JP2023110255A - Extraction method of efficient protein, and efficient production method of protein extract liquid - Google Patents

Abstract

To maintain pH and temperature of reaction mixture liquid containing enzyme and a protein-extracting subject within an appropriate range, and efficiently extract protein from the protein-extracting subject.SOLUTION: A protein extraction method of the present invention includes: a first mixing process of mixing electrolytic water having the temperature 30°C to 70°C and pH4 to 5, cellulase of the genus Aspergillus origin, and cellulase of the genus Trichoderma origin to obtain enzyme mixture liquid; a second mixing process of mixing the enzyme mixture liquid and protein-extracting subject to obtain reaction mixture liquid; and an extraction process of maintaining the temperature and pH of the reaction mixture liquid at 30°C to 70°C and 4 to 5 respectively and extracting protein from the protein-extracting subject, where the protein-extracting subject is any one or more kinds of tea leaves, used tea leaves, coffee beans, coffee chaff, or seeds.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a method for extracting protein and a method for producing a protein extract.

Most of the proteins contained in tea leaves are poorly soluble or insoluble in water, and are hardly extracted during the production of tea beverages, making it difficult to ingest them in the normal usage of tea beverages. According to the Standard Tables of Food Composition in Japan 2020 Edition (8th Edition), tea leaves, which are raw materials for green tea beverages and black tea beverages, contain about 20 g/100 g to 30 g/100 g of protein. On the other hand, tea leaf infusions such as green tea beverages and black tea beverages extracted using these tea leaves as raw materials contain only about 0 g/100 g to 1.3 g/100 g of protein. A lot of protein remains in tea leaves (used tea leaves).

Like tea leaves, coffee beans, which are the raw material of coffee beverages, also contain protein, but coffee beverages, which are coffee bean infusions, contain almost no protein, and the A large amount of protein remains in coffee husks.

Moreover, these used tea leaves and coffee leaves are mostly discarded as they are without being effectively utilized.

As a method for increasing the extraction efficiency of components contained in tea leaves, a method of enzymatically treating tea leaves has been proposed (see, for example, Patent Document 1). In Patent Document 1, crushed raw tea leaves and an enzyme solution are mixed, and the tea leaf tissue is destroyed and extracted by the enzyme, so that the umami component of the tea leaves can be efficiently extracted. is disclosed.

Japanese Unexamined Patent Application Publication No. 2004-113090 JP 2009-77668 A

However, in the method of enzymatically treating the tea leaves in order to utilize the components contained in the tea leaves, etc., the pH of the reaction mixture of the tea leaves, etc. and the enzyme during the enzymatic treatment reaches a value at which the enzymes are not sufficiently activated. In order to maintain the pH of the reaction mixture during the enzymatic treatment within a range suitable for the characteristics of the enzyme, a buffering agent, which is one of the pH adjusting agents, may be added as the enzyme treatment may not progress due to changes over time. In some cases, a buffer solution containing is used as a solvent for the reaction mixture, or a pH adjuster such as an acid, a base, or a salt is added to the reaction mixture as appropriate. When chemicals such as buffers and pH adjusters are added, acids, bases, and salts that are not originally contained in tea leaves are added to the reaction mixture. When ingested, these drugs, acids, bases, and salts are also ingested, which is not preferable.

In addition, in order to increase the efficiency of the enzymatic reaction, it is necessary to keep the temperature of the reaction mixture during enzymatic treatment within a range appropriate for the activity of the enzyme, in accordance with the properties of the enzyme.

Therefore, the present invention focuses on the above problems, and maintains the pH and temperature of the reaction mixture containing the enzyme and the protein extraction target in an appropriate range to extract the protein from the protein extraction target. With the goal.

In the protein extraction method of the present invention, electrolyzed water at a temperature of 30° C. to 70° C. and a pH of 4 to 5, cellulase originating from Aspergillus, and cellulase originating from Trichoderma are mixed to obtain an enzyme mixture solution. a second mixing step of mixing the enzyme mixture and the protein extraction target to obtain a reaction mixture; and setting the temperature of the reaction mixture to 30° C. to 70° C., and and an extraction step of extracting protein from the object for protein extraction while maintaining a pH of 4 to 5, wherein the object for protein extraction is tea leaves, used tea leaves, coffee beans, coffee leaves, or nuts and seeds. or one or more.

Further, in the protein extraction method of the present invention, the extraction step includes continuously or intermittently bringing high-humidity hot air into contact with the reaction mixture to raise the temperature of the reaction mixture to 30° C. to 70° C., and A step of maintaining the pH at 4-5 may also be used.

