JP2002119209A - Green tea beverage and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a green tea beverage without losing flavor and taste essential to a green tea extract liquid, without turbidity or precipitates over a long period and without requiring complicated processes, especially a green tea beverage filled in a transparent hermetically sealed container. SOLUTION: This method for producing the green tea beverage with suppressed turbidity or formation of precipitates is characterized in that the green tea extract liquid is treated with β-mannanase (preferably the β-mannanase derived from Penicillium multicolor) or an enzyme preparation mainly comprising the β-mannanase. The use of the β-mannanase or the enzyme preparation mainly comprising the β-mannanase for producing the green tea beverage. The green tea beverage obtained by the method for production or use.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the production of a green tea beverage in which turbidity or precipitation is suppressed, and more particularly to treating a green tea extract with β-mannanase or an enzyme agent containing β-mannanase as a main component. By doing
A new height that can suppress the generation of secondary precipitates such as floc (flock) caused by hemicellulose and the like during storage and can further improve the original flavor of green tea beverages compared to conventional products. The present invention relates to a method for producing a quality green tea beverage.

[0002]

2. Description of the Related Art In recent years, the market for green tea beverages in sealed containers, which are filled in sealed containers such as cans and plastic bottles and distributed and sold for a long period of time, has been expanding. However, tea beverages, particularly green tea beverages, may cause turbidity or precipitation during long-term storage after production. Secondary precipitation, such as floc (flock), caused by hemicellulose, etc., has become a major factor, particularly in the case of beverage products packed in transparent containers, which impairs the appearance and significantly lowers the commercial value. It has been reported that the turbidity or precipitation of the green tea extract is caused by polymerization of polysaccharides into complexes of catechins (for example, catechin gallate) and caffeine to form turbid particles.
It is also said that components such as caffeine, protein, pectin, polysaccharides and calcium ions are involved in a complex manner.

[0003] The mechanism of precipitation formation is complicated, and since it is not unique, there is no shortage of various techniques for suppressing precipitation. Examples of the prior art include a method of physically removing a precipitation-causing substance to prevent precipitation, a method of adding a stabilizer to prevent coagulation and precipitation, and a method of enzymatically decomposing a precipitation-causing polysaccharide to prevent precipitation. For example, the following method is known.

Japanese Patent Application Laid-Open No. 4-45744 discloses a water-soluble green tea component obtained by extracting green tea or fresh or dried tea leaves.
Organic materials and inorganic materials are separated by ultrafiltration using an ultrafiltration membrane, and high molecular components with a molecular weight of about 10,000 or more are removed to remove high molecular compounds soluble in green tea beverages. It describes that white filamentous and flocculent solids in green tea beverages are prevented from crystallizing.

Japanese Patent Application Laid-Open No. 4-31348 discloses that green tea is extracted with warm water, preferably about 1 to about 100 ° C.
After extraction for 10 to 10 minutes, the tea husk is removed with a metal net, cloth, or the like, and the pH of the extract is adjusted to about 4.0 to 5.0 in an acidic range using L-ascorbic acid. By quenching below room temperature, preferably below 20 ° C,
After promoting the formation of turbidity and ori, and taking various polymer compounds into the turbidity and ori to form particles, the turbidity and ori components containing the particles are removed by centrifugation or any other means, and then Then, a filter aid is added to the supernatant, and the remaining polymer is removed by filtration with a filter cake, and then the pH of the extract is adjusted to a neutral range of about 5.5 to 7.0. ,
It describes a method for producing a green tea beverage which is packed in a plastic container or the like, preferably a transparent container, sterilized by an ordinary method, and free from turbidity or generation of sediment over time.

[0006] JP-A-6-269246 discloses that tea is extracted with warm water, the resulting extract is cooled, tannic acid is added and the mixture is allowed to stand, and then fine tea particles and the like are centrifuged. There is described a method for producing a tea beverage having a long-term storage property, which comprises removing a turbid substance and clarifying the extract by diatomaceous earth filtration.

[0007] JP-A-2-100632 describes a method of adding a water-soluble flavonoid or a glycoside of a flavonoid to a green tea extract. Although there is no decrease in taste components, this method is effective in preventing tannin, caffeine, proteins and the like from being aggregated and precipitated, but is not effective in preventing precipitation due to polysaccharides such as hemicellulose. .

Japanese Patent Application Laid-Open No. 8-228684 discloses that a hot water extract of green tea is clarified and then treated with an enzyme having a hemicellulase activity containing xylanase (xylase) in the presence of ascorbic acid or a salt thereof. Discloses a method for producing a green tea beverage in which heat treatment is further performed to prevent the occurrence of floc. However, when a large amount of an enzyme agent is required to eliminate the precipitation of the tea leaf-derived polysaccharide, there is a problem that protein precipitation of the enzyme is caused. In addition, there is also a problem that the "smell odor" derived from the enzyme agent is imparted, and the taste is remarkably reduced.

As described above, various methods have conventionally been proposed for preventing secondary precipitation of green tea beverages. However, in the method such as filtration at the molecular level as described above, since the precipitation component is removed, the occurrence of secondary precipitation can be suppressed, but at the same time, the taste component is also reduced, and thus the body taste lacks,
There is a problem that the product becomes watery, taste is reduced, and the product becomes inferior in flavor. In addition, in methods such as the addition of a stabilizer and an enzyme agent, the suppression of secondary precipitation is not perfect, and there is a problem that the flavor is affected when the amount added is increased in order to obtain an effect. Therefore, with regard to green tea beverages as favorite beverages, there is a strong demand for the development of a new production method that does not have the above-mentioned disadvantages and can easily produce high-quality products having excellent flavor and taste. Was in

[0010]

According to the above-mentioned prior art relating to suppression of turbidity or sedimentation of green tea beverages, the operation of removing turbidity or sediment by physical filtration also removes some of the flavor and taste components in the tea extract. There is a problem. Further, the addition of a water-soluble flavonoid was not completely effective, and the addition of a xylanase-containing hemicellulase was not completely effective due to formation of a protein-derived precipitate. In particular, in high-grade tea leaves, since a large amount of polysaccharides cause precipitation, a large amount of enzymes are required, and the above-mentioned protein-derived precipitates and undesired "earthy odor" are generated. Therefore, formulate a green tea beverage using high-grade tea leaves. I couldn't do that. An object of the present invention is to provide a green tea beverage without turbidity or sediment, particularly a green tea beverage in a transparent closed container, for a long period of time without requiring complicated steps and without losing the original flavor and taste of the green tea extract. Is to do.

Further, the present invention can effectively suppress the occurrence of secondary precipitation which has been inevitably observed during storage, even if the green tea beverage is stored for a long period of time. It is an object of the present invention to provide a method for producing a high-quality green tea beverage in which the tea feeling (green tea original flavor and taste) of the green tea beverage is further improved as compared with conventional products, and a product thereof.

