JP2000333693A - Isolation of saccharide from glycoside - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a variety of physiologically active components by allowing an enzyme composition to act on a glycoside thereby liberating monosaccharides from the glycoside and by allowing the liberated monosaccharides to act on a precursor of perfume components, pigment components or physiologically active components, for example, β-primeveroside. SOLUTION: An enzyme composition containing β-xylosidase and/or β- glucosidase which can be obtained from microorganisms and/or plants is allowed to act on β-primeveroside and/or similar glycosides, for example, a disaccharide glycoside that are precursors of perfume components, pigment components or physiologically active precursor. Consequently, saccharides are liberated from the glycoside in the form of monosaccharides and the perfumes, pigments and physiological components are enhanced or weakened by the aglycones- liberating action. This enzyme composition can be widely applied to a variety of foods, medicines, quasi drugs and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to various physiologically active ingredients which can be obtained from microorganisms or plants and act on .beta.-primeveroside or an analog thereof, which is a precursor of an aroma component, a pigment component or a physiologically active component. The present invention relates to an enzyme composition containing, as an active ingredient, an enzyme having an ability to produce lipase. More specifically, an enzyme composition containing β-xylosidase and / or β-glucosidase which has a disaccharide glycoside β-primeveroside or an analog thereof cleaved by a monosaccharide unit and has a physiologically active ingredient releasing action. Related to the application used.

[0002]

2. Description of the Related Art For example, alcohol-based aromas such as geraniol, linalool, benzyl alcohol, 2-phenyl ether and C13-norterpenoid alcohol, which are aroma components of plants, are important for generating aroma of flowers, tea, fruits, wines and the like. Working.

[0003] Among these odor components, monosaccharide glycosides such as β-D-glucopyranoside have been isolated and identified as odor precursors of benzyl alcohol and (Z) -3-hexenol.

[0004] Recently, β-glycoside of the disaccharide glycoside has been considered as an alcohol-based odor precursor such as geraniol or linalool, which is thought to play an important role in the odor of flowers.
-Primeveroside (6-O-β-D-xylopyranosyl-β
-D-glucopyranoside) or its analog was confirmed. Further, β-primeveros of disaccharide glycoside is also used as a precursor of other alcohol-based flavor components described above.
The existence of ide and its analogs is becoming apparent.

[0005] In addition to the aroma, the disaccharide glycoside β-primeveros is also used in some physiologically active substances such as pigments and pharmacological components.
It has become clear that it exists as ide or its analogs. For example, it is known that macrozamine present in cycads and the like is cleaved by β-primeverosidase at disaccharide units to produce methylazoxymethanol, which is a physiologically active substance.

[0006]

As described above, the enzyme having the action of releasing a bioactive component by acting on a precursor of a bioactive component which is a disaccharide glycoside β-primeveroside or an analog thereof is tea leaf. It has been slightly confirmed and refined. Therefore, there has been a strong demand for the development of an enzyme that acts on β-primeveroside, which is a disaccharide glycoside, or an analog thereof to generate a physiologically active component, without limiting the source to conventional tea leaves and the like.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, the enzyme composition containing a conventionally known enzyme was unexpectedly replaced with the disaccharide glycoside. The present inventors have found that a bioactive ingredient can be produced by acting on a certain β-primeveroside or an analog thereof, and completed the present invention. That is, the present invention relates to β-xylosidase and / or
Alternatively, using β-glucosidase to act on β-primeveroside, which is a disaccharide glycoside that is a precursor of a physiologically active component, and / or an analog thereof, to release the physiologically active component by cleaving at a monosaccharide unit. The present invention relates to a method for releasing a physiologically active ingredient.

In the present invention, various enzyme activities are indicated by values obtained by the methods described below unless otherwise specified.

