9
Heat at 0 ° C. for 30 minutes to inactivate the enzyme, then cool,
According to a conventional method, decolorization with activated carbon, filtration, desalting with H-type and OH-type ion exchange resins for purification, and further concentration were carried out to obtain syrup having a concentration of about 75% with a solid yield of about 95%.

This product contains about 35% of xylosylfructoside per solid matter, has a good taste sweetness, moderate viscosity, moisturizing property and low cariogenicity, and is a sweetener and taste improver. Agent,
It can be advantageously used in various compositions such as foods and drinks, cosmetics and pharmaceuticals as a stabilizer, a bifidobacteria growth promoter, a mineral absorption promoter and the like.

Example A-8 A sugar solution containing 30% sucrose and 15% xylose was adjusted to pH 6.0, and the β-fructofuranosidase prepared in Example A-1 was added thereto.
It was added at a ratio of 0.5 unit per gram of sucrose and reacted at 50 ° C. for 40 hours. Sodium hydroxide was added to the reaction solution, and the mixture was heated at 100 ° C. while keeping it alkaline at pH 10 or higher, then cooled, decolorized with activated carbon according to a conventional method, filtered, and desalted with H-type and OH-type ion exchange resins. Purification and further concentration gave syrup with a concentration of about 75% with a solids yield of about 55%.

This product contains about 60% of xylosylfructoside per solid matter, has a sweetness with a good taste, an appropriate viscosity and a moisturizing property, and is a sweetener, a taste improver, a stabilizer, It can be advantageously used in various compositions such as food and drink, cosmetics and pharmaceuticals as a bifidobacteria growth promoter, mineral absorption promoter and the like.

Example A-9 A sugar solution containing 20% each of sucrose and trehalose was adjusted to pH 5.5, and 1 g of sucrose was prepared using β-fructofuranosidase prepared by the method of Example A-1. It was added so as to be a ratio of 4 units per 1 hour, and reacted at 55 ° C. for 16 hours. 90 times the reaction solution
After heating at ℃ for 30 minutes to inactivate the enzyme, it is cooled, decolorized with activated carbon according to the usual method, filtered, desalted with H-type and OH-type ion exchange resins for purification, and further concentrated to a concentration of about 75% syrup was obtained with a solids yield of about 95%.

This product contains about 24% of fructosyl trehalose per solid substance, has a good taste sweetness, moderate viscosity, moisturizing property and low caries property, and is a sweetener and taste improver. , Stabilizers, bifidobacteria growth promoters, mineral absorption promoters, etc. can be advantageously used in various compositions such as food and drink, cosmetics and pharmaceuticals.

Example A-10 A sugar solution containing 20% each of sucrose and trehalose was adjusted to pH 5.5, and the β-fructofuranosidase prepared in Example A-1 was added thereto at 4% per gram of sucrose. Units and invertase-deficient yeast are added so as to be 5% in terms of wet weight per solid weight, and the reaction solution is adjusted to pH 6 to 7 with 1N sodium hydroxide solution, and at 35 ° C. for 20 hours. It was made to react. The reaction solution was heated at 90 ° C. for 30 minutes to deactivate the enzyme, then cooled, decolorized with activated carbon according to a conventional method, filtered, desalted with H-type and OH-type ion exchange resins, and further purified. Concentration gave syrup with a concentration of about 75% with a solids yield of about 70%.

This product contains about 34% of fructosyl trehalose per solid matter, has a good taste sweetness, an appropriate viscosity and a moisturizing property, and has a sweetener, a taste improver, a stabilizer,
It can be advantageously used in various compositions such as food and drink, cosmetics and pharmaceuticals as a bifidobacteria growth promoter, mineral absorption promoter and the like.