[0012]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and surprisingly, β-mannanase (Japanese Patent No. 3043560, Japanese Patent Application No. 11-330558),
It has been found that green tea also exhibits a precipitation preventing effect.
More specifically, it has been found that β-mannanase is about 5 to 10 times more effective than xylanase, which has been conventionally said to have a precipitation inhibiting effect. As a result, β-
In the case of an enzyme preparation containing mannanase as a main component, the threshold addition amount can be small, so that an unpleasant taste such as "earthy smell" derived from the enzyme does not occur. Also, even when using advanced tea leaves,
It has been found that secondary precipitation (cotton-like precipitation) is completely suppressed without producing protein-derived precipitates. The present invention has been completed based on the above findings. INDUSTRIAL APPLICABILITY As described above, the present invention relates to the production of a high-quality green tea beverage having good flavor and taste without secondary precipitation. That is, the present invention is a method for producing a green tea beverage in which turbidity or precipitation is suppressed, wherein the green tea extract is treated with β-mannanase or an enzyme agent containing β-mannanase as a main component. The present invention also relates to the use of β-mannanase or an enzyme preparation containing β-mannanase activity as a main component for the production of green tea beverages, particularly for the suppression of turbidity or precipitation. Accordingly, the present invention also relates to a green tea beverage obtained by the above-mentioned production method or use.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. As described above, the method for producing a green tea beverage according to the present invention is characterized in that the green tea extract is treated with β-mannanase or an enzyme agent containing β-mannanase as a main component. In a basic preferred embodiment of the present invention, the hot water extract of green tea is clarified by ordinary centrifugation or filtration, and an antioxidant (for example, L-ascorbic acid or a salt thereof) is added to the extract,
If necessary, the pH is adjusted at least once in an optional step with a pH adjuster (for example, sodium bicarbonate (sodium bicarbonate) or the like), and the mixture is treated with an enzyme agent containing β-mannanase as a main component. This is a method for producing a green tea beverage that does not cause secondary precipitation (cotton-like precipitation). However, the filtration treatment shown here does not indicate filtration at a molecular level as described later, but refers to a filtration treatment capable of avoiding the removal of the taste component.

In the present invention, green tea is the first step in the production of tea leaves or tea buds as a raw material, by steaming or roasting the tea leaves or tea buds in a kettle to stop the enzymatic activity in the tea leaves or tea buds and to ferment them. It refers to unfermented tea made without making it, and does not include fermented tea, black tea, or semi-fermented tea, oolong tea. Further, as the type of green tea in the present invention, sencha,
Gyokuro, matcha, bancha, roasted green tea, steamed green tea, kamasen green tea and the like can be specifically exemplified. In the present invention, green tea is sometimes referred to as green tea leaf.

The hot water extract of green tea used in the present invention is prepared by extracting the above green tea leaves with an aqueous medium (usually hot water) at an appropriate temperature by a conventional method (for example, see Example 1) and centrifuging. What separated tea shell etc. by means may be sufficient. At this time, the type of tea leaves, the concentration of the extract, and the like can be used regardless of the concentration. However, it is usual to obtain a concentrated extract with respect to the concentration.

The conditions for the extraction of the concentrated green tea extract are, for example, 20 to 50 times, preferably 3 times the amount of tea leaves.
An aqueous medium having a weight of 0 to 40 times, particularly an aqueous medium of 60 to 70 ° C., may be subjected to extraction treatment with stirring for about 3 to 10 minutes, preferably once to several times. As a method for removing solids and insoluble components after the extraction treatment, after the completion of the above-described extraction treatment, the tea leaves are filtered off by net filtration, and then 10 to 20 times.
For example, a method of cooling to about ° C. and removing insoluble components by centrifugation at 3000 rpm for about 10 minutes can be exemplified. However, the centrifugation and filtration of the extract do not mean separation at the molecular level as in the ultrafiltration described above, but mean removal of insoluble components without removing taste components.

To the clarified liquid obtained by centrifugation or filtration as described above, for example, 0.1 to 3% by weight of ascorbic acid is added as an antioxidant to tea leaves. Generally, in the production of green tea beverages, L-ascorbic acid and sodium ascorbate are added to prevent oxidation, and in the present invention, the addition of ascorbic acid is
For example, L-ascorbic acid, sodium ascorbate, or those having the same effect as the above may be used in accordance with a conventional method.

In general, green tea beverages are produced by contacting green tea with an aqueous medium such as hot water, subjecting the tea leaves to an extraction treatment and then removing the green tea extract from which solids and insoluble components have been removed into a green tea beverage as it is, A method is known in which a green tea extract obtained in a concentrated state is diluted with a preparation water such as ion-exchanged water to obtain a green tea beverage.
From the viewpoint of easy quality control and the like, a method of diluting a concentrated green tea extract into a green tea beverage has been adopted. The green tea beverage in which the formation of precipitates is suppressed according to the present invention is characterized in that it is treated with β-mannanase or an enzyme agent containing β-mannanase as a main component as described above. The blending of β-mannanase or an enzyme agent containing β-mannanase as a main component may be an addition blending to an aqueous medium for green tea extract,
It is preferable to add and mix the green tea extract. In the case of addition and mixing to such green tea extract, β-mannanase or an enzyme agent containing β-mannanase as a main component, by directly adding and dissolving to the green tea extract, or by adding and dissolving to the preparation water. It may be added to the green tea extract.

In the present invention, β-mannanase refers to β-mannanase.
-1,4-D-mannopyranoside bond-specifically acts on a mannan, galactomannan, glucomannan and non-specifically hydrolyzes a β-1,4-D-mannopyranoside bond to produce mannose and mannooligo. It means the enzyme β-mannanase that produces sugar. This β-mannanase does not act on arabinoxylan, xylan, tragacanth gum, cellulose or carboxymethylcellulose.

In the present invention, the term "enzyme containing β-mannanase as a main component" means that the β-mannanase activity per gram of the enzyme is usually 5,000 U or more, and preferably 1 000 U or more.
It means an enzyme agent that contains not less than 000 U and has an activity ratio (β-mannanase / xylanase) of usually 5 or more, preferably 20 or more, to xylanase contained as a contaminating enzyme. The method for measuring the activity of β-mannanase and xylanase and the definition of the activity are as described in detail in Examples below. As the β-mannanase or the enzymatic agent containing the same as a main component in the present invention, any one can be used as long as it has the above-mentioned properties, but as described above, the β-mannanase / xylanase activity ratio is Those having 5 or more or 20 or more are preferable. Specifically, for example, a penicillium multicolor mch13-2 (FERM BP-) of an application already filed by the present inventors (Japanese Patent Application No. 11-330558).
6831) -derived β-mannanase, β-mannanase derived from cellulosin GM5 (manufactured by Hankyu Bioindustry),
Β-mannanase derived from hemicellulase GM “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.) and the like (see Examples below).
Among them, the β-mannanase preparation derived from penicillium multicolor mch13-2 (FERM BP-6831) of the above-mentioned application by the present inventors has a β-mannanase / xylanase activity ratio of 806 and is the most crude enzyme preparation. It can be used as suitable.

Β-mannanase is by definition a member of the hemicellulase enzyme family. This does not refer to hemicellulase as an enzyme that acts on a single substrate, but generally refers to a group of enzymes that act on a wide variety of polysaccharides extracted from plant tissues with alkali. Because. In a strict definition, the term refers to a group of enzymes that act on polysaccharides (arabinoxylan, xylan, xyloglucan, glucomannan, arabinan, β-glucan) capable of firmly hydrogen bonding to insoluble cellulose (Japanese Patent Application Laid-Open No. Hei 8 (1996)). -228684). However, the definition of hemicellulase is too broad, and the present inventors have examined the green tea precipitation inhibitory effect of a commercially available hemicellulase preparation, but not all hemicellulase preparations have a green tea precipitation inhibitory effect. . Regarding the ineffective enzyme preparation, even when the added concentration was increased, the formation of the precipitate could not be suppressed, and on the contrary, the precipitate derived from the protein was formed. Although not belonging to hemicellulase, the effect of the present invention could not be obtained only with an enzyme that decomposes plant-derived polysaccharide, a pectinase preparation or a cellulase preparation.

Specifically, as shown in Example 2, the effect of various hemicellulases on inhibiting green tea precipitation was examined.
Enzymes examined as hemicellulases include β-mannanase, β-xylanase (xylanase), endo-arabinanase, α-L-arabinofuranosidase, endo β-1,4-galactanase, exo-β-glucanase, endo- β-glucanase. Among these, the effect of suppressing precipitation was confirmed and the highest was β-mannanase. Xylanase was also confirmed to have a green tea precipitation inhibitory effect, but to a lesser extent than β-mannanase.