Β-xylosidase activity The activity was measured using an automatic chemical analyzer (TBA-20R, manufactured by Toshiba Corporation).
This was performed using 45 µl of enzyme sample and 2 parts of paranitrophenyl (pNP) xyloside (Sigma) as substrate
Mix with 200 µl of 2 mM dissolved in 0 mM acetate buffer (pH 5.5), 37 ° C, cycle time 30.0s
After the reaction with ec, 250 μl of sodium carbonate was added and 412 nm
Was measured for absorbance. The blank from the sample was measured in the same manner using a 20 mM acetate buffer (pH 5.5) instead of the substrate solution. The amount of the enzyme that increases the absorbance by 1 under these conditions was defined as 1 unit (1 AU).

[0010] β-glucosidase activity The activity is measured by an automatic chemical analyzer (TBA-20R, manufactured by Toshiba Corporation).
This was performed using 45 μl of enzyme sample and 2 parts of paranitrophenyl (pNP) glucoside (manufactured by Merck) as substrate
Mix with 200 µl of 2 mM dissolved in 0 mM acetate buffer (pH 5.5), 37 ° C, cycle time 30.0s
After the reaction with ec, 250 μl of sodium carbonate was added and 412 nm
Was measured for absorbance. The blank from the sample was measured in the same manner using a 20 mM acetate buffer (pH 5.5) instead of the substrate solution. Under these conditions, the amount of the enzyme that increases the absorbance by 1 was defined as 1 unit (1 AU).

Next, the present invention will be described in detail. The present inventors studied β-primeveroside or a similar disaccharide glycoside at a monosaccharide unit and studied an enzyme having an aglycone releasing action, and a commercially available enzyme preparation having an aglycone releasing action by combining the enzymes. Furthermore, screening was also examined for microorganisms having the ability to produce such enzyme preparations. In addition, glycosides that are the subject of the present invention include not only β-primeveroside or similar disaccharide glycosides as described above, but also glycosides in which more than two sugars are bonded. Can be. In the present invention, the disaccharide glycoside similar to β-primeveroside is a disaccharide having glucose on the aglycone side, for example, apiofranosyl-β-D-glucopyranoside, arabinofuranosyl-β-D -Glucopyranoside and the like.

An enzyme having the properties of the present invention and a microorganism producing the enzyme which can be used in the present invention can be screened as follows. That is, the strain is cultured in an appropriate liquid medium if the strain is used, and the enzyme product is suspended and / or diluted in an appropriate buffer, and the production microorganism is determined using pNP-primeveroside or the like as a substrate and the amount of released pNP as an index. Can be screened.

The pNP-primeveroside serving as a substrate is, for example,
pNP-glucoside (manufactured by Merck) is reacted with xylo-oligosaccharide (manufactured by Wako Pure Chemical Industries) using the enzyme xylosidase (Sigma) to transfer one residue of xylose to pNP-glucoside by β-1,6 bond. Can be synthesized.

As a result of screening a commercially available enzyme preparation, pectinase G (Asperg, manufactured by Amano Pharmaceutical Co., Ltd.)
illus pulverulentus), which is obtained by cleaving β-primeveroside, which is a disaccharide glycoside, or an analog thereof with a monosaccharide unit, and finding that an enzyme having a physiologically active ingredient releasing action is present.

The present inventors have confirmed that β-xylosidase and / or β-glucosidase are involved in this action. That is, xylose is released from β-primeveroside which is a disaccharide glycoside by β-xylosidase activity, and then glucose is released from monosaccharide glycoside by β-glucosidase activity, resulting in release of a physiologically active substance. The mechanism of action was confirmed.

In the present invention, the enzyme preparation having β-xylosidase activity and the enzyme preparation having β-glucosidase activity can be used by mixing those prepared from different sources.

The β-xylosidase and β-glucosidase constituting the enzyme composition that can be used in the present invention can be of any origin such as animals, plants, and microorganisms, and are not particularly limited. For example, β-xylosidase is known to be derived from the genus Penicillium, derived from the apple snail (jumbo snail), and β-glucosidase is derived from apricot seed, cycad seed, Asperg.
It is known from illus niger. Of course, it is also possible to use those obtained by separating and purifying from a commercially available enzyme preparation used by the present inventors in Examples and the like.

