JPH08140675A - Enzyme forming tea flavor component and its production - Google Patents

Abstract

PURPOSE: To obtain a new enzyme which can efficiently produce tea flavors effective for improving the flavor of food products using tea as a raw material by acting on the precursors of tea flavor to generate the tea flavor, which is obtained by extracting raw tea leaves with a buffer solution under agitation. CONSTITUTION: This new enzyme named as β-primeverosidase producing tea flavor, acts on the extract containing tea flavor precursors extracted from raw tea leaves suspended and stirred in a buffer solution, where the enzyme acts on a tea flavor glycoside having primeverose (6-O-β-xylosylglucose) or glucose in its saccharide moiety to hydrolyze them to give primeverose or glucose. Further, it acts on p-nitrophenyl β-glucoside or p-nitrophenyl β-xyloside to liberate glucose or xylose, is stable by the treatment at pH4-7 at 37 deg.C for 1 hour, the optimal pH is 4-6, and stable at pH6 for 1 hour at a temp. lower than 45 deg.C, the optimal temperature is near 50 deg. and has 61,000 molecular weight according to SDS-PAGE.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to .beta.-primevelosidase, which is a tea aroma component-producing enzyme, and a method for producing the same. This enzyme acts on the tea aroma component precursor to produce a tea aroma component and primeverose.

[0002]

2. Description of the Related Art In foods, aroma is one of the very important factors together with taste and color. For example, if a food does not have a palatable scent, a person does not have an appetite and is sensitive to a scent different from the conventional one. However,
The fragrance, which plays an important role as a constituent of food, that is, the flavor component, is present in food in a very small amount, and is highly volatile and often unstable.

Tea drinks are preferred by people, various products are on the market, and they are one of the recent hit products. However, the tea flavor is volatilized or changed in the manufacturing process, and it is not always satisfactory from the viewpoint of flavor of the tea beverage. On the other hand, research on aroma components of tea has progressed rapidly recently, and linalool, geraniol, benzyl alcohol, methyl salicylate, 2-
It has been suggested that major tea flavors such as phenylethanol exist as their glycosides (precursors) in tea leaves [Phytochemistry, vol.20, p2145 (1981), Agri.
Biol. Chem., vol. 54, p1023 (1990)], and in Yabukita species, (z) -3-hexenol β-D-glucoside and benzyl alcohol β-D-glucoside were isolated [Agr.
ic. Biol. Chem., vol.55, p1205 (1991), Agric. Biol.
Chem., Vol.58, p592 (1994)], from oolong tea, geraniol, benzyl alcohol, linalool and 2-
Each β-primeveroside (6-O- of phenylethanol
β-xylosyl β-D-glucoside) has been isolated
[Phytochemistry, vol.33, p1373 (1993), Biotec. Bioc
hem., vol.58, p1532 (1994)].

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a tea aroma component-producing enzyme capable of producing a tea flavor from a nonvolatile tea aroma component precursor which can be sufficiently utilized in a tea beverage, and a method for producing the same. Is to provide.

[0005]

Means for Solving the Problems As a result of repeated studies on a method for producing a tea flavor in a well-balanced manner from a tea aroma component precursor, the present inventors have found that β-prime which is one of glycosidases present in raw tea leaves. We have found that berosidase efficiently produces a tea flavor, and completed the present invention.

[0006] That is, the present invention is characterized in that a tea aroma component-forming enzyme having the following physicochemical properties, β-primeverose, and fresh tea leaves are added with a buffer solution, stirred and extracted. The present invention relates to a method for producing sidase. (1) Action: A tea aroma component is produced by acting on the tea aroma component precursor. (2) Substrate specificity: Primeverose (6-O-
β-xylosyl glucose) or a tea aroma component glycoside having glucose is acted on and hydrolyzed to produce primeverose or glucose. Further, it acts on p-nitrophenyl β-glucoside and p-nitrophenyl β-xyloside to release glucose or xylose. (3) Optimum pH: pH 4 to 6 (4) pH stability: pH at 37 ° C for 1 hour treatment
Stable at 4 to 7 (5) Optimum temperature: around 50 ° C (6) Thermal stability: pH 6, 45 ° C in 1 hour treatment
Stable below (7) Molecular weight: 61,000 (SDS-polyacrylamide gel electrophoresis)

A method for producing the tea aroma component-producing enzyme, β-primeverosidase, of the present invention having the above physicochemical properties will be described. Regardless of the variety, the raw tea leaves may be any of Assam, Chinese, Japanese and the like. The tea leaves to be used may be new shoots, old leaves or a mixture thereof, but it is preferable to use the new shoots because of easy extraction of the present enzyme from the tea leaves.

The extraction of this enzyme from tea leaves is carried out by a general method for extracting plant enzymes (Ikuzo Uritani, Kensuke Shimura, Michinori Nakamura, Masaru Funatsu, Biochemistry Experimental Method 14 Secondary Metabolism Research Method for Higher Plants (1981). ) Academic Society Publishing Center; Takeichi Horio, Nihei Yamashita, Basic Experiments on Proteins and Enzymes (1981) Nankodo). For example, 1-2 with respect to the weight of raw tea leaves
To extract the enzyme by adding 0 times the amount of buffer solution and grinding the raw tea leaves at 0 to 4 ° C. in several steps using a stirring means such as a household mixer, homogenizer, blender, etc. You can In order to minimize the interference of polyphenols contained in a large amount in tea leaves, substances used for adsorbing and removing polyphenols during grinding of fresh tea leaves, such as insoluble polyvinylpyrrolidone (trade name:
Polycla AT, manufactured by Gokyo Sangyo Co., Ltd.) is preferably added in an amount equal to or more than the weight of tea leaves. Alternatively, the polyphenols in the tea leaves can be removed by grinding the raw tea leaves together with an organic solvent such as acetone or butanol at a low temperature of about 4 ° C to -20 ° C.