Then, the purified β-mannanase was compared with the purified xylanase, and the threshold addition amount of green tea precipitation inhibition was compared. Hemicellulase is a general term for enzymes that act on a wide variety of polysaccharides, and there is no uniform value indicating the activity. Therefore, when comparing both enzymes, the threshold addition concentration was compared in terms of protein mass. As a result, at the used tea leaves and tea leaf concentrations, xylanase was found to inhibit green tea floc.
12 mg / ml (final concentration) was required, whereas β-
Mannanase required 0.013-0.022 mg / ml (final concentration) (when stored at 40 ° C. for two weeks). Therefore, when compared with the amount of protein, if the precipitation-inhibiting enzyme is xylanase, about 5 to 10 times that of β-mannanase is required. That is, among the hemicellulase enzymes, β-mannanase was found to be the most effective in inhibiting green tea precipitation.

Therefore, when comparing the commercially available hemicellulase enzyme preparation, the higher the ratio of β-mannanase activity, the higher the green tea precipitation inhibitory effect. Therefore, in the enzyme preparation having a high ratio, the amount of addition required for suppressing green tea precipitation can be reduced. That is, in the enzyme preparation of the actual embodiment, it has been confirmed in Example 12 described later that the enzyme preparation having a higher β-mannanase activity ratio requires a smaller amount of added protein. For example, an application filed by the present inventors (Japanese Patent Application No. 11-330558)
Multicolor mch13-2 strain (FE
It was found that β-mannanase derived from RM BP-6831) had the highest ratio of β-mannanase activity among the tested hemicellulase enzyme agents, and therefore had the highest effect of inhibiting green tea precipitation. Further, it has been found that the β-mannanase contained in the present β-mannanase preparation has a high specific activity even when compared with other β-mannanase. Therefore, the present β-mannanase preparation, which is most effective for inhibiting green tea precipitation and has a β-mannanase activity of high specific activity as a main component, requires a smaller amount of green tea precipitation to be added than other β-mannanase preparations. It turned out to be enough.

In general, it is known that green tea floc is more likely to be generated as the tea leaves become more advanced. This is because the higher the tea leaves, the higher the polysaccharide content in the green tea extract. Therefore, in the case of high-grade tea leaves, the amount of enzyme agent required for suppressing precipitation is inevitably increased. However, if the amount of the enzyme agent is increased, the formation of a protein-derived precipitate is promoted, and the enzyme agent cannot play its original role. However, β-mannanase has an effect of inhibiting green tea precipitation about 5 to 10 times higher than that of xylanase.
-By treating with a mannanase preparation, it is possible to suppress secondary precipitation (cotton-like precipitation) without generating protein-derived precipitates even when a threshold enzyme amount for high-grade tea leaves is added. In particular, an application filed by the present inventors (Japanese Patent Application No. 11-33055).
The β-mannanase of No. 8) is more advantageous because it has a high specific activity and requires only a small amount of protein. This makes it possible to formulate a green tea beverage in a PET bottle using high-grade tea leaves, which was conventionally considered to be difficult to achieve due to the occurrence of flocculent sedimentation, and is extremely useful industrially. It can be said.

In recent years, it has been said that secondary precipitation of green tea is mainly caused by water-soluble polysaccharides having a molecular weight of 20,000 or more. The green tea water-soluble polysaccharide has a molecular weight of 100,000 or more and an average molecular weight of 1
It has been reported that 10,000 high molecular polysaccharides exist. [Tadakazu Takeo, "Journal of Japan Society for Food Science and Technology", Vol.4
5, No. 4, p270-272 (1998)]. The following hypothesis is considered as a mechanism by which β-mannanase suppresses secondary precipitation dominantly.・ Green tea precipitated polysaccharide contains a single polysaccharide, β
-Both mannanase and xylanase can degrade green tea polysaccharide, but β-mannanase shows higher degradability.・ The green tea precipitated polysaccharide contains multiple types of polysaccharides,
Polysaccharides degradable by β-mannanase are quantitatively higher.
The amount of polysaccharide degradable by xylanase is low in quantity.・ The green tea precipitated polysaccharide contains multiple types of polysaccharides,
Certain polysaccharides have the property of being easily degraded by β-mannanase. On the other hand, another polysaccharide has the property of being hardly decomposed by xylanase. -Polysaccharides that can be degraded by β-mannanase can suppress precipitation only by decomposing a part of them, whereas polysaccharides that can be degraded by xylanase cannot suppress precipitation unless all are degraded. Required.

In the present invention, the usual treatment conditions with β-mannanase are as follows. The optimal pH of β-mannanase is generally between 4.5 and 6.0. However, even when the enzyme treatment is not performed at the optimum pH, the pH is 5.0 to 6.5.
Within the range, the effect is the same. In the production of green tea beverages, usually ascorbic acid or a salt thereof is added as an antioxidant, and then, during enzymatic treatment, pH is appropriately adjusted by adding usually sodium bicarbonate, disodium hydrogen phosphate or the like as a pH adjuster. Adjustment is easy.
As described above, the pH adjustment of the green tea extract can be performed by at least one or more additions of the pH adjuster in any step (enzyme agent treatment, antioxidant addition, centrifugation, etc.) in the above production method. preferable.

Next, the suitable temperature range for the action of the β-mannanase preparation is 20 to 70 ° C. β-mannanase treatment
The enzyme agent can be added to the aqueous medium for green tea extraction to perform green tea extraction at a high temperature of 60 to 70 ° C simultaneously with the green tea extraction. However, β-mannanase treatment can also be performed at 20 to 40 ° C with a green tea extract. Alternatively, it is also possible to perform both.
In any case, if the time exceeding 40 ° C. is long, the deterioration of the green tea extract may be observed, which is not preferable. In any case, if the temperature is too low, the enzyme treatment time becomes longer, and the green tea is rather deteriorated. It is not preferable because the extract may be deteriorated. The time of the enzyme treatment is generally required to be 30 minutes or more, but may be appropriately set depending on the amount of the enzyme to be used, the type of tea leaves, the concentration of tea leaves, and the like. However, in order to prevent the extract from deteriorating, it is necessary to make appropriate adjustments so that the treatment can be performed in a short time as possible.

The amount of the enzyme to be added is usually 0.05 U to 10 in terms of β-mannanase activity per ml of green tea extract.
0 U, preferably 0.8 U to 41 U (when stored at 40 ° C. for one month). However, the amount of addition varies depending on the type of tea leaves to be used, the concentration of the green tea extract, the type and properties of the enzyme to be used, various conditions, and the like.

The green tea beverage obtained by the above production method has a remarkable effect of further improving the flavor and taste as compared with conventional green tea beverage products. This is an enzymatic treatment with an enzyme having β-mannanase activity as described above, in which polysaccharides contained in the tea extract are decomposed, and a wide variety of saccharides are decomposed into small molecules, and these saccharides are
It is based on the action of further improving the flavor and taste of the green tea beverage as a moderate flavor component or taste component. In addition, since a small amount of the enzyme is required, there is an advantage that there is no unpleasant taste derived from the enzyme agent.

After the above enzyme treatment, the enzyme reaction is usually stopped simultaneously with the heat sterilization treatment. For these processes, for example, Embodiment 15 and the like can be referred to. Further, as described above, the pH can be easily adjusted with an edible alkali such as sodium hydrogen carbonate at least once before and / or after any of the processing steps in the present invention. PH in the method of the present invention
The pH of the adjustment is preferably adjusted to 6.0 to 6.5 in the beverage after sterilization in order to maintain the flavor and improve the quality.

