JPS5846316B2 - Method for removing bitterness from fruit juice containing naringin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for removing bitterness from fruit juice containing naringin, and more particularly to a method for removing bitterness from fruit juice by decomposing the bitter substance naringin contained in the fruit juice.

Citrus fruits such as summer mandarin oranges, grapefruit, and honeysuckle contain the bitter substance naringin, so when processing these citrus fruits to produce fruit juice products,
It is necessary to remove bitterness.

Conventionally, as a method for removing bitterness from fruit juice containing naringin, a method using a commercially available naringinase agent is known.

However, this method often has a negative effect on the quality of the fruit juice because the naringinase agent contains various contaminants and enzymes in addition to the naringin-degrading enzyme.
In addition, the enzymatic reaction must be carried out at relatively high temperatures and for a long period of time, which may deteriorate the flavor of the fruit juice.Furthermore, it is impossible to recover naringinase after the enzymatic reaction, so it can only be used once. Economically disadvantageous.

In order to solve the above-mentioned drawbacks, attempts have been made in recent years to immobilize naringinase using a carrier having an ion exchange group such as DEAE-cellulose and a protein such as casein, and to perform the enzymatic reaction repeatedly (% However, the immobilized naringinase obtained by this method has a low enzyme activity and is not necessarily satisfactory for industrial use in removing bitterness from fruit juice.

In view of this situation, the present inventors have conducted various studies and found that a water-insoluble tannin preparation in which a water-insoluble and hydrophilic polymer and a tannin component are covalently bonded to each other has been developed by combining amino acids, organic acids, sugars, It does not act on small molecules such as nucleobases, but has a specific affinity for oxygen proteins.If naringinase was adsorbed to this water-insoluble tannin preparation, the enzyme activity would be strong and it would have excellent stability. We discovered that immobilized naringinase can be obtained, and that if the immobilized naringinase thus obtained is applied to fruit juice containing naringin, only the bitterness can be removed without affecting the flavor of the fruit juice, resulting in scaling. , we have completed the present invention.

That is, the present invention involves contacting immobilized naringinase, in which naringinase is bound by adsorption to a water-insoluble tannin preparation in which a water-insoluble and hydrophilic polymer and a tannin component are covalently bound, with a naringin-containing fruit juice. This is a method for removing bitterness from fruit juice containing naringin, which is characterized by decomposing naringin in the fruit juice.

The method of the present invention will be specifically explained below.

The immobilized naringinase according to the present invention is a water-insoluble tannin produced by covalently bonding a tannin compound selected from tannins, aminoalkylated tannins, carboxyalkylated tannins, and hydroxyalkylated tannins to a water-insoluble and hydrophilic polymer. It can be produced by preparing a formulation and then binding naringinase to the water-insoluble tannin formulation by adsorption.

The tannin used in the present invention may be pyrogallo-JL4 tannin or catechol tannin.

Examples of pyrogallol tannins include gallic tannins and pentagram tannins, and examples of catechol tannins include catechol polymers obtained from tea, cacao, and the like.

Further, these tannins do not necessarily have to be purified, and may be impure tannins such as those commercially available as persimmon tannins.

In addition, aminoalkylated tannins, carboxyalkylated tannins, or hydroxyalkylated tannins can be produced by reacting tannins with cyanogen halides (e.g., cyanogen bromide) or epoxy compounds (e.g., epichlorohydrin) to produce cyanogen halide-activated tannins or epoxy A compound-activated tannin is prepared and then combined with an alkylene diamine (e.g. ethylenediamine, tetramethylene diamine, hexamethylene diamine, dodecamethylene diamine), an aminoalkyl carboxylic acid (e.g. epsilon-aminocaproic acid), or an aminoalkanol (e.g. ε-aminocaproic acid).
For example, those obtained by reacting 2-aminoethanol) are preferably mentioned.

On the other hand, the polymer used in the present invention is one selected from the group consisting of hydroxyl group, amino group and carboxyl group.
Examples include water-insoluble and hydrophilic polymers having more than one type of functional group in the molecule.