<Example A-11> A solution containing fructosyltrehalose of about 24% based on the solid matter reacted and purified by the method of Example A-9 was used as a raw sugar solution and concentrated to a concentration of about 45%. did. In order to increase the fructosyl trehalose content of the present sugar solution, a salt type strongly acidic cation exchange resin (sold by Dow Chemical Co., trade name “Dowex 50W × 4”, Ca type) was used as a fractionation resin, except that Example A
Column chromatography was carried out in accordance with the method of No. 4 to collect the fructosyltrehalose-rich fraction.
Further, purification, concentration, vacuum drying, and pulverization were performed to obtain powder having a high content of fructosyltrehalose with a solid yield of about 20%.

This product contains about 80% of fructosyl trehalose per solid matter and has low reducing property, good taste sweetness, moisturizing property and low caries property, and is a sweetener and taste improver. It can be advantageously used in various compositions such as food and drink, cosmetics and pharmaceuticals as an agent, stabilizer, excipient, bifidobacteria growth promoter, mineral absorption promoter and the like.

Example A-12 A sugar solution containing 30% sucrose and 15% isomaltose was adjusted to pH 5.5, and β-fructofuranosidase prepared by the method of Example A-1 was added thereto. It was added at a ratio of 0.5 unit per gram of sucrose and reacted at 50 ° C. for 40 hours. This reaction solution was heated at 90 ° C. for 30 minutes to inactivate the enzyme, then cooled, decolorized with activated carbon, filtered, desalted and purified with an H-type and OH-type ion exchange resin according to a conventional method, Further concentration gave syrup with a concentration of 75% with a solids yield of about 95%.

This product contains about 25% of isomaltosyl fructoside per solid matter, has a good taste sweetness, moderate sugar content, moisturizing property and low cariogenicity, and is a sweetener It can be advantageously used in various compositions such as food and drink, cosmetics and pharmaceuticals as an improving agent, a stabilizer, a bifidobacteria growth promoter, a mineral absorption promoter and the like.

Example B-1 Mixed Sweetener Example A-
1 part by weight of the powder having a high content of lactosucrose obtained by the method of 4 was uniformly mixed with 0.05 part by weight of α-glycosyl stevioside (manufactured by Toyo Seika Sugar Co., Ltd., trade name α-G Sweet) to prepare a powder sweetener. . The product has an elegant sweetness, about twice the sweetness of sugar. In addition, this product exerts a bifidobacteria growth-promoting effect and is used for beauty and health maintenance and promotion, prevention of adult diseases, promotion of recovery during and after disease, treatment and prevention of hyperammonemia, hepatic encephalopathy, etc. It can be used to advantage. Further, it can be advantageously used for prevention of infection of domestic animals such as livestock and poultry, prevention of diarrhea, promotion of appetite, promotion of fattening and suppression of odor of feces.

<Example B-2 Hard Candy> 80 parts by weight of reduced maltose starch syrup (water content 25%) was added to Example A-2.
30 parts by weight of isomaltosyl fructoside-containing syrup obtained by the method of 12 is dissolved by heating and concentrated under reduced pressure until the water content is less than 2%, and 1 part by weight of citric acid and an appropriate amount of lemon flavor and colorant are added thereto. And were mixed and then molded according to a conventional method to produce a hard candy.

This product is a low caries hard candy having an elegant sweetness. In addition, this product exerts the effect of promoting the growth of bifidobacteria and the effect of promoting the absorption of minerals.
It can be advantageously used for maintaining and promoting health, preventing adult diseases, promoting recovery during and after illness, and treating and preventing various diseases.

Example B-3 Chewing Gum 2 parts by weight of a gum base were melted by heating so as to be soft, and 4 parts by weight of the lactosucrose-rich powder obtained by the method of Example A-4, 3 parts by weight of glucose and After mixing an appropriate amount of mint flavor and color, the mixture was kneaded by a roll according to a conventional method and molded to produce a chewing gum.

The product has good texture and sweetness. In addition, the product exerts a bifidobacteria growth-promoting effect and can be advantageously used for beauty and health maintenance and promotion, prevention of adult diseases, promotion of recovery during and after illness, and treatment and prevention of various diseases.