As the mixing ratio, each activity ratio is such that β-xylosidase activity: β-glucosidase activity is 10: 0 to
0:10, preferably 8: 2 to 2: 8, more preferably 6: 4 to 4: 6. In addition, these enzyme compositions can be sequentially reacted to release the desired physiologically active substance from the glycoside. Furthermore, the present inventors have also found in the present invention that β-xylosidase and β-glucosidase each alone can act on β-primeveroside or an analog thereof and cleave at a monosaccharide unit to release a physiologically active component. .

Furthermore, the enzyme composition that can be used in the present invention can be newly obtained from nature by the above-described screening method. The microorganism thus obtained can be cultured and extracted by ordinary means. Thus, an enzyme composition that can be used in the present invention can be obtained. Also,
An enzyme composition that can be used in the present invention can also be produced by various genetic manipulation techniques.

That is, the production method that can be used in the present invention includes a mutant strain of a strain having β-xylosidase and / or β-glucosidase producing ability, or β-xylosidase and / or β-glucosidase by a recombinant DNA method. Various microorganisms or various cells, such as yeast cells, bacterial cells, higher plant cells, animal cells, etc., which have been modified so as to be able to produce E. coli are also included. When the β-xylosidase and / or β-glucosidase production ability is imparted by introducing the β-xylosidase and / or β-glucosidase gene, the host microorganism is β-xylosidase and / or β-glucosidase.
The ability to produce xylosidase and / or β-glucosidase may not be required.

In order to produce β-xylosidase and / or β-glucosidase using various microorganisms, etc.,
Methods and conditions suitable for the culture of the microorganism can be set, and these methods and conditions are not particularly limited. For example, the method for culturing the various bacterial species described above may be either liquid culture or solid culture, but preferably liquid culture is used. The liquid culture can be performed, for example, as follows.

As the medium that can be used, any medium can be used as long as a microorganism capable of producing β-xylosidase and / or β-glucosidase can grow. For example, glucose, sucrose, soluble starch, glycerin, dextrin, molasses, carbon sources such as organic acids, and also ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein hydrolysis , Bran, meat extract and other nitrogen sources, as well as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts,
What added an inorganic salt, such as a zinc salt, can be used. Furthermore, various inducers can be added to the medium in order to produce and accumulate β-xylosidase and / or β-glucosidase.

The pH of the medium is adjusted, for example, to about 3 to 8, preferably about 5 to 6, and the culture temperature is usually about 10 to 50 ° C., preferably about 30 ° C., for 1 to 15 days, preferably 4 to 15 days. Culture under aerobic condition for about 7 days. As the culture method, for example, a shaking culture method or an aerobic submerged culture method using a jar fermenter can be used. However, the above-mentioned various culture conditions and the like are, of course, appropriately changed depending on the microorganism or cell to be cultured, and β-
The conditions and the like are not limited as long as xylosidase and / or β-glucosidase are produced.

To isolate and purify β-xylosidase or β-glucosidase from the obtained culture, centrifugation,
Purified β was obtained by combining various chromatographies such as UF concentration, salting out, ion exchange resin, etc.
-Xylosidase or β-glucosidase can be obtained.

The enzyme composition of the present invention can be used as it is in the culture solution cultured as described above. Of course, the degree of purification of the main culture solution can be appropriately changed depending on the purpose of use of the present invention.

Next, various uses using the enzyme composition of the present invention will be described. The enzyme composition that can be used in the present invention can be used to enhance various components, for example, the aroma, color, and physiologically active content of plant materials, and to adjust the extraction efficiency of these components. Therefore, it can be used for the production of more fragrant foods, beverages and higher fragrance spices and fragrances, perfumes, etc., and can also be used for the early release of undesirable fragrances by appropriately using it in the above-mentioned production process . As for the color, it can be used for improving the color tone of plant materials, foods and beverages, coloring, or producing pigments.