Hydrophilicity here means that the polymer has the property of being wetted or swollen in water.

Examples of water-insoluble and hydrophilic polymers having hydroxyl groups in the molecule include polysaccharides such as cellulose, agarose, and crosslinked dextran, and hydroxyalkylated polysaccharides (e.g., human xyglobylated crosslinked dextran). Examples include polysaccharides into which spacers have been introduced.

Next, water-insoluble and hydrophilic polymers having an amino group in the molecule include, for example, aminoalkylated polysaccharides (for example, aminoethylated cellulose, aminohexylated cellulose, aminohexylated agarose), aminoaralkylated polysaccharides, Suitable examples include polysaccharides into which a spacer having an amino group is introduced, such as saccharides (for example, aminobenzylated cellulose).

Further, water-insoluble and hydrophilic polymers having a carboxyl group in the molecule include, for example, carboxyalkylated polysaccharides (e.g., carboxymethylated cellulose, carboxyhexylated agarose, carboxypentylated agarose, carboxymethylated crosslinked dextran). Preferred examples include polysaccharides into which a spacer having a carboxyl group is introduced.

In order to produce a water-insoluble tannin preparation by covalently bonding a tannin compound as described above to a water-insoluble and hydrophilic polymer, a known method employed for producing an immobilized enzyme by a covalent bonding method [ For example, Chibata (ed.), “Immobilized Enzyme J, pH~40,” published by Kodansha (March 20, 1975)
``Affinity Chromatography'', edited by Yamazaki et al.
.

p19-32. Published by Kodansha (February 1975 IB)] can be used.

For example, in order to covalently bond a water-insoluble and hydrophilic polymer (e.g., cellulose, agarose, polysaccharide such as cross-linked dextran) having a hydroxyl group in the molecule to a tannin compound, the following procedure can be carried out. I can do it.

That is, (1) a cyanogen halide (e.g., cyanogen habromide) is reacted on a polysaccharide to produce a cyanogen halide-activated polysaccharide, and this is reacted with an aminoalkylated tannin, or (2) a polysaccharide to react with an epoxy compound (e.g. epichlorohydrin, 1,2,7,8-jepoxyoctane) to produce an epoxy compound-activated polysaccharide, which is then reacted with tannin, aminoalkylated tannin or hydroxyalkylated tannin. A water-insoluble tannin preparation can be produced by this method.

In addition, in order to covalently bond a water-insoluble and hydrophilic polymer having an amino group in the molecule and a tannin compound,
This can be done as follows.

That is, (1) the polymer is reacted with a tannin activated with cyanide rhodanide, (2) the polymer is diazotized and then reacted with a tannin, or (3) the polymer and (4) activating the polymer with an epoxy compound and then reacting it with a tannin, an aminoalkylated tannin or a hydroxyalkylated tannin, or conversely, a tannin, Water-insoluble tannin preparations can be prepared by activating aminoalkylated tannins or hydroxyalkylated tannins with an epoxy compound and then reacting this with the polymer.

Furthermore, covalent bonding of a water-insoluble, hydrophilic polymer having a carboxyl group in its molecule and a tannin compound can be carried out as follows.

That is, a water-insoluble tannin preparation can be produced by peptide condensation of the polymer and aminoalkylated tannin.

Immobilized naringinase can be easily produced by adsorbing naringinase to the water-insoluble tannin preparation obtained as described above by bringing the water-insoluble tannin preparation into contact with a naringinase-containing solution.

This adsorption operation can be carried out by either a batch method or a column method.

For example, in the case of a batch method, by adding a water-insoluble tannin preparation to a naringinase-containing solution and stirring, naringinase is adsorbed to the water-insoluble tannin preparation to obtain immobilized naringinase.

In the case of a column method, a column is filled with a water-insoluble tannin preparation, and then a naringinase-containing solution is passed through the column, whereby naringinase is adsorbed to the water-insoluble tannin preparation to obtain immobilized naringinase.