A typical green tea beverage of the present invention is prepared by diluting a concentrated green tea extract with a compounding water such as ion-exchanged water, and then adding ascorbic acid or a salt thereof, and adjusting the pH with sodium bicarbonate or the like. And β-mannanase or β
After being treated with an enzyme agent containing mannanase as a main component, it is filled in a sealed container. The sealed container is not particularly limited, and examples thereof include a poly container, a can, a paper container, and a glass bottle. Filling and sterilization may be performed by a usual processing method, and are not particularly limited. For example, in the case of cans, reheating, filling, and high-pressure sterilization (retort sterilization) are performed, and in the case of PET bottles, the instant sterilization method (13) is used.
0 to 135 ° C. for about 30 seconds) to produce a green tea beverage product in a sealed container by a conventional method (for example, see Example 15). Since the green tea beverage of the present invention produced in this manner suppresses the formation of precipitates, it is advantageous to be able to produce a green tea beverage in a sealed container, particularly a green tea beverage in a transparent sealed container such as a PET bottle.

[0033]

EXAMPLES Next, the present invention will be described specifically with reference to examples, but the present invention is not limited to the examples. Example 1 Method for Evaluation of Green Tea Precipitation Inhibition Green tea precipitation inhibition evaluation for obtaining an enzyme threshold addition amount was performed by the following method. 10 g of Shibuya-produced "Yabukita-seed" green tea leaves of the highest grade (referred to as tea leaves 1) were added to 350 g of ion-exchanged water at 65 ° C., and the mixture was allowed to stand for 5 minutes and extracted (immediately after tea leaves were introduced, 2.5 minutes). After stirring for 10 seconds each). After roughly filtering tea leaves using a 30-mesh and a 120-mesh stainless steel filter, the extract solution cooled to about 20 ° C. is centrifuged (3000 rpm) for 10 minutes, and the extract solution is further filtered using a 200-mesh stainless filter. Then, a clear liquid was obtained. To stabilize the quality, dilute 3.7-fold with ion-exchanged water, add sodium ascorbate to a final concentration of 0.04%, and then sterilize sodium bicarbonate to pH 6.25. And adjusted. Next, 10 ml was dispensed into a heat-resistant test tube with a cap having a size of 10 ml. Then, each enzyme agent was added at each concentration, and the enzyme treatment was performed at 30 ° C. (water bath) for 30 minutes. Next, sterilization treatment was performed at 121 ° C. for 10 minutes.
Stored at 40 ° C.

Example 2 : Green tea precipitation of various hemicellulases
Evaluation of the inhibitory effect on green tea The inhibitory effect on green tea precipitation of various commercially available purified hemicellulase enzyme agents and crude enzyme agents other than hemicellulase shown in Table 1 below was examined (preserved at 40 ° C. for one week). The evaluation of green tea precipitation inhibition was performed according to the method described in Example 1. Table 1 Types of enzyme preparations Enzyme activity Precipitation (U / ml tea liquor) Evaluation ^{(Note 2)} -Various purified hemicellulases ^{(Note 1)} -Endo-arabinanases 0.0812 5 0.406 5 2.035 α-L-arabinofuranosidase 0.290 5 0.145 5 0.725 5 Endo-1,4-β-galactanase 0.042 5 0.211 5 1.05 5 Endo-1,3-β-glucanase 0.0097 5 0.0485 5 0.243 5 Exo-1,3-β-glucanase 0.0506 5 0.253 5 1.265 5 Lichenase 0.211 5 1.05 5 5.27 5 Cellobiohydrolase 0.0213 5 0.107 5 0.533 5 β-xylanase 0.481 5 2.41 5 12.0 1 β-mannanase 0.0792 5 0.535 5 3.21 0-Crude enzyme preparation other than hemicellulase ^{(Note 3)} - all Celluclast 1.5 L 0.035 5 0.35 5 3.5 5 cellulase a "Amano" 3 1.50 5 15.0 5 150 5 pectinase GL Amano 0.045 5 0.45 5 4.5 5 (1) commercially available purified enzyme agent was used Megazyme Corporation. (Note 2) The degree of floc generation of the enzyme concentration is indicated by a numeral (0; no flocculent sedimentation, 1 to 5; the higher the value, the more flocculent sediment). (Note 3) Cell crust 1.5 L and cellulase A “Amano” 3 are indicated by CMCase activity, and pectinase GL amano are indicated by endo-polygalacturonase activity. From this result, it was found that among various hemicellulases, β-xylanase and β-mannanase have an effect of suppressing green tea precipitation. However, the effect of β-xylanase is
It was weaker than β-mannanase. Although it does not belong to hemicellulase, an enzyme that decomposes plant-derived polysaccharide, for example, a cellulase preparation or a pectinase preparation, did not have the effect of suppressing green tea precipitation.

Example 3 Penicillium Multicolor
β-mannanase of mch13-2 strain β-mannanase -producing bacterium, penicillium multicolor The mch13-2 strain was isolated from soil in Gunma Prefecture. 1
10 g / l locust bean gum dispensed into a 5 ml test tube, 10 g / l bactopeptone (Difco), 1 g / l yeast extract (Difco), 2 g / l phosphoric acid The cells were inoculated into a liquid medium consisting of potassium dihydrogen and 0.5 g / l of magnesium sulfate heptahydrate, and shake-cultured. Β-mannanase activity was measured, and mch13-2 was identified as the most active strain. Got the strain. This strain is Penicillium multicolor
The strain was named ch13-2 strain and deposited at the National Institute of Advanced Industrial Science and Technology, Ministry of International Trade and Industry (accession number) FERM BP-6831. The activity of β-mannanase was measured by the following method. 3 ml of a 10 g / liter locust bean gum solution and 150 mM sodium acetate buffer (p
H5.0) 1 ml of the test solution was added to 2 ml, and
Minutes. The reducing sugar generated in the reaction solution was quantified by the Somogyi-Nelson method. β-mannanase activity in test solution 1
Enzyme activity in which ml produces a reducing power corresponding to 1 μmole of mannose per minute is shown as 1 Unit (U). In addition, locust bean gum (No.G-075 manufactured by Sigma)
3) is dissolved at 100 ° C. with stirring for 3 minutes,
The supernatant centrifuged at 500 rpm for 10 minutes was used.

Example 4 : Penicillium multicolor
Purification of β-mannanase derived from mch13-2 30 g / liter copra meal (Fuji Oil Co., Ltd.), 2
g / l potassium dihydrogen phosphate, and 0.5 g
1.5 liter of a medium containing 1 / liter of magnesium sulfate heptahydrate was placed in a 2.5 L mini-jar fermenter and sterilized at 121 ° C. for 40 minutes. Penicillium
The multicolor mch13-2 strain was inoculated into the mini-jar fermenter prepared as described above,
The cells were cultured at 500 rpm and 0.5 vvm for 7 days. The culture solution was subjected to filtration and separation of the cells using a Nutsche, and about 1.
3 L were obtained. All subsequent operations were performed at 4 ° C.
The β-mannanase activity was measured by the method described in Example 3. The culture supernatant obtained as described above was concentrated with an ultrafiltration membrane (molecular weight cut: 13000), and then hydrolyzed to desalinate UF to obtain a 52 ml concentrated liquid.
The solution obtained by centrifugation as above has a final concentration of 50
Tris / hydrochloric acid buffer solution (pH 8.0) was added so as to be mM. The half volume was divided into two portions and ion exchange chromatography (column: TOSO) equilibrated with the same buffer
H TSK-gel DEAE-TOYOPEARL 650S 120 ml). Wash the column with the same buffer,
Β-mannanase was eluted with a linear gradient of 0 to 0.3 M saline. The active fraction is passed through an ultrafiltration membrane (molecular weight cut 1000
0) and concentrated to a final concentration of 50 mM Tris-HCl (pH
8.0) and 1.5 M ammonium sulfate to give a 10 ml solution. This solution was subjected to hydrophobic chromatography equilibrated with the same buffer (column: TOSOH TS
K-gel Phenyl-TOYOPEARL 650S (120 ml).
The column is washed with the same buffer, then 300 ml of 1.5
Β-mannanase was eluted with a linear gradient of M to 0 M ammonium sulfate. Ultrafiltration membrane (molecular weight cut 10,000) for active fraction
And then 10 mM Tris-HCl, 0.15
UF washing with M NaCl buffer (pH 7.5), desalting and concentration were performed to obtain a 2 ml solution. Next, gel filtration chromatography (column: Phar
macia HiLoad 16/60 Superdex 200pg)
Β-mannanase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10,000),
ml solution was obtained. Finally, a purified enzyme showing a single band was obtained by SDS polyacrylamide gel electrophoresis and isoelectric focusing. The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 2 below. The protein amount was measured by the Foreign-Lowry method using BSA (bovine serum albumin) as a standard substance. Table 2 Purified fraction Total enzyme activity Total protein Specific activity Recovery Purity (U / ml) (mg) (U / mg) (%) (fold) Culture supernatant 197600 1815 109 100 1 DEAE Toyopearl 30120 164 184 15.2 1.69 Phenyl Toyopearl 16018 72 222 8.1 2.03 Gel filtration 6354 27 235 3.2 2.16