<Example B-4 Chocolate> 40 parts by weight of cocoa paste, 10 parts by weight of cocoa butter, Example A
15 parts by weight of the erulose-rich powder obtained by the method of -6, 10 parts by weight of sucrose and 15 parts by weight of whole milk powder were mixed and passed through a refiner. After reducing the particle size, the mixture was placed in a conche, to which 0.5 part by weight of lecithin was added, and kneaded at 50 ° C. for two days and nights. Subsequently, it was poured into a molding machine according to a conventional method, and was molded and solidified to be manufactured.

This product has no fear of fat bloom or sugar bloom, and has a good melting condition and flavor when placed on the tongue.

<Example B-5 Milk Beverage> 70 parts by weight of coffee brewed liquid, 100 parts by weight of milk, 24 parts by weight of lactosucrose-rich syrup obtained by the method of Example A-3, and an appropriate amount of coffee flavor and colorant were added. After mixing and homogenizing,
A milk drink was produced by sterilization, cooling, filling and packaging according to a conventional method.

This product is coffee milk having a good scent and taste. In addition, this product has a bifidobacteria growth promoting effect,
It exerts a mineral absorption promoting effect and can be advantageously used for beauty and health maintenance and promotion, prevention of adult diseases, promotion of recovery during and after illness.

Example B-6 Custard Cream 500 parts by weight of cornstarch, 400 parts by weight of maltose (Hayashibara Shoji Co., Ltd., registered trademark of San Marto) and 5 parts by weight of salt were thoroughly mixed through a sieve to prepare 1400 eggs of chicken.
Parts by weight and 600 parts by weight of syrup having a high content of fructosyl trehalose obtained by the method of Example A-10 were added and stirred, and 5000 parts by weight of boiled milk was gradually added thereto,
Further, this was heated over a simmer to continue stirring, and when the cornstarch was completely gelatinized and the whole became translucent, the heat was turned off, the cornstarch was cooled, and an appropriate amount of vanilla flavor was added to produce a custard cream.

This product has a smooth luster and is delicious without being too sweet. In addition, the product exhibits a bifidobacteria growth-promoting effect, and can be advantageously used for beauty and health maintenance and promotion, prevention of adult diseases, and promotion of recovery during and after illness.

Example B-7 Instant Corn Potage Soup> 30 parts by weight of α-modified corn powder, 5 parts by weight of α-modified starch, 4 parts by weight of α-modified potato starch, 12 parts by weight of α-modified waxy cornstarch. Grinding 8 parts by weight of high-fructosyltrehalose-containing powder obtained by the method of Example A-11, 5 parts by weight of sodium glutamate, 8.5 parts by weight of salt, 7 parts by weight of skimmed milk powder, and 0.5 parts by weight of onion powder. After mixing well, add sorbitan fatty acid ester 0.
Heat-melted 5 parts by weight and hydrogenated vegetable oil 9 parts by weight were added and mixed, and further 10 parts by weight of lactose were added and mixed, and this was charged into a fluidized bed granulator, and a small amount of water was added. After spraying and granulating, it was dried with hot air at 70 ° C. and sieved to produce instant corn potage soup.

This product can be easily dissolved and dispersed by pouring hot water into a soup with an excellent flavor. In addition, this product exerts a bifidobacteria growth promoting effect and mineral absorption promoting effect,
It can be advantageously used for beauty and health maintenance and promotion, prevention of adult diseases, promotion of recovery during and after illness.

Example B-8 Emulsion 0.5 part by weight of polyoxyethylene behenyl ether, 1 part by weight of polyoxyethylene sorbitol tetraoleate, 1 part by weight of lipophilic glyceryl monostearate, behenyl alcohol of 0. 5 parts by weight, avocado oil 1 part by weight, erulose-containing syrup 3.5 parts by weight obtained by the method of Example A-5, 1 part by weight of α-glycosyl rutin, vitamin E and an appropriate amount of preservative were heated according to a conventional method. After dissolution, 5 parts by weight of 1,3-butylene glycol, 0.1 part by weight of carboxyvinyl polymer and 85.3 parts by weight of purified water were added, and the mixture was homogenized and emulsified to prepare an emulsion.