The above adsorption operation is preferably carried out at a pH of 3 to 8 and a temperature of 5 to 45°C.

The immobilized naringinase obtained as described above is brought into contact with a naringin-containing fruit juice to decompose the naringin in the fruit juice and remove bitterness by a column method, a fluidized bed method, or a batch method. can also be implemented.

For example, in the case of using a column method, a column is filled with immobilized naringinase, and then naringin-containing fruit juice is allowed to flow down the column, and the fruit juice that has passed through the column is collected.

Although the flow rate of fruit juice can be arbitrarily selected, it is generally preferable that the space velocity (SV) is 0.01 to 30 (hr').

Furthermore, when the fluidized bed method is used, it can be carried out by passing the naringin-containing fruit juice through a fluidized bed in which immobilized naringinase is dispersed.

The flow rate of fruit juice is generally a space velocity (SV) of 0.1 to 2.
It is preferable that it is 50 (hr').

Furthermore, when carrying out the batch method, immobilized naringinase is added to the naringin-containing fruit juice and stirred.

A stirring time of about 1 hour is sufficient, but stirring several times a day is enough to achieve the desired purpose.

The operation temperature in the above column method, fluidized bed method and batch method is preferably 5 to 55°C, preferably 20 to 40°C, and the naringin-containing fruit juice applicable to the above method is citrus fruit juice such as summer mandarin orange, grapefruit, and honeysuckle. Examples include fruit juices of citrus fruits, transparent fruit juices obtained by clarifying these juices using pectinase, and solutions containing naringin eluted from the pulp of citrus fruits, sand sacs, and the like.

According to the method of the present invention as described above, bitterness can be removed in a short time by a simple operation of bringing immobilized naringinase into contact with naringin-containing fruit juice.

Furthermore, according to the method of the present invention, bitterness can be effectively removed even at a relatively low temperature of about 25° C., so fruit juice of good quality can be produced without impairing the flavor of the fruit juice.

In the Reference Examples and Examples described later, enzyme activity was measured as follows.

That is, in the case of naringinase, naringin 100
η in 0.02M acetic acid/sodium acetate buffer (pH 4,5)
Prepare a substrate solution by heating and dissolving in 80 ml, add 1 rnl of enzyme solution to 4wLl of this substrate solution, and react at 40°C for 300 minutes.

Next, the reaction was stopped by heating in boiling water for 10 minutes, and the resulting reducing sugar (rhamnose) was immediately determined colorimetrically by the Somogyi-Nernon method.

On the other hand, in the case of immobilized naringinase, naringin 10
01v to 0.02M acetic acid/sodium acetate buffer (pH 4,
5) A substrate solution was prepared by heating and dissolving in 100 rrtl, and a predetermined amount of immobilized naringinase was added to 35 ml of this substrate solution, and the mixture was shaken at 40° C. for 30 minutes to react.

Then, the reaction solution was passed through the mouth, and the oral solution was immediately diluted in boiling water for 10 minutes.
After heating for a minute, the reducing sugar produced was colorimetrically determined using the Somogyi-Nernon method.

Under the above conditions, the enzyme activity that produced 1 µ of reducing sugar was defined as 1 unit.

Hereinafter, the method of the present invention will be specifically explained with reference to Reference Examples and Examples, but the method of the present invention is not limited to these Reference Examples and Examples.

Reference example 1 Cellulose Powder C (product manufactured by Toyo Kuchigashi & cotton cellulose with fiber length of 300 mesh or more) 20i
3 in 200 ru of 5% sodium hydroxide aqueous solution at 10℃
After being immersed for 0 minutes, it was thoroughly washed with water.

The alkali-treated cellulose thus obtained was suspended in 400 ml of a 0.1M aqueous sodium bicarbonate solution, and the pI (
was adjusted to 11.5.

Cyanogen bromide 4i was added to this suspension to activate the cellulose.

Since a decrease in pH was observed during the reaction, the pH was adjusted to 11 to 11.5 with a 5N aqueous sodium hydroxide solution.