Example 5 : Penicillium multicolor
Properties of β-mannanase derived from mch13-2 The enzymatic properties of the purified enzyme obtained in Example 4 were measured. The enzyme activity was measured by the method described in Example 3 above. (1) Action The enzyme of the present invention was added to a 0.2% solution of locust bean gum having β-1,4-D-mannopyranoside bond in an amount of 2.0%.
The solution was added to a concentration of U / ml, a sample was taken with time, and the reaction product was analyzed by liquid high-performance chromatography (Column Aminex 42A, Bio-rad). As a result, the high-molecular locust bean gum disappeared 60 minutes after the start of the reaction, and mainly enzymatic degradation products of medium to low molecular weight were observed, but after 5 hours from the start of the reaction, they were further reduced in molecular weight. . In the early stage of the reaction, the macromolecule disappeared promptly, and the enzyme degradation product of medium to low molecular weight was observed. Thus, the enzyme acts non-specifically in an endo-type on the β-1,4-D-mannopyranoside bond, It is considered that mannooligosaccharide and mannose were produced. (2) Substrate specificity The present enzyme 2.0 was added to each 0.2% solution of locust bean gum (galactomannan), glucomannan (derived from konjac), arabinoxylan (derived from barley), xylan, tragacanth gum, cellulose and carboxymethylcellulose.
The mixture was mixed to U / ml and reacted at 45 ° C. for 66 hours. Thereafter, after stopping the reaction at 100 ° C., the reaction product was subjected to liquid high performance chromatography (column Bio-ra).
The analysis was performed using d company Aminex 42A). as a result,
Low molecular weight was observed in locust bean gum and glucomannan, but no change was observed in other arabinoxylan, xylan, tragacanth gum, cellulose and carboxymethylcellulose. (3) Molecular weight The molecular weight was measured by SDS polyacrylamide gel electrophoresis. 200,000, 11 as marker proteins
6,300, 97,400, 66,300, 55,400, 36,500, 31,000, 21,
500 and 14,400 were used. As a result, this enzyme was considered to have a sugar chain, and the band of SDS polyacrylamide gel electrophoresis was smeared. The molecular weight range is 42,0
The molecular weight of the main band is 4 to 45,000.
It was 3,000. (4) Isoelectric point A solution was prepared by adding one pharmalite pH 4.5 to 5.4 (Pharmacia) to 3 pharmalite pH 2.5 to 5.0 (Pharmacia). further,
Isoelectric focusing was performed using an agarose gel swollen with a solution obtained by adding 15 parts of pure water to the solution 1. As a result, the isoelectric point was 3.2. (5) Stability The enzyme was stable (100% residual activity) at 50 ° C. for 1 hour, and 70% residual activity at 60 ° C. for 1 hour. In addition, the treatment was stable at 20 ° C. at a pH of 3.0 to 10.0 for 20 hours. (6) Reactivity The enzyme has a reaction optimum temperature around 70 ° C. and a reaction optimum pH around pH 5.5. (7) Influence of various activators and inhibitors In the method for measuring β-mannanase activity in Example 3, the substances shown in Table 3 below were added together with the substrate, and the activity in each case was measured to determine the activation or The presence or absence of inhibition was examined. As a result, it was found that manganese ion and N-bromosuccinimide inhibited the reaction. Table 3 Activator / Inhibitor Concentration (mM) relative to no addition control ( %) control (no addition) 100.0 AlCl ₃ 5 88.5 AgNO ₃ 5 42.7 CaCl ₂ 5 98.2 CoCl ₂ 5 92.7 CuSO ₄ 5 33.0 FeCl ₂ 5 80.7 MnCl ₂ 5 0.0 ZnSO ₄ 5 74.0 EDTA 5 86.3 SDS 5 76.3 N-bromosuccinimide 5 0.0 Iodine acetic acid 5 88.2

Example 6 : Hemicellulase GM "Amano"
Purified hemicellulase GM "Amano" derived from (Amano Pharmaceutical Co., Ltd.) is a crude enzyme preparation produced by a microorganism belonging to the genus Aspergillus. All purification operations were performed at 4 ° C. The β-mannanase activity was measured by the method described in Example 3. Hemicellulase G
Tris / hydrochloric acid buffer (pH 7.0) was added to 12 g of M “Amano” to a final concentration of 50 mM to prepare a 40 ml solution. This solution was equilibrated with the same buffer for ion exchange chromatography (column: TOSOH TSK-gel DEAE
-TOYOPEARL 650S 120ml). The column was washed with the same buffer, and then β-mannanase was eluted with a linear gradient of 300 ml of 0 to 0.3 M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10,000),
Final concentration 10 mM Tris-HCl (pH 7.5), 1.5
M ammonium sulfate was added to obtain a solution of 11 ml. The solution was subjected to hydrophobic chromatography equilibrated with the same buffer (column: TOSOH TSK-gel Phenyl-TOYOP
EARL 650S 120 ml). The column was washed with the same buffer, and then β-mannanase was eluted with a linear gradient of 300 ml of 1.5 M to 0 M ammonium sulfate. The active fraction was concentrated by an ultrafiltration membrane (molecular weight cut: 10,000), followed by UF washing with 10 mM Tris-HCl, 0.15 M NaCl buffer (pH 7.5), desalting and concentration, and 2.5 ml
Solution. Next, gel filtration chromatography (column: Pharmacia HiLoad 1) in which the concentrated solution was equilibrated with the same buffer solution
6/60 Superdex 200pg) and apply β
-Mannanase was eluted. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut off: 10,000) to obtain 14.4 ml of a solution. Finally, a purified enzyme showing a single band was obtained by SDS polyacrylamide gel electrophoresis and isoelectric focusing. Table 4 below shows the total enzyme activity, total protein amount and specific activity in each purification step. The amount of protein is
BSA (bovine serum albumin) was used as a standard substance and measured by the Foreign-Lowry method. Table 4 Purified fraction Total enzyme activity Total protein Specific activity Recovery Purity (U) (mg) (U / mg) (%) (fold) Culture supernatant 279960 3588 78 100 1 DEAE Toyopearl 165341 1283 129 59.1 1.65 Phenyl Toyopearl 57 117 376 152 20.4 1.95 Gel filtration 32 184 223 144 11.5 1.85

Example 7 : Hemicellulase GM "Amano"
Properties of β-mannanase derived from The enzymological properties of the purified enzyme obtained in Example 6 were as follows. The enzyme activity was measured by the method described in Example 3 above. (1) Action It has a galactomannanase activity that acts in an endo-type on locust bean gum or the like linked to β-1,4-D-mannopyranoside. (2) Substrate specificity Although it acted on locust bean gum, glucomannan, and guar gum and was reduced in molecular weight, no change was observed in arabinoxylan, xylan, tragacanth gum, cellulose, and carboxymethyl cellulose. (3) Molecular weight The molecular weight was measured by SDS polyacrylamide gel electrophoresis. 200,000, 11 as marker proteins
6,300, 97,400, 66,300, 55,400, 36,500, 31,000, 21,
500 and 14,400 were used. As a result, this enzyme was considered to have a sugar chain, and the band of SDS polyacrylamide gel electrophoresis was smeared. The molecular weight range is 41,0
It was 00-47,000. (4) Reactivity The obtained purified enzyme has an optimum reaction temperature and pH around 70 ° C.
It has a reaction optimum pH around 4.0 to 5.0.