This product is a moisturizing emulsion, which is a sunscreen,
It can be advantageously used as a whitening agent.

Example B-9 Skin Cream 2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glyceryl monostearate, 2 parts by weight of α-glycosyl rutin, 1 part by weight of liquid paraffin,
10 parts by weight of glyceryl trioctanoate, 4 parts by weight of syrup containing fructosyltrehalose obtained by the method of Example A-9 and an appropriate amount of preservative were dissolved by heating according to a conventional method,
To this, 5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water were added and emulsified by applying a homogenizer,
Furthermore, an appropriate amount of flavor was added and mixed with stirring to produce a cream.

This product is a cream with good elongation and can be advantageously used as a sunscreen, skin beautifying agent, skin lightening agent and the like.

Example B-10 Toothpaste 45 parts by weight of dibasic calcium phosphate, 1.5 parts by weight of sodium lauryl sulfate, 25 parts by weight of glycerin, 0.5 parts by weight of pooxyethylene sorbitan laurate, Example A-8. 15 parts by weight of xylopyl fructoside-rich syrup obtained by the method of
0.02 parts by weight of saccharin and 0.05 part by weight of a preservative were mixed with 13 parts by weight of water to obtain a toothpaste.

The product has good gloss and detergency and is suitable as a toothpaste.

Example B-11 Tube Feeding Agent Example A
20 parts by weight of powder containing a high amount of erulose obtained by the method of No. 6, 1.1 parts by weight of glycine, 1 part by weight of sodium glutamate, 0.4 part by weight of calcium lactate, 0.1 part by weight of magnesium carbonate, 0.01 part by weight of thiamine. And 0.01 parts by weight of riboflavin are prepared. Each 24 g of this mixture was filled in a laminated aluminum package and heat-sealed to obtain a product.

This product contains about 300 to 500 ml per bag.
It is used as a nutritional supplement by dissolving it in water and administering it to the nasal cavity, gastrointestinal tract, etc. by the tube method.

This product can be advantageously used as a parenteral nutritional supplement liquid for domestic animals as well as humans.

Example B-12 Tube Feeding Agent Example A
580 parts by weight of lactosucrose-rich powder obtained by the method of No. 4, 190 parts by weight of dried egg yolk, 209 parts by weight of skim milk powder,
Sodium chloride 4.4 parts by weight, potassium chloride 1.85 parts by weight, magnesium sulfate 4 parts by weight, thiamine 0.01 parts by weight, sodium ascorbate 0.1 parts by weight, vitamin E acetate 0.6 parts by weight and nicotinic acid amide. A formulation consisting of 0.04 parts by weight is prepared. Each 25 g of this mixture was filled in a laminated aluminum pouch and heat-sealed to produce a solid tube feeding agent.

This product maintains stable quality for a long time,
Good solubility and dispersibility. This drug can be used for about 15 bags.
It is dissolved in 0 to 300 ml of water to prepare a nutritional supplement liquid, which is administered to the nasal cavity, esophagus, stomach, etc. by a tube method for use.
In addition, this product exerts the effect of promoting the growth of bifidobacteria and the effect of promoting the absorption of minerals, and improves the maintenance of beauty and health, the prevention of adult diseases, the promotion of recovery during and after illness, the treatment and prevention of various diseases,
Further, it can be advantageously used for promoting recovery after and after illness of livestock, poultry and the like, and promoting fattening.

Example B-13 Tablets Example A-11
Fructosyltrehalose-rich powder 40 obtained by the method
1 part by weight, maltose 10 parts by weight, tricalcium phosphate 1 part by weight, sugar ester 1 part by weight and an appropriate amount of powdered flavor are uniformly mixed, and then, in accordance with a conventional method, 1 tablet approximately 350
Tablets were obtained by tableting with a tableting machine so as to give mg.