Further, the reaction temperature was kept not to exceed 30°C.

After about 8 minutes, the activated cellulose was taken out and washed with pre-cooled 0 and 1M aqueous sodium bicarbonate solutions and water, respectively.

The thus obtained cyanogen bromide-activated cellulose was dissolved in an aqueous solution (pH 9,
5) The mixture was suspended in 400 ml and reacted at 25°C for 16 hours.

After the reaction was completed, the produced aminohexylated cellulose was taken out and washed with a 0.1M aqueous sodium bicarbonate solution and water, respectively.

On the other hand, 5x tannin 26? was dissolved in 400 ml of 0.1M aqueous sodium carbonate solution, and the pH was adjusted to 11.5 and the temperature was adjusted to 20°C.

Cyanogen bromide 41 was added to this solution and reacted for 15 minutes while keeping the pH at 10.5.

The thus obtained cyanogen bromide-activated pentad tannin solution was added to the above aminohexylated cellulose and reacted at 25° C. for 16 hours.

After the reaction is completed, take out the solid content and take 0. Water-insoluble tannin preparation 45-11 (wet weight) was obtained by thorough washing with IN aqueous sodium bicarbonate solution and water.

Reference example 2 (1) Naringinase (15000u/P) at 20
0 containing ■, 40■, 60■, 80■ or 100■
, 005M acetic acid/sodium acetate buffer (pH 4,5) 5m
1 into five test tubes, and added the water-insoluble tannin preparation 11 obtained in Reference Example 1 to the husband's test tube, and heated to room temperature (21"
The mixture was shaken in step c) for 2 hours to immobilize naringinase.

The enzyme activity and immobilization yield of the immobilized naringinase thus obtained were as shown in Table 1 below.

(2) Sephatex G-25 (manufactured by Pharmacia) 2.48f was mixed with the immobilized naringinase 0.02S' obtained using naringinase 60η in (1) above, and a column (column volume: 3, 4 m) was filled.

A 0.02% naringin solution dissolved in 0.02M acetate buffer (pH 4,5) was added to this column at a temperature of 25℃ or 37℃ for 53 days at a space velocity (SV) of -1,76 (hr-1). The enzyme reaction was carried out by continuously flowing down.

During this time, the effluent was sampled over time, and the sugar produced by the decomposition of naringin was measured using the Somogyi-Nelson method to examine the stability of the immobilized naringinase.

As a result, the enzyme half-life of immobilized naringinase was 198 days for the reaction at 25°C, and 37 days for the reaction at 25°C.
The reaction time at ℃ was 87.7 days.

Reference example 3 (1) -)-+)-/Kif-ze (15000u/f
) to 601nI? Containing pH 3, 5, 4, 0, 4, 5
, 5,0 or 5.5 in 0.005M acetic acid/sodium acetate buffer into 5 test tubes, add the water-insoluble tannin preparation 1i obtained in Reference Example 1 to each test tube, and let the mixture cool to room temperature. The mixture was shaken for 2 hours to immobilize naringinase.

The enzyme activity and immobilization yield of the immobilized naringinase thus obtained were as shown in Table 2 below.

From the table above, it can be seen that it is best to adsorb naringinase at pH 4.5.

(2) In (1) above, 20μ of immobilized naringinase immobilized at pH 4.5 was added to 35WL of 0.1% naringin solution dissolved in 0.02M acetic acid/sodium acetate buffer (pH 4.5). The mixture was then shaken at 40°C for 30 minutes to perform an enzyme reaction.

After the reaction, the reaction solution 5rI'Ll was sampled, immediately filtered, and the oral fluid was heated for 10 minutes, and the naringin content in the oral fluid was determined by Davis' modified method [Science and Industry, vol. 43, p. 6
42-649 (1969)).

As a result, the content of naringin remaining in the reaction solution was 0.
．． It was 0.023%.

Furthermore, as a result of sensory examination of the intensity of bitterness of the reaction solution, it was confirmed that no bitterness was felt.