Example 8 : Cellulase Y-NC (Yakult)
Purified cellulase Y-NC derived from Aspergillus is an enzyme produced by a microorganism belonging to the genus Aspergillus. All purification operations were performed at 4 ° C. The activity of the xylanase of the present invention was measured by the following method. 1 ml of the test solution was added to 4 ml of the substrate solution (detailed preparation method is shown below) and reacted at 40 ° C. for 10 minutes. The reducing sugar generated in the reaction solution was quantified by the Somogyi-Nelson method. Preparation of substrate solution 1) 0.625 g of xylan (Oat Spellt)
s-derived, Lot. GG01, manufactured by Tokyo Chemical Industry)
Add water. 2) Add 5 ml of 2N sodium hydroxide and add 10 ml
Add water and warm in a boiling water bath to dissolve. 3) Add about 40 ml of water and cool to room temperature. 4) Add 20 ml of 1M acetic acid gradually over 10 to 30 minutes. 5) Add 1M acetic acid or 1N sodium hydroxide to pH
Set to 4.5. 6) Add water to make the total volume 100 ml. The solution prepared according to the above procedure is used as a substrate solution for xylanase activity measurement. Xylanase activity is 1 ml of test solution
Shows the enzyme activity that produces a reducing power equivalent to 1 μmole xylose per minute as 1 Unit (U). 5.0 g of cellulase Y-NC was added to a final concentration of 10 mM sodium acetate buffer (pH 4.0) and 1.0 M ammonium sulfate to obtain a 50 ml solution. This solution is
Hydrophobic chromatography equilibrated with the same buffer (column: TOSOH TSK-gel Phenyl-TOYOPEARL 650S 120m
1). Wash the column with the same buffer, then
Xylanase was eluted with a linear gradient of 0 ml of 1.0 M to 0 M ammonium sulfate. Ultrafiltration membrane (molecular weight cut 10)
000), followed by UF washing with 20 mM Tris / HCl buffer (pH 6.0), desalting and concentration, and
1 solution. Ion exchange chromatography (column: TOSOH TSK-gel) equilibrated with this buffer
DEAE-TOYOPEARL 650S 120 ml). The column was washed with the same buffer, and xylanase eluted in the non-adsorbed fraction was eluted. Ultrafiltration membrane (molecular weight cut 1)
0000), followed by UF washing with 50 mM Tris-hydrochloric acid and 0.2 M NaCl buffer (pH 6.0), desalting and concentration to obtain a 2.0 ml solution. Next, gel filtration chromatography in which the concentrated solution was equilibrated with the same buffer (column: Pharmacia HiLoad 16/60 Superdex 200pg)
And xylanase was eluted with the same buffer.
The active fraction was concentrated by an ultrafiltration membrane (molecular weight cut: 10,000) to obtain 17.6 ml of a solution. Finally, a purified enzyme showing a single band was obtained by SDS polyacrylamide gel electrophoresis and isoelectric focusing. The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 5 below. The amount of protein was determined by BSA (bovine serum albumin).
Was used as a standard substance and measured by the Foreign-Lowry method. Table 5 Purified fraction Total enzyme activity Total protein Specific activity Recovery Purity (U) (mg) (U / mg) (%) (fold) Culture supernatant 4575 825 5.54 100 1 DEAE Toyopearl 3254--71.1- Phenyl Toyopearl 2491 − − 54.4 − Gel filtration 743.1 41.1 18.1 11.5 3.27

Example 9 : Xyl derived from cellulase Y-NC
Properties of Lanase The enzymological properties of the purified enzyme obtained in Example 8 were as follows. The enzyme activity was measured by the method described in Example 8 above. (1) Action It has xylanase activity that acts in an endo-type on xylan or the like linked to β-1,4-D-xylopyranoside. (2) Substrate specificity It acted on arabinoxylan and xylan, and a reduction in molecular weight was observed, but no change was observed in locust bean gum, glucomannan, guar gum, and tragacanth gum. (3) Molecular weight The molecular weight was measured by SDS polyacrylamide gel electrophoresis. 205,000, 11 as marker proteins
6,000, 97,400, 69,000, 55,000, 36,500, 29,000, 20,
100 and 14,300 were used. As a result, the molecular weight is about 20,0.
00. (4) Reactivity The obtained purified enzyme has a reaction optimum temperature and pH around 60 ° C.
It has a reaction optimum pH around 4.0 to 5.0.

Example 10 : β-mannanase purified enzyme
Comparative effect of xylanase purified enzyme on green tea sedimentation Penicillium multicolor mc obtained in Example 4
Purified β-mannanase (β-mannanase 1) derived from h13-2 strain, hemicellulase GM obtained in Example 6
The effect of purified β-mannanase derived from “Amano” (β-mannanase 2) and the purified xylanase derived from cellulase Y-NC obtained in Example 8 on green tea precipitation was evaluated. The method is described below. Each enzyme was added at the following concentrations, and the threshold addition amount of each enzyme after storage at 40 ° C. for two weeks was determined. In addition, the threshold addition amount or the threshold addition concentration means a minimum amount that becomes “no flocculent precipitation” in the evaluation of precipitation generation. Since there is no unified value indicating the hemicellulase activity, both enzymes were compared in terms of the amount of protein for comparison (Table 6). The evaluation of the green tea precipitation inhibiting effect was performed according to the method described in Example 1. Table 6 Purified enzyme Protein concentration (mg protein / ml tea liquor) β-mannanase ゛ 1 0.0327 0.0131 0.00653 0.003265 0.00196 0.00131 0.000653 0.000327 0.000131 0.0000653 β-mannanase ゛ 2 0.0556 0.0222 0.0111 0.00556 0.00333 0.00222 0.00111 0.000556 0.000222 0.000111 β-xylanase 0.201 0.161 0.121 0.0803 Table 7 shows the results of 0.0401 0.0263 0.0105 0.00526 0.00263 0.00210 or more. Table 7 Purified enzyme Threshold addition concentration (mg protein / ml tea liquor) β-mannanase {1} 0.0131 β-mannanase {2 0.0222 β-xylanase} 0.121 Table 4 shows the results of storage at 40 ° C for 4 weeks in the same manner. Table 8 Purified enzymes Threshold addition concentration (mg protein / ml tea liquor) β-mannanase ゛ 1 0.0326 β-mannanase ゛ 2 0.0556 β-xylanase ゛> 0.201 (precipitation could not be suppressed even at the maximum concentration) Mannanase inhibited green tea precipitation by adding 1 / 9.24 to 1 / 5.45 compared to xylanase, that is, β-mannanase was found to have about 5 to 10 times higher precipitation inhibiting effect. .

