JPH11100395A - New disaccharide, sweetener, production of the disaccharide and enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new disaccharide having more refreshed sweetness than sugar, leaving no aftertaste, useful in the field of foods, medicines, etc., as a noncaloric sweetener, etc., comprising 1-O-α-D-fructofuranosyl-D-fructose. SOLUTION: This new disaccharide comprises 1-O-α-D-fructofuranosyl-D- fructose (αFF) of the formula, has more refreshed sweetness than sugar, characteristics of no remaining aftertaste, is a homobiose of fructose having no calorie or calorie close to approximately to zero, suitable as an active ingredient for a sweetener useful in the field of foods and medicines. The compound is obtained by treating di-α-D-fructorfuranose 1, 2':2, 1' dianhydride (α-DFA) with an enzyme for hydrolyzing one α1, 2 fructofuranoside bond of α-DFA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a specific saccharide, a sweetener containing the same, a method for producing the specific saccharide, and an enzyme used for the production thereof, which can be used particularly in the food and pharmaceutical fields.

[0002]

2. Description of the Related Art Heterobiose containing fructose as a constitutional unit includes non-reducing sucrose (1-α-D-glucopyranoside-2-β-D-fructofuranoside) and other reducing maltulose (Glcα1 → 4Fruf), lactulose (Galβ1 → 4Fruf) and the like are known. As a homobiose containing fructose as a constituent unit, reducing inurobiose (1-O-β-D-fructofuranosyl-D-fructose) and the like are known. With respect to the taste of disaccharide containing fructose as a constituent unit, it is known that sucrose and inurobiose have sweetness.

In recent years, various tastes have been demanded due to diversification of food preferences. A variety of oligosaccharides have been developed, and among them, there are those with special characteristics in taste, such as nigello oligosaccharides that have a rich and rich taste. Most of the oligosaccharides are mellow and rich, but there is no known oligosaccharide with a cleaner taste than sucrose.

[0004] The only known non-caloric saccharide is erythritol, which is a kind of sugar alcohol. This carbohydrate is regarded as non-caloric because almost all of it is excreted in urine as it is without being metabolized after being absorbed into the body. However, erythritol exhibits a high endothermic effect when dissolved, so that it feels cool in the mouth when eaten. For this reason, it was sometimes difficult to use the product for which cooling sensation was not desirable. Also, since erythritol is a monosaccharide of tetracarbon sugar, when used in frozen desserts,
There was also the problem that the freezing point was too large. Disaccharide sugar alcohols such as maltitol are not decomposed by digestive enzymes,
It is treated as 2 kcal / g because it is converted into organic acids by intestinal bacteria and absorbed into the body, and non-caloric carbohydrates more than disaccharides are not known at present.

Further, α-D-fructofuranose β-D
-Fructofuranose 1,2 ': 2,1' dianhydride (DFAI) and α-D-fructofuranose β-
With respect to D-fructofuranose 1,2 ': 2,3' dianhydride (DFAIII), an enzyme that hydrolyzes the α bond to produce inulobiose is known (Starch Science Vol. 35, No. 2, p. 113-). p120
(1988)). However, an enzyme that hydrolyzes one α-fructofuranoside bond of di-α-D-fructofuranose dianhydride or an enzyme that hydrolyzes a single α-fructofuranoside bond is not known.

[0006]

DISCLOSURE OF THE INVENTION The present invention has a feature that it has a sweeter taste that is cleaner than sucrose and has no aftertaste, shows a moderate degree of sweetness, and is non-caloric or has almost no calories. And a sweetener containing the same, a method for producing the disaccharide, and an enzyme used for the production thereof.

[0007]

DISCLOSURE OF THE INVENTION The present invention relates to a novel disaccharide which has a sweeter taste that is cleaner than sucrose, has no aftertaste, and is non-caloric or almost zero in calories. 1-O-α-D-fructofuranosyl-D-fructose (hereinafter referred to as αFF)
About.

[0008]

Embedded image

The present invention also relates to a sweetener containing αFF as an active ingredient. The present invention further provides di-α-D-
Fructofuranose 1,2 ': 2,1' dianhydride (hereinafter referred to as α-DFA) (structure will be described later)
-A method for producing αFF, characterized in that an enzyme that hydrolyzes one α1,2 fructofuranoside bond of DFA is acted on, and an enzyme that condenses fructose to produce αFF is acted on fructose. ΑFF manufacturing method, α
The present invention relates to an enzyme which hydrolyzes one α1,2 fructofuranoside bond of DFA and an enzyme which hydrolyzes α-fructofuranoside bond of αFF to produce fructose and condenses fructose to produce αFF.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The method for producing the αFF of the present invention will be described later. α
FF is a sweet substance that dissolves well in water. The specific rotation of αFF is [α] +10.0. FIG. 1 shows the C-NMR spectrum of αFF. The sweetness of αFF is shown in Table 1 in comparison with other disaccharides. The sweetness is 5w / v% or 10w /
The measurement was carried out at 20 ° C. with 10 panelists using v% sucrose solution as a standard. The measurement method is the first report of the Research Society of Starch Sugar Technology, No. 1.
No. 4, p44 (1956).