Reference Example 4 2 oz of Cellulose Powder C (trade name manufactured by Toyo Kushi Co., Ltd., cotton cellulose with a fiber length of 300 mesh or more) was immersed in a 25% aqueous sodium hydroxide solution at 25°C for 30 minutes, and then thoroughly washed with water. did.

The alkali-treated cellulose thus obtained was suspended in 400 ml of a 1N aqueous sodium hydroxide solution, and then 50 ml of epichlorohydrin was added, followed by reaction at 60° C. for 30 minutes with vigorous stirring.

After the reaction, take the epichlorohydrin-activated cellulose, wash it thoroughly with water, and add hexamethylene diamine 4.6
400mA of pH 10 aqueous solution containing 2! Stuck in the middle,
The reaction was allowed to proceed at 60° C. for 2 hours with slow stirring.

After the reaction was completed, the produced aminohexylated cellulose was taken out and thoroughly washed with a 0.1M aqueous sodium bicarbonate solution and water.

On the other hand, 501 parts of five-fold tannins and 11 parts of water! Dissolved in pH
After adjusting the temperature to 11.5, 4.01 l of cyanogen bromide was added and reacted for 8 minutes.

The thus obtained cyanogen bromide-activated pentad tannin solution was added to the above aminohexylated cellulose and heated at 25°C for 1 hour.
The reaction was allowed to proceed for 6 hours.

After the reaction was completed, the solid content was taken out and thoroughly washed with a 0.1 M aqueous sodium bicarbonate solution and water to obtain 43.2 P (wet weight) of a water-insoluble tannin preparation.

Reference Example 5 (1) 0,005M acetic acid/sodium acetate buffer (pH 4,
5) Put 5ml into a test tube, add the water-insoluble tannin preparation 1y obtained in Reference Example 4 to this test tube, and let it cool at room temperature (23cm).
℃) for 2 hours to immobilize naringinase.

The immobilized naringinase thus obtained showed an activity of 587 units.

Immobilization yield: 65.2%. (2) 20μ of the immobilized naringinase obtained in (1) above was added to 35M of 0.1% naringin solution dissolved in 0.02M acetic acid/sodium acetate buffer (pI 44.5), and the mixture was heated at 40°C. The enzyme reaction was performed by shaking for 30 minutes.

After the reaction, sample 5ml of the reaction solution and immediately **
After heating the gastric juice for 10 minutes, the content of naringin in the liquid was measured by a modified Davis method.

As a result, the remaining naringin content in the reaction solution was 0.
．． It was 0.029%.

Furthermore, as a result of sensually examining the intensity of bitterness of the reaction solution, it was confirmed that there was no bitterness.

Example 1 After peeling the outer skin of summer mandarin orange fruit, hassaku fruit, and gray fruit fruit, the fruit was squeezed and crushed using a juicer, and the juice obtained by sipping was heated at 80° C. for 8 minutes.

A bitterness removal reaction was carried out using the three kinds of fruit juices thus prepared as raw materials.

That is, 35 ml of each fruit juice was placed in a 100 ml Meyer vessel, 20 μl of immobilized naringinase immobilized at pH 4 and 5 in Reference Example 3 was added, and a shaking reaction was carried out at 40° C. for 60 minutes.

Immediately after the reaction, the juice was sipped and the immobilized naringinase was removed, and the juice was heated for 10 minutes.

Next, the naringin content in this fruit juice was measured by a modified Davis method.

The results are shown in Table 3 below, and the residual amount of naringin was 40% or less in all cases.

Further, as a result of sensory examination of bitterness intensity and fruit flavor, it was confirmed that no bitterness was felt and the fruit flavor was also satisfactory.

Example 2 The raw material was summer tangerine juice, which was prepared by squeezing summer tangerine fruit using an in-line juicer, heat sterilization, etc. using a conventional method, and stored in a 5-gallon can.