Further, like the fragrance component, it can be used for decomposing and removing a pigment precursor which is unfavorable in quality. Alternatively, it can be used for decomposing and removing undesirable components.

That is, the above-mentioned action can be obtained by causing the β-xylosidase and / or β-glucosidase of the present invention to act on various primeverosides and disaccharide glycoside components which are analogs thereof.

The substance containing primeveroside or an analog thereof which is an object of the present invention includes β
-Any substance that is affected by the enzyme composition containing xylosidase and / or β-glucosidase may be used, for example, food, cosmetics, pharmaceuticals, quasi-drugs, agricultural chemicals, feeds, and the like. Specifically, it can be used for the production of foods having various flavors, toiletry products, industrial products made from plant materials such as woodwork products and tatami mats, and the like.

An object to which the method of the present invention is preferably used is a food having a flavor component. Specifically, in the production of oolong tea, jasmine tea, etc.,
For example, it can be used in the step of "atrophy" or used to enhance the aroma of black tea (eg, tea pack black tea by the CTC method) and the aroma of wine. It can also be used for preserving the fragrance of cosmetics and fragrance, improving the fragrance of pharmaceuticals, and improving the pharmacological effect.

Further, it is useful in the production of dyes.
For example, by using the dye alizarin from ruberitic acid from Western Akane, the pigment can be more efficiently extracted than before.

As a method of using the enzyme composition which can be used in the present invention, the addition method, the addition amount, the reaction method and the like can be appropriately changed depending on the form of the object.

As a specific application method, an enzyme composition is added to a plant extract or fermentation product containing an aroma precursor and incubated. The conditions are not particularly limited as long as the enzyme composition acts on the fragrance, the dye, and the bioactive ingredient precursor to release the fragrance, the dye, and the bioactive ingredient, but those skilled in the art require a great deal of labor for the conditions. Can be set without spending. Under these conditions, the concentration of the component can be increased.

The enzyme of the present invention can also be used for increasing the concentration of aroma, pigment, and physiologically active component present in plants. That is, since the plant has precursors of these components, an effective amount of the enzyme composition is given to the plant, and the fragrance of the plant is obtained by cultivating the plant under conditions in which the precursor in the plant can be hydrolyzed.
Dyes and physiologically active ingredients can be increased. Also,
By using the enzyme composition of the present invention, it is possible to control the generation time of a fragrance, a pigment, a physiologically active component, and the like in a target plant.

The reaction conditions of the method of the present invention in which the enzyme composition is allowed to act on glycosides and the sugars are released from the glycosides in monosaccharide units are not particularly limited as long as the above reaction occurs. The reaction may be carried out in an aqueous solution state, in an organic solvent such as methyl acetate, ethanol, or methanol, or in a mixed solvent thereof, wherein the substrate or enzyme is solid and the medium is liquid. The solid-liquid heterogeneous state may be used, but it is preferably carried out in an aqueous solution state. The concentration of the glycoside is preferably about 1 M, more preferably 0.1 M or less, and most preferably 4 mM or less. The amount of each enzyme used is preferably 1 to glycoside.
It is used in an amount of at least 10% by mass, more preferably at least 10% by mass (preferably 0.01-1 unit / mg substrate, more preferably 0.1-1 unit / mg substrate). The reaction temperature is preferably 0 to 65 ° C, more preferably 20 to 65 ° C, most preferably 30 to 50 ° C, and the reaction time is preferably 1 hour to 3 days.

Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to the following Examples.