[0011]

[Table 1]

The taste of αFF was evaluated as follows. That is, 5 w / v% sucrose, 10 w / v% αF
F, 12.5 w / v% maltose and 4 w / v% fructose were each prepared, and after confirming that the sweetness at 20 ° C. was the same, each of the items in Table 2 below by 12 panelists Is 0 if it is about the same as sucrose, +1 if slightly strong, +2 if strong, + if slightly weak
1. If weak, it was evaluated with +2, and the average score was calculated. As a result, as shown in Table 2, αFF was “clean”, “plain”, and “no aftertaste”
Items were clearly rated highly.

[0013]

[Table 2]

From the above, it can be understood that αFF is an oligosaccharide having an unprecedented characteristic that it is cleaner than sucrose and fructose and has no aftertaste.

Since αFF is an α-conjugate of fructose, which has not been known so far, it was not decomposed at all when its digestibility by various digestive enzymes was examined (Example 5). In addition, as a result of examining assimilation by intestinal bacteria in the large intestine, none of the bacteria was assimilated (Example 6). Normally, maltitol, which is an indigestible saccharide, is fermented by intestinal bacteria to produce organic acids, which are absorbed into the body and become calories. However, αFF is not degraded by digestive enzymes and is not assimilated by intestinal bacteria. Therefore, unlike normal indigestible carbohydrates, αFF is understood to be non-caloric or a carbohydrate with almost zero calories. You. This property is due to the fact that αFF bound only by α-fructofuranoside bond
Other polymers of fructose (i.e., oligosaccharides and polysaccharides) are considered to have properties in common. Therefore,
For example, α-D-fructofuranosyl-D- other than αFF
Fructose is also considered a non-caloric sugar.

Utilizing such properties of αFF, the present invention also relates to a sweetener containing αFF as an active ingredient. The sweetener may consist solely of αFF or may be in a form mixed with auxiliary or other active ingredients, for example other sweeteners.

As the auxiliary components, water, commonly used excipients and binders and the like can be used. As an excipient, in addition to sucrose, lactose, fructose, sorbitol, maltitol, etc., which are also used as sweeteners, polysaccharides such as soluble starch, dextrin, gum mannan, pectin, alginic acid, gelatin, low molecular weight polypeptide, etc. Protein,
Salts such as calcium carbonate and calcium phosphate, citric acid,
It can be used by appropriately selecting from acids such as malic acid and fumaric acid. As the binder, guar gum, gum arabic, dextrin and the like can be appropriately selected and used as needed. In addition, flavors, essences, vitamins, seasonings and the like can be appropriately selected and used as needed. As other sweet components, sugars such as glucose, fructose, and sucrose used for imparting sweetness, oligosaccharides such as isomaltooligosaccharide, sugar alcohols such as sorbitol and maltitol, aspartame, stevioside, and saccharin can be used. . In addition, other taste components include components that impart a taste, astringency, umami, bitterness, and the like from sourness and salt.

The sweetener of the present invention can be used in various forms, for example, as powders, granules, syrups and the like.

The present sweetener can be widely used for imparting sweetness and / or increasing the sugar content of foods and drinks or drugs taken or administered by humans and other animals. Examples of the foods and drinks include various seasonings (for example, soy sauce, miso, mayonnaise, dressing, tentsuyu, ketchup, grilled meat sauce, curry roux, stew ingredients, soup ingredients, dash ingredients, etc.), and various Japanese sweets (eg, , Senbei, hail, mochi, bun, uiro, sheep, jelly, castella,
Candy, etc., various Western confectionery (eg, biscuit, cracker, cookie, pie, pudding, cream puff, sponge cake, donut, chocolate, chewing gum, etc.), breads, ice confectionery (eg, ice cream, sherbet, etc.), syrups (Eg, fruit syrup pickles, etc.), pastes (eg, fruit paste, peanut paste, etc.), jams, marmalades, pickles (eg, Fukugami pickles, senmai pickles, lucky pickles, etc.), and meat and meat products (eg, ham, sausage, etc.) ), Fish paste products (eg, kamaboko, bamboo, etc.), various delicacies, boiled foods, alcoholic drinks, coffee, cocoa, juice, carbonated drinks, stamina drinks, lactic acid drinks, lactic acid drinks, instant foods and drinks (eg, instant juice, Instant coffee etc.) It is. Examples of the drug include powders, tablets, solutions, syrups, and the like, as well as dentifrices and mouthwashes.

The amount of the present sweetener can be arbitrarily used to the extent necessary for imparting sweetness to the intended food or drink or medicine. Although the specific amount of use differs depending on the food and drink or medicines, usually, the present sweetener is used in individual foods and drinks or medicines imparted with sweetness by the present sweetener, as αFF of about 0.5 to about 70 w / w.
%, More preferably from about 5.0 to about 30% w / w.