This product is an easy-to-drink tablet with no cracks and good stability. It is taken by an adult for about 1 to 40 tablets, preferably about 2 to 20 tablets, and it has a bifidobacteria growth promoting effect and mineral absorption. It exerts a promoting effect and can be advantageously used for beauty and health maintenance and promotion, prevention of adult diseases, promotion of recovery during and after illness, and treatment and prevention of various diseases.

<Example B-14 Interferon tablet> Human natural interferon-α preparation (manufactured by Hayashibara Biochemical Laboratory Co., Ltd., sold by Cosmo Bio Co., Ltd.)
The immobilized anti-human interferon-α was
Apply the antibody to an antibody column, adsorb the human natural interferon-α contained in the sample, remove the stabilizer bovine serum albumin by passing it through, and then change the pH to carry out human natural interferon-α. The lactosucrose-rich powder obtained by the method of Example A-4 was eluted with physiological saline containing 5%. This solution is microfiltered to give about 2
Dehydrated and powdered with 0 times the amount of anhydrous crystalline maltose powder (trade name "Fine Tose (registered trademark)" sold by Hayashibara Shoji Co., Ltd.), and tableted with a tableting machine to give 1 tablet (about 2 tablets).
A tablet containing about 150 units of human natural interferon-α per (00 mg) was obtained.

This product is used as a sublingual tablet, etc.
About 1 to 10 tablets for adults are orally administered and can be advantageously used for treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., for which the number of patients is increasing rapidly in recent years. In this product, maltose acts as a stabilizer together with lactosucrose, and maintains its activity for a long period of time even when left at room temperature.

Example B-15 Compounded feed 40 parts by weight of flour, 38 parts by weight of skimmed milk powder, 12 parts by weight of powder containing high fructosyltrehalose obtained by the method of Example A-11, 10 parts by weight of vitamin preparation, 5 parts by weight of fish meal, 5 parts by weight of dibasic calcium phosphate, 3 parts by weight of liquid oil and fat, 3 parts by weight of calcium carbonate, 2 parts by weight of salt and 2 parts by weight of mineral agent were mixed to produce a compounded feed.

The product is a feed for domestic animals, poultry and the like with improved palatability, and is particularly suitable as a feed for piglets. In addition, this product exerts a bifidobacteria-proliferating effect and a mineral absorption promoting effect, and can be advantageously used for prevention of infection of domestic animals, prevention of diarrhea, promotion of appetite, promotion of fattening, suppression of odor of feces, and the like. In addition, this product is used, if necessary, in combination with other feed ingredients, for example, concentrated feed ingredients such as grains, wheat flour, starch, oil meal and rice bran, and roughage ingredients such as straw, hay, bagasse and corn cob. Then, other mixed feed can be used.

[0117]

INDUSTRIAL APPLICABILITY As described above, the present invention relates to a novel β-fructofuranosidase derived from a microorganism having a high fructosyl transfer ability, which is advantageous in industrial practice, a method for producing the same and use thereof. When β-fructofuranosidase of the present invention is used with sucrose as a donor and a reducing or non-reducing sugar as an acceptor, a fructosyl group is transferred to the acceptor to produce a non-reducing sugar. . The newly produced fructosyl-transferred sugar has a good taste and sweetness, and also has an osmotic pressure regulating property,
It has properties such as moisturizing properties, light-imparting properties, anti-crystallization properties, and starch anti-aging properties, and also has functions such as caries resistance, bifidobacteria growth-promoting properties, and mineral absorption-promoting properties. It is advantageously used for various compositions such as products, cosmetics, pharmaceuticals and molded products. Therefore, the industrial significance in the fields of food, cosmetics, pharmaceuticals, etc. is extremely great.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of pH on β-fructofuranosidase activity of the present invention.

FIG. 2 is a graph showing the effect of temperature on the β-fructofuranosidase activity of the present invention.