[0037]

EXAMPLES Example 1 Pectinase G (trade name: Asperg, manufactured by Amano Pharmaceutical Co., Ltd.)
illus pulverulentus) (3.6 kg) was dissolved in 20 mM phosphate buffer (pH 6.0) to make 17.5 L. Ammonium sulfate 7.0
kg (65% saturation), centrifuge after stirring, and 17.8 L of supernatant
Was recovered. Add 3.65 kg of ammonium sulfate to the supernatant
(95% saturation), and the mixture was stirred and centrifuged to collect 16.7 L of the supernatant. The supernatant was concentrated to 210 ml with an ultrafiltration membrane having a molecular weight of 6,000 cuts, and then lyophilized. The lyophilized product was measured for β-xylosidase activity and β-glucosidase activity.

As a result, β-xylosidase activity was 7.12
Unit / mg, β-glucosidase activity was 0.12 unit / mg.

50 mg of the above freeze-dried product was dissolved in 10 ml of 25 mM bis-Tris buffer (pH 7.1), and the solution was dissolved in 25 mM bis-Tris buffer (pH 7.
Anion exchange Mono-P column (5 x 200m) equilibrated in 1)
m) and eluted with Polybuffer (Pharmacia) to obtain a partially purified enzyme preparation of β-xylosidase. As a result, β-xylosidase activity was 82.2 units / ml,
Glucosidase activity was 1.01 units / ml.

Example 2 Pectinase G (manufactured by Amano Pharmaceutical Co., Ltd., Aspergillus pu
lverulentus) 1.2 g in 20 mM phosphate buffer (pH 6.0) 12
Dissolved in ml and centrifuged. 28 ml of 100% saturated ammonium sulfate / 20 mM phosphate buffer (pH 6.0) is added to 12 ml of the supernatant.
Was added (70% saturation), and the mixture was stirred and centrifuged to collect a precipitate. This precipitate was dissolved in 12 ml of 35% saturated ammonium sulfate / 20 mM phosphate buffer (pH 6.0), and the supernatant after centrifugation was recovered. The β-glucosidase activity and β-xylosidase activity of this supernatant were measured.

As a result, β-glucosidase activity was 66.6
Unit / ml, β-xylosidase activity was 2.42 units / ml.

10 ml of the above supernatant was developed on a phenyl sepharose column (16 × 100 mm) equilibrated with 35% saturated ammonium sulfate / 20 mM phosphate buffer (pH 6.0), and the solution was added to a 20 mM phosphate buffer (pH 6.0). And a partially purified enzyme preparation of β-glucosidase was obtained. As a result, the β-glucosidase activity was 24.1 units / ml, and the β-xylosidase activity was 0.18 units / ml.

Example 3 The partially purified β-xylosidase obtained in Example 1 (β
-Xyl) was prepared by diluting with β-xylosidase activity 5.0 units / ml with 20 mM acetate buffer (pH 5.5).
Further, the partially purified β-glucosidase (β-Glc) obtained in Example 2 was converted to a β-glucosidase activity of 5.0 units / m 2.
The solution was diluted with a 20 mM acetate buffer (pH 5.5) to a volume of 1 l. Using these enzyme solutions and pNP-primeveroside, a β-xylosidase alone reaction, a β-glucosidase alone reaction, and a reaction of adding β-glucosidase after each hour after the β-xylosidase alone reaction are performed as follows,
The amount of released pNP was compared.

The β-xylosidase alone reaction was performed by mixing 0.2 ml of a pNP-primeveroside solution adjusted to 10 mM with a 20 mM acetate buffer (pH 5.5) and 0.2 ml of partially purified β-xylosidase, sampling 0.05 ml after 5 minutes, 0.025 ml of 20 mM acetate buffer (pH 5.5) and 0.075 ml of 0.5 M sodium carbonate were added and mixed, and the absorbance at 420 nm was measured (reaction for 5 minutes). Same
Five minutes later, 0.15 ml of a sample was sampled, and a 20 mM acetate buffer solution (pH 5.
0.075 ml of 5) was added, and the reaction was continued. Further, after 5 and 15 minutes, 0.1 ml of the sample was sampled, 0.1 ml of 0.5 M sodium carbonate was added and mixed, and the absorbance at 420 nm was measured (reaction for 10 and 20 minutes).