The method of using the present sweetener in a target product for imparting sweetness and / or increasing the sugar content may be carried out in the same manner as a usual sweetener. For example, at the time of production of foods or beverages or medicines, or at the time of their ingestion, they can be incorporated into the target product by using an appropriate method such as mixing, kneading, dissolving, dipping, penetrating, spraying, spraying, or pouring. .

Next, a method for producing αFF will be described. αF
F is (1) α-DFA, α1, one of α-DFA,
An enzyme that hydrolyzes 2 fructofuranoside bonds (hereinafter referred to as
α-DFA-degrading enzyme) or (2) an enzyme capable of condensing fructose to produce αFF (hereinafter referred to as αFF-degrading enzyme) on fructose.

First, the method (1) will be described. The enzyme used in this method has (1) an action of hydrolyzing one α1,2 fructofuranoside bond of α-DFA.

The above enzyme has the following enzymatic properties in more detail. (2) Substrate Specificity α-D-Fructofuranose β-D-Fructofuranose 1,2 ′: 2,1 ′ Dianhydride (DFAI) and α-D-Fructofuranose β-D-Fructofuranose 1,2 ′: 2,3 'dianhydride (DFAII
It has no effect on I). Inurobiose is intramolecularly condensed to produce DFAI. (3) Optimum pH The reaction was performed using McIlvine buffer (pH 4.5-8.0), and the relative activity was examined.
Was 6.0 to 6.5 (FIG. 2). (4) Stable pH range After incubation at 40 ° C. for 1 hour using McIlvine buffer (pH 4.5 to 8.0) and measuring the residual activity, the enzyme was stable at pH 5.0 to 7.5. (FIG. 2).

(5) Optimum temperature The reaction was carried out at various temperatures (30 to 60 ° C.) in a 20 mM potassium phosphate buffer (pH 7.0), and the relative activity was determined. (FIG. 3). (6) Temperature stability Incubation for 1 hour at various temperatures (30 to 60 ° C) in 20 mM potassium phosphate buffer (pH 7.0) and measurement of the residual activity revealed that the temperature at which 100% activity was maintained was 50%. ° C (Figure 3).

(7) Molecular weight HiLoad 16/60 Superdex200p
The molecular weight was determined by gel filtration chromatography using g (Pharmacia Biotech Co., Ltd.) from the relative elution retention time with various standard proteins. As a result, the molecular weight of the enzyme was about 210,000. The value of the molecular weight determined from the relative mobility with various standard proteins by SDS gel electrophoresis was about 51,000. From the results of the above gel filtration chromatography and SDS gel electrophoresis, it is considered that the present enzyme forms a tetramer. (8) Isoelectric point The enzyme was subjected to isoelectric focusing on a Multiphor II electrophoresis system (Pharmacia Biotech Co., Ltd.) using Ampholine PAG plate (Pharmacia Biotech Co., Ltd.) as a gel for isoelectric point measurement. After staining, the gel was cut out at intervals of 5 mm, extracted with a 20 mM potassium phosphate buffer (pH 7.0), and the activity was measured. The position where the activity was detected coincided with the position of the stained band, and the isoelectric point was determined to be 5.0.

The α-DFA-degrading enzyme of the present invention is α-DFA-degrading enzyme.
Not only α1,2 fructofuranoside bond of DFA,
There is also a possibility of hydrolyzing the α-fructofuranoside bond of other difructose dianhydride having an α-bond (hereinafter referred to as DFA).

In determining the enzymatic properties, the activity of the α-DFA-degrading enzyme of the present invention was measured as follows. 0.5 ml of 1 w / v% α-DFA (in 20 mM potassium phosphate buffer (pH 7.0)) and 0.5 ml of enzyme solution
After mixing at 40 ° C. for 30 minutes, the mixture was treated in a boiling water bath for 3 minutes to stop the reaction. This solution is HPL
C was analyzed to determine the amount of α-DFA degradation. One unit of the enzyme was the amount of the enzyme that hydrolyzes 1 μmol of α-DFA per minute. The HPLC conditions are shown below. (HPLC conditions) Column: YMC-Pack ODS-AQ (4.6 × 2
50 mm) (manufactured by YMC) Mobile phase: distilled water Flow rate: 0.5 ml / min Detection: RI.

The α-DFA-degrading enzyme of the present invention comprises α-DF
The bacterium belonging to the genus Bacillus having the ability to produce A-degrading enzyme can be cultured in a nutrient medium, and α-DFA-degrading enzyme produced from the culture can be collected.

The microorganism used in the production may be any microorganism as long as it belongs to the genus Bacillus and has the ability to produce α-DFA degrading enzyme. Specifically, the A1 strain isolated from the soil by the present inventors can be mentioned. The bacteriological properties of this strain are as shown in Table 3. Due to these properties, "The Genus Bacillus" (1973) U.S.A. S.
Department of Agriculture
And the A1 strain was named Bacillus brevis () A1. The A1 strain has been deposited with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, as a FERM P-16342. The microorganisms used in the present invention are not limited to wild strains, and wild strains such as the above wild strains can be prepared by ultraviolet, X-ray, radiation, drug [NTG (N-methyl-N'-nitro-N-nitrosoguanidine), EMS (ethyl methanesulfonate). )
And the like, a microorganism mutated by known artificial mutation means using α-DFA degrading enzyme gene,
-DFA-degrading enzyme-producing ability can be used.