Add 0.0% pectinase (manufactured by Tanabe Seiyaku Co., Ltd.) to genuine mandarin orange juice.
The gastric juice was added at a rate of 10%, clarified at 40°C for 2 hours, passed through the mouth, and the gastric juice was heat sterilized at 85°C for 5 minutes.

The thus prepared clarified peeled mandarin juice (pH 3, 4,
Naringin content: 49.0%) was added to the immobilized naringinase prepared at pH 4.5 in Reference Example 3.
A column packed with a mixture of IP and cellulose C (manufactured by Toyo Kuchushi Co., Ltd.) 2.4 S' (wet weight) was heated at a temperature of 25°C or 37°C and a space velocity (SV') of 1.2 (hr').
) to perform an enzymatic reaction to decompose bitterness.

The residual naringin content in the effluent fruit juice was measured by a modified Davis method** and the stability of the enzyme activity of the immobilized naringinase was investigated, and the results shown in Table 4 below were obtained.

Furthermore, the half-life of the enzymatic activity of immobilized naringinase is 25
The reaction time was 44.6 days for the reaction at 37°C, and 14.7 days for the reaction at 37°C.

Furthermore, as a result of sensory examination of the intensity of bitterness and flavor of the fruit juice, it was confirmed that no bitterness was felt and the flavor of the fruit was satisfactory.

Example 3 After peeling off the outer skin of a summer mandarin orange fruit, the fruit juice obtained by squeezing and crushing the fruit was heated at 80°C for 3 minutes to prepare a summer mandarin juice. Near 8.6%)
Take 35rfLl into a 100ml Meyer and use Reference Example 5.
Add immobilized naringinase 201v prepared in
A shaking reaction was performed at 40°C for 60 minutes.

Immediately after the reaction, the fruit juice from which the immobilized enzyme was removed was passed through the mouth.
Heated for minutes.

The naringin content in the fruit juice thus obtained was measured by a modified Davis method and was found to be 30.27%.

In addition, no bitterness was perceived sensually, and no deterioration of flavor was detected.

Claims (1)

[Scope of Claims] 1. Immobilized naringinase, in which naringinase is bound by adsorption to a water-insoluble tannin preparation in which a water-insoluble and hydrophilic polymer and a tannin component are bound by covalent bonds, is combined with naringin-containing fruit juice. A method for removing bitterness from a fruit juice containing naringin, which comprises contacting the fruit juice to decompose naringin in the fruit juice. 2. The method for removing bitterness according to claim 1, wherein the operation of bringing the immobilized naringinase into contact with the naringin-containing fruit juice is carried out by a column method in which the naringin-containing fruit juice is passed through a column packed with immobilized naringinase. 3. The method for removing bitterness according to claim 1, wherein the operation of bringing the immobilized naringinase into contact with the naringin-containing fruit juice is carried out by a fluidized bed method in which the naringin-containing fruit juice is passed through a fluidized bed in which the immobilized naringinase is dispersed. 4. The method for removing bitterness according to claim 1, wherein the operation of bringing the immobilized naringinase into contact with the naringin-containing fruit juice is carried out by a batch method of adding and dispersing the immobilized naringinase to the naringin-containing fruit juice in a reaction tank. 5. A water-insoluble and hydrophilic polymer in which the immobilized naringinase has one or more functional groups selected from the group consisting of a hydroxyl group, an amino group, and a carboxyl group in its molecule;
A substance in which naringinase is bound by adsorption to a water-insoluble tannin preparation in which a tannin compound selected from the group consisting of tannin, aminoalkylated tannin, carboxyalkylated tannin, and hydroxyalkylated tannin is bound by a covalent bond. A method for removing bitterness according to claim 1.2.3 or 4. 6 Claim 5, wherein the polymer described in Claim 5 is a polysaccharide or a polysaccharide into which a spacer having a hydroxyl group, an amino group, or a carboxyl group is introduced.
De-bittering method described in section. 1. The method for removing bitterness according to claim 6, wherein the polysaccharide is cellulose, agarose, or crosslinked dextran.