In the β-glucosidase single reaction, 0.15 ml of a pNP-primeveroside solution adjusted to 10 mM with 20 mM acetate buffer (pH 5.5) and 0.15 ml of partially purified β-glucosidase were mixed, and after 5 minutes, 0.05 ml was sampled. 0.025 ml of 20 mM acetate buffer (pH 5.5) and 0.075 ml of 0.5 M sodium carbonate were added and mixed, and the absorbance at 420 nm was measured (reaction for 5 minutes). Similarly, after 5 minutes, 0.2 ml was sampled, and a 20 mM acetate buffer solution (pH 5.
5) was added and the reaction was continued. After another 5, 15 minutes
0.1 ml was sampled, 0.1 ml of 0.5 M sodium carbonate was added and mixed, and the absorbance at 420 nm was measured (reaction for 10 and 20 minutes).

After the β-xylosidase alone reaction,
In the reaction for adding glucosidase, 0.15 ml was sampled 5 minutes after the β-xylosidase alone reaction, 0.075 ml of partially purified β-glucosidase was added, and the reaction was continued. Further, after 5 and 15 minutes, 0.1 ml of a sample was sampled, 0.1 ml of 0.5 M sodium carbonate was added and mixed, and the absorbance at 420 nm was measured (10, 20).
Minute reaction).

The same experiment was performed on the sample which had been heat-treated (100 ° C., 10 minutes) in advance for each reaction.
Blank values were used. Table 1 shows the results.

[0048]

[Table 1]

As is clear from the table, in the reaction of adding β-glucosidase after the reaction of β-xylosidase alone, the amount of released pNP was larger than that of each enzyme alone. From this, pNP-primeveroside is decomposed into pNP-glucoside and xylose by β-xylosidase, and β-glucosidase is used as a substrate of βN-glucosidase.
It was confirmed that P-glucoside was decomposed into pNP and glucose.

Example 4 Activity of partially purified β-xylosidase obtained in Example 1
It was prepared by diluting with 20 mM acetate buffer (pH 5.5) to 5.0 units / ml. Furthermore, the partially purified β-
Glucosidase was prepared by diluting it with a 20 mM acetate buffer (pH 5.5) to an activity of 5.0 units / ml. Using this enzyme solution, the activity ratio of β-xylosidase activity: β-glucosidase activity was 10: 0, 9: 1, 8: 2, 7:
3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8, 1:
A sample was prepared so as to be 9, 0:10.

0.05 ml of each sample and 20 mM acetate buffer (pH 5.5)
Was mixed with 0.05 ml of a pNP-primeveroside solution adjusted to 10 mM in the above, and incubated at 37 ° C. for 20 minutes. 0.1 ml of 0.5 M sodium carbonate was added and mixed, and the absorbance at 420 nm was measured. The same experiment was performed on a sample that had been heat-treated (100 ° C., 10 minutes) in advance, and a blank value was obtained. Table 2 shows the results.

[0052]

[Table 2]

As is clear from the table, the mixture ratio of β-xylosidase activity: β-glucosidase activity at which the amount of released pNP was highest was 5: 5, that is, 1: 1.

Example 5 Method for Preparing Eugenyl Primeveroside, a Precursor of Scent Components About 2 kg of fresh leaves of mountain tea was extracted with hot water at 100 ° C. for 10 minutes, and the extracted solution was Diaion HP20 (manufactured by Mitsubishi Chemical Corporation). ) Was applied to the column, and eugenyl primebeside was adsorbed. After washing the column with about twice the bed volume with deionized water and 20% methanol, eugenyl primeveroside adsorbed with 100% methanol was recovered. The recovered methanol solution containing eugenyl primeveroside was then concentrated to crystallize eugenyl primeveroside, and collected by a glass filter.