[0031]

[Table 3]

As a nutrient medium for culturing a microorganism capable of producing α-DFA-degrading enzyme, a carbon source, a nitrogen source,
Either a natural medium or a synthetic medium may be used as long as it contains an inorganic substance and, if necessary, micronutrients required by the strain used. As a carbon source, carbohydrates such as maltose, glucose, fructose, lactose, starch, dextrin, and glycerin are used. Since the present enzyme is an inducing enzyme, α-DFA is preferably added to the medium as a part of the carbon source so as to be 0.01 to 0.5 w / v%. Amino acids such as ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate and glutamic acid, and inorganic organic nitrogen compounds such as uric acid are used as the nitrogen source. As the nitrogen source, nitrogen-containing natural products such as peptone, polypeptone, meat extract, yeast extract, corn steep liquor, soybean flour, soybean meal, dried yeast, casamino acid, soluble vegetable protein and the like can also be used. As the inorganic substance, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium chloride, potassium chloride, calcium chloride and the like are used. In addition, trace nutrients such as biotin and thiamine are used as needed.

As a culturing method, a liquid culturing method (a shaking culturing method or an aerated stirring culturing method) is preferable, and the aerated stirring culturing method is most suitable industrially. The culturing temperature can be in the range of 20 to 40 ° C. The pH is preferably 6.5 to 7.5. The culturing time varies depending on the culturing conditions, but is usually about 15 to 48 hours. When the production of α-DFA-degrading enzyme is confirmed, preferably when the production reaches the maximum, the culturing is stopped.

In order to collect the α-DFA-degrading enzyme of the present invention from the culture thus obtained, the culture is first separated into a culture solution fraction and a cell fraction by centrifugation or filtration. . α-DFA-degrading enzyme is detected in both of the above-mentioned fractions. However, since it is mainly obtained from the cell fraction, the cells are subjected to physical treatment such as ultrasonic crushing, glass bead crushing, French press, and enzymes such as lysozyme. It is destroyed by the treatment to obtain a cell extract, which is further subjected to ultrafiltration, salting out, dialysis, solvent precipitation, ion exchange chromatography, hydrophobic chromatography, gel chromatography, adsorption chromatography, isoelectric point Well-known isolation such as chromatography
By applying the purification method alone or in combination, α-D
A concentrated or purified preparation of the FA-degrading enzyme can be obtained. Example 1 shows a specific example of the isolation and purification of the α-DFA-degrading enzyme of the present invention.

An αFF can be produced by allowing α-DFA degrading enzyme to act on α-DFA.
More specifically, the α-DFA-degrading enzyme is added to α-DFA in an aqueous medium at 30 to 50 ° C. and pH 5.0 to 7.0.
By acting in step 5, αFF can be manufactured.

The α-DFA used for producing the αFF
The degrading enzyme may be a purified enzyme or a crude enzyme containing another enzyme that does not adversely affect the production of αFF. As the crude enzyme, α-
A crude enzyme precipitated from a DFA-degrading enzyme-containing fraction by salting out or solvent precipitation, or a crude enzyme in the middle of purification, which is further purified by the above-mentioned purification means, may be used.
Further, it is also possible to use an immobilized enzyme obtained by immobilizing these enzymes on a carrier by a conventional method.

Α-DFA has a chemical name of di-α-D-fructofuranose 1,2 ′: 2,1 ′ dianhydride and is represented by the following formula.

[0038]

Embedded image

Α-DFA is prepared by converting a high-concentration aqueous fructose solution of 70 to 90% by weight to pH 2.5 to 3.5, 110 to 150 ° C., preferably 130 to 140 ° C. under atmospheric pressure for 1 to 180 minutes, preferably 20 to 90 ° C. By holding the mixture for 60 minutes, a condensate of which DFA accounts for about 50% of the condensate is formed. Then, the treatment solution is adjusted to pH 2 and then kept in a boiling water bath for 30 to 60 minutes to remove the condensate other than the DFA. It is decomposed into fructose, and the treated liquid is passed through a Na-type cation exchange resin column to obtain a fraction of DFAs and a fraction of fructose. Then, the fractions of DFAs are passed through an ODS column to obtain each peak. Separated, one of them
It can be produced by obtaining a fraction of α-DFA as one peak fraction. Reference Example 1 shows a more specific production example of α-DFA.

Note that Carbohydrate Res
search, 174 (1988), 323-329,
The obtained syrup-like substance 9 is α, α-diffruct
offuranose diahydrate or β, β
-Difructofuranose dianhyd
rate is unknown, but β, β-diffructof
It is described that the compound is assumed to be uranose diahydrate (p. 325).
NMR substantially agrees with α-DFA obtained in Reference Example 1 of the present application, and α, α-diffructofuranose
diahydrate, that is, α-DFA.