Example 11 : Preparation of β-mannanase crude enzyme solution
Examination of pH during enzyme treatment The pH at which green tea was treated with β-mannanase enzyme was examined. 1. When sodium bicarbonate is added after sterilization to pH 6.25, adjusted, and then subjected to enzyme treatment. Performed according to the method described in Example 1. This is referred to as a one-stage sodium bicarbonate addition method. 2. When sodium hydrogen carbonate is added to adjust the pH to 5.0, the mixture is treated with an enzyme, and sodium hydrogen carbonate is further added so that the pH is adjusted to 6.25 after sterilization. This is the sodium bicarbonate two-stage addition method. The details are shown below. 10 g of "Yabukita Seed", the most medium-grade green tea leaf (tea leaf 1) from Shizuoka, was added to 350 g of ion-exchanged water at 65 [deg.] C. and extracted for 5 minutes. Stirring). After roughly filtering tea leaves using a 30 mesh and 120 mesh stainless steel filter,
The extract cooled to about 20 ° C. is centrifuged (3000 rpm /
And 10 minutes), and the extract was filtered using a 200-mesh stainless steel filter to obtain a clear solution. To stabilize the quality, dilute 3.7-fold with ion-exchanged water, add sodium ascorbate to a final concentration of 0.04%, and then add sodium bicarbonate to pH 5.
It was adjusted to be 0. Next, 10 ml was dispensed into a heat-resistant test tube with a cap having a size of 10 ml. Using a penicillium multicolor mch13-2-derived β-mannanase of the present applicant's already-filed application (Japanese Patent Application No. 11-330558) as a crude enzyme preparation having β-mannanase activity as a main component, the mixture was heated at 30 ° C. (water bath). ) For 30 minutes. After the enzyme treatment, sodium hydrogencarbonate was added again after sterilization to adjust the pH to 6.25. Next, a sterilization treatment was performed at 121 ° C. for 10 minutes. Through the above operation, the threshold addition concentration was obtained in the same manner as in the above example. The enzyme treatment was carried out by the above two methods, and the threshold addition concentrations at the time of storage at 40 ° C. for two weeks are summarized in Table 9. Table 9 Formulation of sodium bicarbonate Threshold addition concentration (U / ml tea liquor) One-step addition method 1.6 Two-step addition method Turned out to be the same.
And it turned out that the same effect is recognized even if sodium hydrogen carbonate is added once or twice.

Example 12 : Protein content of various crude enzyme preparations and green
Evaluation of Threshold Addition Concentration of Tea Precipitation Prevention Effect Penicillium Multicolor m obtained in Example 3
The β-mannanase activity and xylanase activity of the crude enzyme solution derived from the ch13-2 strain and various crude enzyme preparations shown in Table 10 below were measured by the methods shown in Examples 3 and 8. Further, the respective protein amounts were measured by the Foreign-Lowry method. Table 10 Crude enzyme preparation β-mannanase xylanase Type of protein activity (U / g) Activity (U / g) (mg protein / g) Sumiteam 2000 740 9180 210 Sumiteam C 400 2400 85 Sumiteam AC 2700 1300 139 Sumiteam ACH 11000 480 213 Sumiteam X 250 6700 77 Cellulosin HC 1800 1010 63 Cellulosin TP25 230 24000 333 Cellulosin HC100 740 7000 138 Cellulosin GM5 14800 360 372 GODO-TXL 62 2070 91 Cellulase "Onozuga" 3S 310 1300 80 Cellulase Y-NC 3400 1210 165 Cellulase "Amano" 90 610 6050 213 Hemicellulase GM "Amano" 23000 490 264 Penicillium multicolor mch13-2 16360 20 119 Then, according to the method of Example 1, a crude enzyme preparation for 10 ml of tea solution (used as a 10% solution) ) Is 2, 5, 10, 20, 50, 100, 20 , 500μ
After the enzyme treatment was performed by providing an experimental plot to obtain l, and stored at 40 ° C for 2 weeks, the threshold addition amount at which flocculent precipitation did not occur was measured. For each crude enzyme preparation, the β-mannanase / xylanase activity ratio and The relationship was evaluated. Table 11 shows the amount of protein added at the threshold for suppressing flocculation and the β-mannanase / xylanase activity ratio. In addition, the thing which did not have the flocculent precipitation inhibitory effect was excluded. Table 11 Crude enzyme preparation β-mannanase / xylanase Type of protein addition amount at threshold Activity ratio (mg protein / ml tea liquor) Cellulosin HC100 0.11 0.069 Cellulosin HC 1.8 0.063 Sumizyme AC 2.1 0.070 Cellulase Y-NC 2.8 0.083 Sumizyme ACH23 0.043 Cellulosin GM5 41 0.037 Hemicellulase GM "Amano" 47 0.026 Penicillium multicolor mch13-2 806 0.013 As can be seen from the results, the higher the β-mannanase / xylanase activity ratio, the smaller the amount of protein added at the threshold. I understood that. That is, this phenomenon was confirmed not only for the purified enzyme but also for the crude enzyme preparation. And it was also confirmed that the β-mannanase / xylanase activity ratio became remarkable at 20 or more. Penicillium multicolor mch13-2 consisting mostly of β-mannanase
Was a very high value of 806, and the amount of protein added at the threshold was 0.013 mg protein / ml tea liquor, which was very small. When the amount of the xylanase contained and the value of the threshold addition amount were compared, it was found that there was a tendency to have an additive effect with β-mannanase.

Example 13 : β-mannanase purified enzyme
Synergistic effect of xylanase purified enzyme on green tea precipitation prevention
Evaluation of effect Penicillium multicolor mc obtained in Example 4
Using the purified β-mannanase derived from the h13-2 strain and the purified xylanase derived from the cellulase Y-NC obtained in Example 8, the presence or absence of a synergistic effect in preventing green tea precipitation was evaluated. According to the method of Example 1, as shown below, 10 ml of tea liquor was subjected to enzymatic treatment by adding β-mannanase at each concentration diluted in 10 steps, and purified xylanase was added thereto. (0.121 mg / ml in Example 10 = 2.18)
U / ml), 3.3% and 6.7%
Three series of experimental plots were prepared in which the enzyme treatment was performed by further adding a relatively purified xylanase. This was stored at 40 ° C. for two weeks, and the threshold addition concentration at which flocculation did not occur was measured. Experimental plot series 1 (upper row: β-mannanase activity, lower row: xylanase activity, U / ml) 6.4 3.2 1.6 0.80 0.40 0.20 0.10 0.050 0.025 0.012 0 0 0 0 0 0 0 0 0 0 0 Experimental series 2 (upper row: β-mannanase activity, lower row: xylanase activity, U / ml) 6.4 3.2 1.6 0.80 0.40 0.20 0.10 0.050 0.025 0.012 1.8 0.91 0.46 0.23 0.11 0.057 0.029 0.014 0.0071 0.0036 Experimental series 3 (upper row: β-mannanase activity, lower row: xylanase activity, U / ml) 6.4 3.2 1.6 0.80 0.40 0.20 0.10 0.050 0.025 0.012 3.7 1.8 0.91 0.46 0.23 0.11 0.057 0.029 0.014 0.0071 As a result, in any experimental series, the purified β-mannanase to which 0.8 U / ml or more had been added had a flocculent precipitate. Elimination was observed, and xylanase was added at the threshold amount of 6.
It was found that the addition of 70% had no effect on the flocculent-elimination effect of β-mannanase, and it was considered that there was no synergistic effect. This result was consistent with the observed additive effect of both enzymes of Example 12.