It is also possible to extract a target enzyme by adding a buffer solution to the tea leaf powder from which the polyphenols have been removed in this way and stirring the mixture at 0 to 4 ° C. for about 1 to 3 hours. Here, various buffers can be used, for example, citrate buffer, acetate buffer, tartrate buffer, succinate buffer, maleate buffer, phosphate buffer, imidazole-buffer.
Examples thereof include hydrochloric acid buffer solution, Tris-HCl buffer solution, borate buffer solution, and citric acid-phosphate buffer solution.

In order to remove the residue from the extract obtained by the above method, solid-liquid separation means such as filtration and centrifugation is applied to obtain a crude enzyme extract. A known separation / purification method can be applied to the purification of the present enzyme from the crude enzyme extract. For example, a crude enzyme protein can be obtained from a crude enzyme extract by an ammonium sulfate salting-out method, an organic solvent precipitation method and the like, and a purified enzyme can be obtained by appropriately combining this with various chromatographies such as ion exchange, gel filtration and affinity. it can.

[0011]

EXAMPLES The present invention will be described below with reference to examples.
The present invention is not limited to these. Example 1 To 1 kg of fresh tea leaves (Yabukita seeds) was added 4 liters (L) of acetone that had been cooled to -20 ° C in advance, and the mixture was ground with a homogenizer for 3 minutes. The residue was collected by filtration, washed three times with 1 L of acetone, and then dried in a vacuum desiccator to obtain 200 g of acetone powder. This acetone powder was added to 1 L of 0.1 M citrate buffer (pH 6.0).
Then, the enzyme was extracted by suspending the solution in 4 times and stirring at 4 ° C. for 3 hours. The extract was centrifuged to remove the residue, and then the same amount of acetone was added to the supernatant to precipitate the protein. Then, the precipitate is collected by centrifugation and the citrate buffer solution 30 described above is used.
After dissolution in 0 ml, salting out with ammonium sulfate was performed.

The precipitate deposited in the 40-80% saturated ammonium sulfate fraction was collected by centrifugation, and this precipitate was collected in a small amount of 20 mM.
After dissolving in a citrate buffer (pH 6.0), it was dialyzed against the same buffer at 4 ° C. overnight. 2 of the obtained crude enzyme solution
CM equilibrated with 0 mM citrate buffer (pH 6.0)
-It was developed on a Toyopearl 650M column and subjected to ion exchange chromatography. After washing the column with the above buffer, the enzyme was eluted with the same buffer containing 0-0.5 M sodium chloride.

The enzyme activity was examined by using p-nitrophenyl β-glucoside as a substrate and measuring the released p-nitrophenol by a spectrophotometer. The enzyme activity was defined as 1 unit of the amount of enzyme that liberates 1 μmole of p-nitrophenol per minute. FIG. 1 shows the elution pattern of the enzyme in ion exchange chromatography using CM-Toyopearl. As is clear from the figure, glycosidases I to III were obtained. These three fractions are reacted with a crude fraction containing a tea aroma component precursor at 37 ° C. for 90 minutes,
The tea fragrance composition produced was compared with the fragrance composition produced when acetone powder was used. As a result, the composition of aroma components produced by using glycosidase II was the most similar to that of the acetone powder. That is, the glycosidase II fraction contained in the tea aroma component-producing enzyme, β-
It was confirmed that prime berosidase was present.

Next, the glycosidase II fraction was concentrated by ultrafiltration, and the obtained concentrate was equilibrated with 20 mM citrate buffer (pH 6.0), and the cation exchange Mono-S was mixed.
It was developed on a column (5 × 50 mm) and eluted with the above-mentioned buffer solution to obtain a purified enzyme preparation of the tea aroma component-forming enzyme and β-primeverose. The enzyme preparation obtained by the above purification procedure was homogeneous by SDS-polyacrylamide gel electrophoresis. In addition, it was confirmed that this enzyme has the physicochemical properties described above.

[0015]

EFFECTS OF THE INVENTION The tea aroma component-producing enzyme, β-primeverose, of the present invention can be obtained from fresh tea leaves by a simple operation. Since this enzyme can efficiently produce a balanced tea flavor from a non-volatile tea flavor precursor,
It can be effectively used to improve the flavor of tea-based foods such as tea beverages.

[Brief description of drawings]

FIG. 1 is an elution pattern of an enzyme in ion exchange chromatography using CM-Toyopearl.

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Claims (2)

[Claims] 1. A β-primemoversidase, which is a tea aroma component-forming enzyme having the following physicochemical properties. (1) Action: A tea aroma component is produced by acting on the tea aroma component precursor. (2) Substrate specificity: Primeverose (6-O-
β-xylosyl glucose) or a tea aroma component glycoside having glucose is acted on and hydrolyzed to produce primeverose or glucose. Further, it acts on p-nitrophenyl β-glucoside and p-nitrophenyl β-xyloside to release glucose or xylose. (3) Optimum pH: pH 4 to 6 (4) pH stability: pH at 37 ° C for 1 hour treatment
Stable at 4 to 7 (5) Optimum temperature: around 50 ° C (6) Thermal stability: pH 6, 45 ° C in 1 hour treatment
Stable below (7) Molecular weight: 61,000 (SDS-polyacrylamide gel electrophoresis) 2. The method for producing β-primeverosidase according to claim 1, wherein a buffer solution is added to fresh tea leaves, and the mixture is stirred and extracted.