Examples of the aqueous medium include water and a buffer. As the buffer, an acetate buffer, a potassium phosphate / citrate buffer, a citrate buffer, a succinate buffer, a Tris / hydrochloride buffer, or the like can be used. There is no particular limitation on the amount of the enzyme used.
It is appropriate to use 0.5 to 30 units, preferably 1 to 10 units. The reaction is terminated when a sufficient amount of αFF is generated under the above conditions, and the reaction is usually completed in 10 to 48 hours.

After completion of the reaction, the reaction is stopped by an appropriate means such as deactivation of the enzyme by heating the reaction solution, or deactivation of the enzyme by lowering the pH (addition of an acid such as hydrochloric acid). By appropriately combining isolation and purification means such as treatment,
αFF can be obtained. Example 2 shows a specific example of production of αFF by hydrolysis of α-DFA.

Next, the method (2) will be described. The enzyme used in this method has (1) the action of hydrolyzing the α-1,2 fructofuranoside bond of αFF to produce fructose and condensing fructose to produce αFF.

The above enzymes have the following enzymatic properties in more detail. (2) Substrate specificity It does not act on inurobiose. (3) Optimum pH The reaction was performed using McIlvine buffer (pH 4.5-8.0), and the relative activity was examined.
Was 6.5 to 7.0 (FIG. 4). (4) Stable pH range After incubation at 40 ° C. for 1 hour using McIlvine buffer (pH 4.5 to 8.0) and measuring the residual activity, the enzyme was stable at pH 5.0 to 7.5. (FIG. 4).

(5) Optimum temperature The reaction was carried out at various temperatures (30 to 60 ° C.) in a 20 mM potassium phosphate buffer (pH 7.0), and the relative activity was examined. (FIG. 5). (6) Temperature stability When incubated at various temperatures (30 to 60 ° C) for 1 hour in a 20 mM potassium phosphate buffer (pH 7.0) and the residual activity was measured, the temperature at which 100% activity was maintained was 40 ° C. ° C (Figure 5).

(7) Molecular weight HiLoad 16/60 Superdex 200p
As a result of determining the molecular weight from the relative elution retention time with various standard proteins by gel filtration chromatography using g (Pharmacia Biotech Co., Ltd.), the molecular weight of the present enzyme was about 410,000. The value of the molecular weight obtained from the relative mobility with various standard proteins by SDS gel electrophoresis was about 100,000. From the results of the above gel filtration chromatography and SDS gel electrophoresis, it is considered that the present enzyme forms a tetramer. (8) Isoelectric point The enzyme was subjected to isoelectric focusing on a Multiphor II electrophoresis system (Pharmacia Biotech Co., Ltd.) using Ampholine PAG plate (Pharmacia Biotech Co., Ltd.) as a gel for isoelectric point measurement. After staining, the gel was cut out at intervals of 5 mm, extracted with a 20 mM potassium phosphate buffer (pH 7.0), and the activity was measured. The position where the activity was detected coincided with the position of the stained band, and the isoelectric point was measured at 4.8.

The αFF-degrading enzyme of the present invention may hydrolyze not only the fructofuranoside bond of αFF but also the fructofuranoside bond of other α-D-fructofuranosyl-D-fructose.

In determining the enzymatic properties, the activity of the αFF-degrading enzyme of the present invention was measured as follows. 0.5 ml of 1 w / v% αFF (in 20 mM potassium phosphate buffer (pH 7.0)) and 0.5 ml of the enzyme solution were mixed, reacted at 40 ° C. for 30 minutes, and then mixed in a boiling water bath.
The reaction was stopped by a minute treatment. This solution was analyzed by HPLC to determine the amount of αFF decomposed. One unit of the enzyme was the amount of the enzyme that hydrolyzed 1 μmol of αFF per minute. HP
The LC conditions are shown below. (HPLC conditions) Column: YMC-Pack ODS-AQ (4.6 × 2
50 mm) (manufactured by YMC) Mobile phase: distilled water Flow rate: 0.5 ml / min Detection: RI.

The αFF-degrading enzyme of the present invention can be produced by culturing a bacterium belonging to the genus Bacillus or Pseudomonas having the ability to produce αFF-degrading enzyme in a nutrient medium, and collecting the αFF-degrading enzyme produced from the culture.

The microorganism used in the production may be any microorganism that belongs to the genus Bacillus or Pseudomonas and has the ability to produce αFF-degrading enzyme. Specifically, the above-mentioned A1 isolated from the soil by the present inventors.
Strain, ie Bacillus brevis A1 FERM P-
16342 and 12L strains.

The bacteriological properties of the 12L strain are as shown in Tables 4 and 5. Due to these properties, the 12L strain was named Pseudomonas putida () 12L based on "Burgess Manual of Systematic Bacteriology" VOL1 (1984). 12L shares were transferred to the Institute of Biotechnology and Industrial Technology,
Deposited as RM P-16344. The microorganism used in the present invention is not limited to a wild strain, and a wild strain, for example, the wild strain described above may be subjected to ultraviolet ray, X-ray, radiation, drug [NTG (N
-Methyl-N'-nitro-N-nitrosoguanidine),
EMS (ethyl methanesulfonate) etc.], and a mutant strain mutated by known artificial mutation means, and a microorganism into which an αFF-degrading enzyme gene has been introduced can be used as long as they have αFF-degrading enzyme-producing ability.