Example 14 : Verification of effect on high-grade tea leaves It is known that higher-grade tea leaves contain more polysaccharides that cause secondary precipitation. In addition, when high-grade tea leaves are used, the amount of enzyme added increases, and protein-derived precipitates tend to appear. Therefore, using cellulase Y-NC and a crude enzyme solution of β-mannanase derived from penicillium multicolor mch13-2, which was filed by the present inventors (Japanese Patent Application No. 11-330558), Shizuoka used in the above Examples. The threshold addition amount of Shibuya's "Yabukita", the highest grade of tea from the middle grade (tea leaf 1), which is more advanced than the tea leaves for evaluation of green tea precipitation (the tea leaves 2) An evaluation was performed. The evaluation test was performed in the same manner as in the method described in Example 1 except that the
The test was performed in a heat-resistant test tube of a size. In addition, the tea leaf 2 is a higher quality tea leaf than the tea leaf 1 and has a large amount of precipitate formation. The enzyme concentrations are as shown in Table 12. Table 12 Enzyme name Enzyme concentration ^(Note) (U / ml tea liquor) Cellulase Y-NC 13 11 9.5 3.8 1.9 0.95 0.38 0.19 0.095 0.038 mch13-2 62 41 21 10 4.1 2.1 1.0 0.41 0.21 0.10 Crude enzyme solution (Note) Cellulase Y-NC has xylanase activity and mch13-2 derived β- The crude mannanase enzyme solution is indicated by β-mannanase activity. As a result of storage at 40 ° C. for two weeks, cellulase Y-NC:
The effect of inhibiting green tea precipitation could not be exerted, and even if the amount of added enzyme was further increased, protein-derived precipitates were generated, so that no effect could be obtained with advanced tea leaves (tea leaves 2). On the other hand, in the case of the β-mannanase crude enzyme solution, 41 U / ml
, Precipitation could be completely suppressed. Therefore, the crude enzyme solution of the present β-mannanase was confirmed to have an effect of suppressing green tea precipitation even in advanced tea leaves (tea leaves 2). The absence of a protein-derived precipitate is considered to include β-mannanase, which has a large effect, and to contain almost no contaminating enzymes. It is also considered that the specific activity of the present β-mannanase itself is high and the amount of added protein is small. From the above results, it is considered that the use of a β-mannanase crude enzyme agent containing β-mannanase as a main component enables the production of a green tea beverage using high-grade tea leaves, which was conventionally considered difficult.

Example 15 : Evaluation of taste Taste of Yabukita from Shizuoka, Ichibancha middle-grade green tea leaf (tea leaf 1) 160
g was added to 5.6 kg of ion-exchanged water at 65 ° C., and the mixture was allowed to stand and extracted for 5 minutes (immediately after the introduction of tea leaves and 2.5 minutes after the addition of tea leaves, respectively).
Stirring for 0 seconds). After roughly filtering tea leaves using a 30-mesh and a 120-mesh stainless steel filter, the extract cooled to about 20 ° C. is centrifuged (3000 rpm / min, 10 minutes), and the extract is further filtered using a 200-mesh stainless filter. Then, a clear liquid was obtained. To stabilize the quality, the mixture was diluted 3.7 times with ion-exchanged water, and sodium ascorbate was added to a final concentration of 0.04%.
And adjusted so that As a crude enzyme preparation having β-mannanase activity as a main component, a penicillium multicolor mch13-2-derived β-mannanase crude enzyme solution of the present application (Japanese Patent Application No. 11-330558) or a cellulase as a comparative example Using Y-NC, 3
The enzyme treatment was performed at 0 ° C. (water bath) for 30 minutes. Then 2
The mixture was placed in a 00 ml heat-resistant glass bottle and subjected to a retort sterilization treatment to obtain a green tea beverage. The results are shown in Table 13 below. Table 13 Enzyme name Enzyme concentration ^(Note) (U / ml tea liquor) Cellulase Y-NC 2.0 1.0 0.50 0.25 0.10 mch13-2 derived 12.8 6.4 3.2 1.6 0.80 β-mannanase (Note) Cellulase Y-NC has xylanase activity. , Mch13-2 derived β-mannanase crude enzyme solution is indicated by β-mannanase activity. The above green tea extract was evaluated for suppression of green tea precipitation when stored at 25 ° C. and 40 ° C. for four weeks, and sensory tests were conducted by three experienced panelists for flavor during one week storage to compare the quality. Table 14 shows the results. However, 25 ° C
And storage at 40 ° C. gave the same results. Table 14 (1) Cellulase Y-NC enzyme added amount (U / ml) Flock generation status Flavor 0 Floating a large amount of flocks Good 0.1 Some flocs floating The soil odor derived from the enzyme 0.25 No flocs are found 0.5 No flock is observed.Enzyme-derived earth smell 1.0 No flock is observed.Enzyme-derived earth smell 2.0 No flock is observed.Enzyme-derived earth smell (2) Amount of enzyme added to mch13-2-derived β-mannanase crude enzyme solution (U / ml) Flock generation status Flavor 0 Floc floats in large quantities Good 0.8 Slight flock floats Kokumi and tea taste increased 1.6 No flock is seen. Kokumi taste and brownness are increased. 3.2 Flock is not seen. Kokumi and brownness are increased.6.4 Flock is not seen. Kokumi and brownness are increased. 12.8 Flock is not seen. As described above, the cellulase Y-NC suppressed the formation of the precipitate, but had a low concentration in the flavor and had already generated the enzyme-derived earthy odor. On the other hand, in the case of the crude enzyme solution of β-mannanase derived from mch13-2, precipitation was suppressed, and at the same time, the body taste was increased and the tea feeling was further increased. This clearly showed a quality improvement effect.

[0048]

As described above, according to the present invention,
It does not require complicated processes and has the original flavor of green tea extract.
It is possible to provide a green tea beverage having no turbidity or sediment for a long period of time without loss of taste, particularly a green tea beverage in a transparent closed container. In addition, even if the green tea beverage is stored for a long period of time, the occurrence of secondary precipitation that has been unavoidably observed during storage can be effectively suppressed, and the tea feeling of the green tea beverage as a favorite beverage ( Green tea original flavor and taste)
Can provide a method for producing a high-quality green tea beverage which is much more improved than conventional products, and a product thereof. Furthermore, β-mannanase has an effect about 5 to 10 times higher than that of xylanase which has been conventionally said to have a precipitation inhibiting effect. As a result, in the case of an enzyme preparation containing β-mannanase as a main component, a threshold value is added. Since the amount is small, no unpleasant taste such as "earthy smell" derived from the enzyme is generated. In addition, even when high-grade tea leaves were used, it was possible to completely suppress secondary precipitation (cotton-like precipitation) without causing precipitation due to protein.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masataka Yamada 1620 Kurami, Samukawa-cho, Koza-gun, Kanagawa Prefecture Inside the Research Laboratory, Kirinbi Barrier Co., Ltd. F-term (reference) in R & D Co., Ltd. 4B027 FB13 FC05 FC10 FE06 FK01 FK03 FK07 FR04 4B050 CC07 CC08 DD03 LL02

Claims (11)

[Claims] 1. A method for producing a green tea beverage in which turbidity or precipitation is suppressed, wherein the green tea extract is treated with β-mannanase or an enzyme agent containing β-mannanase as a main component. 2. The method for producing a green tea beverage according to claim 1, wherein the green tea extract is treated with β-mannanase or an enzyme agent containing β-mannanase as a main component, and an antioxidant is added. 3. The method for producing a green tea beverage according to claim 1, wherein the enzyme treatment is performed at a temperature of 20 to 70 ° C. 4. The method for producing a green tea beverage according to claim 1, wherein the pH adjuster is added at least once or more. 5. The method for producing a green tea beverage according to claim 1, wherein the green tea beverage is treated with the enzyme agent having a β-mannanase / xylanase activity ratio of 5 or more. 6. The method for producing a green tea beverage according to claim 1, wherein the green tea beverage is treated with the enzyme agent having a β-mannanase / xylanase activity ratio of 20 or more. 7. The method for producing a green tea beverage according to claim 1, wherein the β-mannanase is penicillium multicolor-derived β-mannanase. 8. The penicillium multicolor is a penicillium multicolor mch13-2 strain (FERM BP-6831).
The method for producing a green tea beverage according to claim 7, which is: 9. Use of β-mannanase or an enzyme preparation containing β-mannanase as a main component for producing a green tea beverage. 10. The use according to claim 9, wherein the enzymatic agent suppresses the formation of cloudiness or precipitation of the green tea beverage. 11. A green tea beverage obtained by the method according to any one of claims 1 to 8 or the use according to claim 9 or 10.