Example 6 The partially purified β-xylosidase and partially purified β-glucosidase obtained in Examples 1 and 2 were diluted with a 20 mM acetate buffer (pH 5.5) to give β-xylosidase activity: β-glucosidase. A sample having an activity = 1: 1 was prepared. That is, β-
Samples with xylosidase activity 10.0 units / ml and β-glucosidase activity 10.0 units / ml were prepared.

0.5 ml of the enzyme solution of this sample and 0.5 ml of eugenyl-primeveroside solution adjusted to 10 mM with 20 mM acetate buffer (pH 5.5) were mixed and incubated at 37 ° C. The samples after 6 and 24 hours were collected, respectively, and after stopping the reaction by heat treatment (100 ° C., 10 minutes), pentanol was added as an internal standard substance and subjected to gas chromatography (manufactured by Shimadzu Corporation). As a result, a peak was confirmed in the eugenol detection time for both 6 and 24 hours. It was also confirmed that the amount of eugenol released increased with the passage of time.

The enzyme solution was previously heat-treated (100 ° C., 10 ° C.
Eugenol was not detected in both cases for 6 and 24 hours.

An organoleptic test (10 panelists) revealed that scent peculiar to eugenol was observed for both 6 and 24 hours. It was confirmed that the fragrance peculiar to eugenol was enhanced over time. Heat treatment (100
℃, 10 minutes) did not feel the scent unique to eugenol

From the above, it was confirmed that aglycone release was also caused by the action of the enzyme composition containing β-xylosidase and β-glucosidase in the case of primeverose glycoside, which is a natural substrate.

Example 7 1 ml of the enzyme solution obtained in Example 6 and 1 ml of grape juice (commercially available product: 100% fruit juice, concentrated and reduced) were mixed and incubated at 37 ° C. overnight (14 hours) to give a fragrance. When I checked,
The aroma was clearly enhanced as compared with the case where acetate buffer was added instead of the enzyme preparation. Also, this action is performed by heating the enzyme solution to 100 ℃.
No heat treatment was found for 10 minutes.

Example 8 1 ml of the enzyme solution obtained in Example 6 and 1 ml of commercially available orange juice (reduced concentrated solution) were mixed, incubated at 37 ° C. for 24 hours, and the generation of aroma was examined by a sensory test. As a result, the effect of enhancing the aroma of orange juice was recognized. Heat treatment (100 ° C., 10 minutes) of the enzyme solution did not show such an effect.

[0062]

Industrial Applicability According to the present invention, an enzyme composition containing β-xylosidase and / or β-glucosidase cleaves β-primeveroside and / or a similar disaccharide glycoside at a monosaccharide unit to inhibit aglycone releasing action. It is clear that the enzyme composition can be used in the present invention, and the enzyme composition can be supplied from microorganisms as a source and can be widely used in various foods, pharmaceuticals, quasi-drugs, and the like. For example, it can enhance or attenuate the flavor, pigment, and physiologically active ingredient in foods and the like.

Claims (7)

[Claims] 1. A method of reacting an enzyme composition with a glycoside to release sugar from the glycoside in monosaccharide units. 2. The method according to claim 1, wherein the glycoside is a disaccharide glycoside. 3. The method according to claim 2, wherein the disaccharide glycoside is β-primeveroside and / or a similar disaccharide glycoside. 4. The method according to claim 1, wherein the enzyme composition can be obtained from a microorganism and / or a plant. 5. The method according to claim 1, wherein the enzyme composition contains β-xylosidase and / or β-glucosidase. 6. The method according to claim 1, wherein the disaccharide glycoside is a precursor of a fragrance component, a precursor of a dye component or a precursor of a physiologically active component. 7. The method according to claim 6, wherein the aroma component precursor, the pigment component precursor or the physiologically active component precursor is derived from a plant.