[0052]

[Table 4]

[0053]

[Table 5]

As a nutrient medium and a culture method for culturing a microorganism capable of producing αFF-degrading enzyme,
A nutrient medium and a culture method similar to the nutrient medium and the culture method for culturing a microorganism having the ability to produce DFA-degrading enzyme can be used. The αFF-degrading enzyme of the present invention can be collected from the obtained culture in the same manner as in the case of α-DFA-degrading enzyme. Example 3 shows a specific example of the production of the αFF-degrading enzyme of the present invention.

ΑFF can be produced by allowing αFF-degrading enzyme to act on fructose. More specifically, the above-mentioned αFF-degrading enzyme is added to fructose in an aqueous medium for 3 hours.
ΑFF can be manufactured by acting at 0 to 50 ° C. and pH 5.0 to 7.5.

The αFF-degrading enzyme used in the production of αFF may be a purified enzyme or a crude enzyme which may contain another enzyme which does not adversely affect the production of αFF. As the crude enzyme, α
A crude enzyme precipitated from the FF-degrading enzyme-containing fraction by salting out or solvent precipitation, or a crude enzyme in the middle of purification, which is further purified by the above-described purification means, may be used. Further, it is also possible to use an immobilized enzyme obtained by immobilizing these enzymes on a carrier by a conventional method.

The isolation and purification of α-FF from the aqueous medium, the amount of the enzyme used, the reaction time, and the reaction-finished solution were performed using α-DF
It can be carried out in the same manner as in the case of producing αFF using A-degrading enzyme. Example 4 shows a specific example of the production of αFF by fructose condensation.

[0058]

Next, the present invention will be described specifically with reference to examples and reference examples. In the following Examples and Reference Examples,% indicating the concentration represents w / v% unless otherwise specified. Example 1 Production of α-DFA-degrading enzyme Bacillus brevis A1 FERM P-16342 was added to 0.3% α-DFA, 0.2% yeast extract, 0.02%
Polypeptone, 0.1% sodium nitrate, 0.05% monopotassium phosphate, 0.02% magnesium sulfate, pH
7.0 medium (100 ml / 500 ml flask x 3
This was shake-cultured at 30 ° C. for 20 hours, and the cells were collected by centrifugation, followed by 20 mM potassium phosphate buffer (pH 7.0).
The suspension was suspended in 15 ml, and the cells were disrupted by ultrasonic treatment to obtain a cell extract. The α-DFA-degrading enzyme activity of the cell extract was 20 units in total. This is fractionated with ammonium sulfate, HiLoad
Gel filtration by 16/60 Superdex 200pg (Pharmacia Biotech Co., Ltd.), monoQ5
Purified singly by electrophoresis with 10% recovery after anion exchange chromatography on / 5 (the company). The enzymatic properties of the obtained purified α-DFA-degrading enzyme are as described above.

Example 2 α by hydrolysis of α-DFA
Production of FF 5 g of α-DFA prepared as in Reference Example 1 was 20 m
Purified α-DFA dissolved in 100 ml of M potassium phosphate buffer (pH 7.0) and prepared by the method described in Example 1.
5 units of a degrading enzyme was added and reacted at 40 ° C. for 30 hours. When the reaction solution was analyzed by HPLC, αFF was formed at 25% w / w in the total saccharide. The reaction solution was subjected to a reversed phase chromatography column (YMC-YMC-
The resulting aqueous solution was concentrated under reduced pressure and 1.6 g of a 75% aqueous solution (syrup) of αFF having a purity of 98% was collected by PackODS-AQ (10 × 100 cm × 2).
I got The aqueous solution was further concentrated to about 80% w / w, and then crystallized by standing at 4 ° C. to obtain 0.5 g of αFF crystals having a purity of 99% or more. The specific rotation of the obtained crystal αFF is as described above, and the C-NMR spectrum is shown in FIG. The degree of sweetness of the obtained αFF is as shown in Table 1 described above.

Example 3 Production of αFF-degrading enzyme Pseudomonas putida 12L FERM P-163
44 in 0.3% αFF, 0.2% yeast extract, 0.02
% Polypeptone, 0.1% sodium nitrate, 0.05%
Monopotassium phosphate, 0.02% magnesium sulfate, pH
7.0 medium (100 ml / 500 ml flask x 3
This was shake-cultured at 30 ° C. for 20 hours, and the cells were collected by centrifugation, followed by 20 mM potassium phosphate buffer (pH 7.0).
The suspension was suspended in 15 ml, and the cells were disrupted by ultrasonic treatment to obtain a cell extract. The αFF-degrading enzyme activity of the cell extract was 10 units in total. This was fractionated with ammonium sulfate, HiLoad16.
/ 60 Superdex200pg (Pharmacia
Gel filtration by Biotech Co., Ltd., monoQ5 / 5
Purified singly electrophoretically with 10% recovery by anion exchange chromatography by the company. The enzymatic properties of the purified αFF-degrading enzyme obtained are as described above.