FIG. 3 pH of β-fructofuranosidase of the present invention
It is a figure which shows stability.

FIG. 4 is a diagram showing the thermostability of β-fructofuranosidase of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A23K 1/16 303 A23K 1/16 303D A23L 1/236 A23L 1/236 A 1/30 1/30 Z 1/40 1/40 2/38 2/38 P A61K 9/20 ACK A61K 9/20 ACKA C12P 19/18 C12P 19/18 // A61K 7/00 A61K 7/00 F 7/16 7/16 7 / 48 7/48 31/70 ADD 31/70 ADD AER AER (C12N 9/24 C12R 1:07)

Claims (13)

[Claims] 1. A β-fructofuranosidase having the following physicochemical properties. (1) Action Hydrolyzes at least β-fructofuranoside bonds of sucrose, raffinose and erulose to release fructose. In addition, the sugar having these β-fructofuranoside bonds is used as a sugar donor, and the transfer of the β-fructofuranosyl group is catalyzed to an acceptor selected from sugars other than sugar donors, sugar alcohols and alcohols. To do. (2) Molecular weight By SDS-gel electrophoresis, 49,000 ± 5,000.
Dalton (3) By isoelectric focusing ampholine-containing electrophoresis method, 4.6 ± 0.5 (4) Optimum pH 40 ° C., 10 minutes reaction, about 5.5 to 6.0 (5) Optimum temperature At pH 6.0 for 10 minutes, around 45 ° C in the absence of calcium ions, around 50 ° C in the presence of calcium ions (6) pH stability At a temperature of 4 ° C for 24 hours, pH of about 5.0 to 5.0 Stable in the range of 8.0 (7) Temperature stability Stable up to around 45 ° C under the condition of pH 6.0 and holding for 1 hour 2. The β-fructofuranosidase according to claim 1, which is derived from a microorganism belonging to the genus Bacillus. 3. The microorganism is Bacillus species V23.
0 (Institute of Biotechnology, Institute of Biotechnology, Contract No. F)
ERM BP-5054), The β-fructofuranosidase according to claim 2. 4. A β-fructofuranosidase characterized by comprising culturing a microorganism capable of producing β-fructofuranosidase, and collecting the β-fructofuranosidase according to claim 1, 2 or 3 from the culture. Production method. 5. The method for producing β-fructofuranosidase according to claim 4, wherein the microorganism capable of producing β-fructofuranosidase is a microorganism belonging to Bacillus species. 6. A microorganism having an ability to produce β-fructofuranosidase, Bacillus species V230 (Institute of Biotechnology, Institute of Industrial Science, Accession No. FERM BP)
-5054). 7. A fructosyl transfer sugar-containing saccharide obtainable by allowing the β-fructofuranosidase according to claim 1, 2 or 3 to act on a solution containing sucrose and other saccharides. 8. The fructosyl-transferred sugar is xylosyl fructoside, erulose, isomaltosyl fructoside,
The fructosyl transfer sugar-containing saccharide according to claim 7, which is a saccharide selected from lactosucrose and fructosyltrehalose. 9. The fructosyl transfer sugar-containing sugar according to claim 7, wherein the content of the fructosyl transfer sugar is increased by a method selected from a yeast fermentation method, an alkali treatment method and a column chromatography. 10. A composition containing the fructosyl-transferring saccharide-containing saccharide according to claim 7, 8, or 9. 11. The composition according to claim 10, which is a food or drink, a cosmetic, a pharmaceutical product, or a molded product. 12. A method for producing a saccharide containing a fructosyl-transferred sugar, which comprises reacting a solution containing sucrose and a saccharide other than sucrose with β-fructofuranosidase according to claim 1, 2 or 3. 13. The fructosyl transfer sugar-containing saccharide according to claim 12, wherein the other sugar is one or more selected from lactose, maltose, xylose, trehalose, isomaltose, galactose, cellobiose, panose and melibiose. Method for producing sugar.