Example 4 Production of αFF by Condensing Fructose 5 g of crystalline fructose was added to a 20 mM potassium phosphate buffer (pH
7.0) Dissolve in 2 ml, add 5 units of purified αFF-degrading enzyme prepared by the method described in Example 3, and add
Allowed to react for hours. When the reaction solution was analyzed by HPLC,
αFF was produced at 5 w / w% of the total sugars. The reaction solution was subjected to a column for reversed phase chromatography (YMC-Pack ODS-AQ, manufactured by YMC), 10 × 100 cm
× 2), and the resulting aqueous solution was concentrated under reduced pressure to obtain 0.3 g of a 98% pure αFF 75 w / w% aqueous solution (syrup). Further, this aqueous solution is 80 w / w%
After concentrating to a degree, crystallize by leaving it at 4 ° C.
0.1 g of αFF crystal having the above purity was obtained. Obtained α
The specific rotation and the C-NMR chemical shift of FF substantially coincided with the αFF obtained in Example 2.

Example 5 Degradability of αFF by Various Digestive Enzymes α-amylase 1% αFF (50 mM maleate buffer containing 1 mM CaCl; pH 6.0 for salivary amylase test, pH 6.9 for pancreatic amylase test) 1 ml of human salivary amylase (manufactured by Sigma), porcine pancreatic amylase (Sigma) After adding 1 unit of each of them and reacting at 37 ° C. for 2 hours, residual αFF was analyzed by HPLC (HPLC conditions were the same as the HPLC conditions already described in the measurement of αFF-degrading enzyme activity). When either enzyme was used, 98% of αFF remained. * 1 unit means the amount of enzyme that produces a reducing power equivalent to 1 μmol of glucose per minute when reacted at 37 ° C. using 1% soluble starch as a substrate.

2. Small intestinal mucosal localization enzyme Rat small intestine acetone powder (manufactured by Sigma) was suspended (100 mg / ml) in physiological saline, homogenized, and centrifuged, and the supernatant was used as an enzyme solution. 1% αFF (25
mM maleate buffer (pH 6.0)), 1 unit of the enzyme solution was added, and reacted at 37 ° C. for 4 hours. αFF is 95%
It remained. * 1 unit means the amount of enzyme that degrades 1 μmol maltose per minute when reacted at 37 ° C. using 1% maltose as a substrate.

Example 6: Assimilation of αFF by enterobacteria Fresh bacteria cultured on an agar medium were cultured in a peptone-yeast-field solution (PYF) medium containing 0.5% of each sugar solution. The cells were inoculated at 10 CFU / tube, cultured at 37 ° C. for 48 hours under anaerobic conditions, and assimilation was determined by the growth degree with respect to a glucose-added medium used as a control. The results are shown in Tables 6 and 7. As is evident from Tables 6 and 7, αFF was not assimilated to all strains tested.

[0065]

[Table 6]

[0066]

[Table 7]

Example 7 Sweetener Formulation Example 1 αFF syrup (water 25 w) prepared by the method of Example 2
/ W%) and a high-purity fructose aqueous solution (water 25w / w)
%) Was mixed with 50 g to prepare a liquid sweetener. This sweetener had the same degree of sweetness (per solid) as sucrose, but had a very clean aftertaste. Example 8 Sweetener Formulation Example 2 20 g of the αFF crystal prepared by the method of Example 2 and 10 g of crystalline xylitol were pulverized and mixed to prepare a powdered sweetener. The sweetener had a sweetness of about 50 and had a refreshing taste.

Example 9 Addition Example 1 to Food and Beverage (Utilization for Cider) In 500 ml of water, 50 g of the sweetener shown in Example 7, 0.1 g of citric acid, 0.25 g of malic acid, 0.05 g of sodium fumarate After adding 0.5 ml of lime flavor and stirring sufficiently with a mixer, the mixture was filtered with a microfilter, sterilized at 65 ° C. for 30 minutes, and then filled with gas and bottled. This product was a clear sweet cider with little aftertaste. Example 10 Addition Example 2 to Food and Beverage (Use in Sherbet) 175 g of orange juice, sweetener 70 shown in Example 7
g, aspartame 0.2 g, sodium alginate 1
g, carrageenan 0.5 g, pectin 0.7 g, gelatin 0.5 g and water 252.1 g to form a solution,
The product was sterilized at 65 ° C for 30 minutes, cooled, and then frozen at -20 ° C to obtain a product. Orange flavor works well,
It was a sweet sorbet with little aftertaste.

Example 11 Examples of Addition to Chemicals (Use in Dentifrice) 50 g of calcium phosphate dihydrate, 10 g of glycerin,
13 g of the sweetener prepared in Example 8, 2.5 g of sodium lauryl sulfate, 1.5 g of spearmint oil, 1.0 g of tragacanth gum and 22 g of water were mixed to obtain a product. This product matched mint flavor with refreshing sweetness.

Reference Example 1 Production of α-DFA 150 g of fructose was dissolved in 40 g of water, and 0.6 ml of a 10% aqueous citric acid solution was added. This is heated with an electric heater while stirring with a mixer, and 130 ° C. while evaporating water.
The reaction was carried out at ℃ 140 ° C. for 20 minutes. The reaction product is sugar concentration 50w
/ W%, adjusted to pH 2 with hydrochloric acid, and treated in a boiling water bath for 30 minutes to hydrolyze condensates other than DFA (difructose dianhydride). This was separated by a strongly acidic Na-type cation exchange resin (UBK-530, manufactured by Mitsubishi Chemical Corporation, resin amount: 11.8 L) to obtain 40 g of a fraction containing α-DFA (19 w / w%) as a solid content. . Then, the solution was concentrated to a concentration of 50 w / w%, and a column for reverse phase chromatography (YMC-Pack ODS-A manufactured by YMC) was 7 g each.
Q, 10 × 100 cm × 2), using demineralized water as a mobile phase, passing the solution at a flow rate of 250 ml / min, fractionating and fractionating, and concentrating the resulting aqueous solution under reduced pressure to 98% purity. 8.8 g of a 75% w / w aqueous solution (syrup) of α-DFA
I got Further, this aqueous solution was concentrated to about 80% w / w, and crystallized by standing at 4 ° C. to obtain 2.5 g of α-DFA crystals having a purity of 99% or more. Α-DF obtained
The specific rotation [α] of A is +146, and the C-NMR chemical shifts are 61.2 (C-1) and 103.9 (C-
2), 83.1, 80.2, 77.0 (above C-3, C
-4, C-5), 61.2 (C-6)
Met.

[0071]

The αFF of the present invention is a fructose homobiose having a characteristic that it has a clearer sweetness than sucrose and no aftertaste. According to the present invention, α-
An enzyme that hydrolyzes one α1,2 fructofuranoside bond of DFA and an industrial method for producing αFF using the enzyme;
And an enzyme for condensing fructose to produce αFF, and an industrial method for producing αFF using the same.

[Brief description of the drawings]

FIG. 1 shows a C-NMR spectrum of αFF.

FIG. 2 shows the optimal p of the α-DFA degrading enzyme obtained in the present invention.
H and stable pH range are shown.

FIG. 3 shows the optimum temperature and temperature stability of α-DFA degrading enzyme obtained in the present invention.

FIG. 4 shows the optimum pH and stable pH range of the αFF-degrading enzyme obtained by the present invention.

FIG. 5 shows the optimum temperature and temperature stability of the αFF-degrading enzyme obtained by the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:07) (72) Inventor Tetsuya Yatake 2-20-2 Hinode, Funabashi-shi, Chiba Pref.

Claims (8)

[Claims] 1. 1-O-α-D-fructofuranosyl-
D-fructose (hereinafter referred to as αFF in the claims). 2. A sweetener containing αFF as an active ingredient. 3. Di-α-D-fructofuranose 1,2
': An enzyme which hydrolyzes one α1,2 fructofuranoside bond of α-DFA is allowed to act on 2,1 ′ dianhydride (hereinafter referred to as α-DFA in the claims). ΑFF manufacturing method. 4. A method for producing αFF, comprising reacting fructose with an enzyme that condenses fructose to produce αFF. 5. An enzyme that hydrolyzes one α1,2 fructofuranoside bond of α-DFA. 6. The enzyme according to claim 5, which further has the following enzymatic properties: (2) substrate specificity α-D-fructofuranose β-D-fructofuranose 1,2 ′: 2,1 ′ dianhydride (DFAI) and α-D-fructofuranose β-D-fructofuranose 1,2 ′: 2,3 ′ dianhydride (DFAII)
It has no effect on I). Inurobiose is intramolecularly condensed to produce DFAI. (3) Optimal pH 6.0 to 6.5 (4) Stable pH range 5.0 at 50 ° C for 1 hour
７7.5. (5) Optimum temperature 55 ° C. (6) Temperature stability The temperature at which 100% activity is maintained at pH 6.0 for 1 hour is up to 50 ° C. (7) Molecular weight The value measured by gel filtration chromatography is about 21
Of about 51,000 as determined by SDS gel electrophoresis. (8) Isoelectric point 5.0 7. An α-fructofuranoside bond of αFF is hydrolyzed to produce fructose, and fructose is condensed to form αFF.
Producing enzymes. 8. The enzyme according to claim 7, which further has the following enzymatic properties: (2) Substrate specificity It does not act on inurobiose. (3) Optimum pH 6.5 to 7.0 (4) Stable pH range 5.0 at 40 ° C for 1 hour
７7.5. (5) Optimum temperature 50 ° C (6) Temperature stability The temperature at which 100% activity is maintained at pH 7.0 for 1 hour is up to 40 ° C. (7) Molecular weight The value measured by gel filtration chromatography is about 41
000, and the value measured by SDS gel electrophoresis is about 100,000. (8) Isoelectric point 4.8