In the method for producing a protein extract of the present invention, a mixed solution containing electrolyzed water at a temperature of 30° C. to 70° C. and a pH of 4 to 5, cellulase originating from the genus Aspergillus, and cellulase originating from the genus Trichoderma is prepared. , while applying high humidity hot air generated using electrolyzed water having a desired pH so that the temperature of the mixed solution during the reaction is 30 ° C. to 70 ° C. and the pH is maintained at 4 to 5. to extract an extract containing protein from the protein extraction target.

Since the present invention uses electrolyzed water at a temperature of 30° C. to 70° C. and a pH of 4 to 5, the electrolyzed water containing no chemicals such as pH adjusters is used to prepare a reaction mixture containing an enzyme and a protein extraction target. By maintaining the pH and temperature of the liquid within appropriate ranges, protein can be extracted from the protein extraction target.

An embodiment of the present invention will be described.

In the protein extraction method of the present embodiment, electrolyzed water having a temperature of 30 ° C. to 70 ° C. and a pH of 4 to 5, cellulase derived from Aspergillus, and cellulase derived from Trichoderma are mixed, and the enzyme is mixed. A first mixing step of obtaining a liquid; a second mixing step of mixing the enzyme mixture and the protein extraction target to obtain a reaction mixture; is maintained at 4-5 to extract the protein from the protein extraction target.

In the extraction step, hot and humid air is continuously or intermittently brought into contact with the reaction mixture to maintain the reaction mixture at a temperature of 30° C. to 70° C. and a pH of 4 to 5. may be

In the method for producing a protein extract of the present invention, a mixed solution containing electrolyzed water at a temperature of 30° C. to 70° C. and a pH of 4 to 5, cellulase originating from the genus Aspergillus, and cellulase originating from the genus Trichoderma is prepared. , while applying high humidity hot air generated using electrolyzed water having a desired pH so that the temperature of the mixed solution during the reaction is 30 ° C. to 70 ° C. and the pH is maintained at 4 to 5. to extract an extract containing protein from the protein extraction target.

[Protein extraction object]
The protein extraction object in the protein extraction method of the present embodiment is any one or more of tea leaves, used tea leaves, coffee beans, coffee leaves, and nuts and seeds.

(Tea leaves and used tea leaves)
The tea leaves in the present embodiment refer to fresh leaves immediately after plucking from tea trees, or the state before processing the fresh leaves and using them as raw materials for tea beverages, and fresh leaves immediately after plucking, crude tea, finished tea , and intermediate products in each tea processing step. In addition, the presence or absence of the fermentation process in the tea production process is not limited, that is, unfermented tea such as green tea and fermented tea such as black tea can be used as tea leaves. Moreover, when fresh leaves are used as an object for protein extraction, it is preferable to subject the fresh leaves to heat treatment in order to inactivate the enzymes contained in the fresh leaves.

Tea leaves in the present embodiment refer to residues remaining after solution extraction using sencha as a raw material. The state after solution extraction has a high water content and is prone to putrefaction. Therefore, if used tea leaves are not immediately used as an object for protein extraction in this embodiment, they may be dried and stored. There are no particular restrictions on the method of drying used tea leaves, and for example, sun drying or mechanical drying may be used.

(coffee beans and coffee husks)
The coffee beans in the present embodiment refer to the state from the green beans harvested from the coffee tree before drying to the state before processing the green beans and using them as raw materials for coffee beverages. Includes beans, roasted beans, and intermediate products in each processing step.

The coffee husks in the present embodiment refer to residues remaining after extracting a coffee beverage using roasted beans as a raw material. The state after extraction of the coffee beverage has a lot of moisture and is easily spoiled. Therefore, if the coffee husks are not used immediately as the protein extraction object of this embodiment, they may be dried and stored. The method for drying the coffee grounds is not particularly limited, and may be, for example, sun drying or mechanical drying.

(seeds and nuts)
The nuts and seeds in this embodiment refer to edible fruits and seeds, and examples thereof include almonds, walnuts, and hazelnuts. Nuts and seeds can be harvested to remove seed coats and shells, processed by drying or heating, or leftovers after extracting some components such as fats and starches contained in nuts and seeds. It may be in a state of residue such as.

In the present embodiment, the plant raw material as described above is used as a protein extraction target. The variety of each plant raw material is not particularly limited.

Also, the shape of the protein extraction target is not particularly limited, and for example, in order to increase the reaction efficiency with cellulase, it may be pulverized before being used in the protein extraction method of the present embodiment. Alternatively, the protein extraction target may be pulverized after the enzyme mixture is sufficiently permeated into the protein extraction target, or the protein extraction target may be pulverized in the extraction step.

[Electrolyzed water]
Electrolyzed water in the present embodiment is an aqueous solution obtained by electrolyzing water, and acidic electrolyzed water with a pH of 4 to 5 is used in the present embodiment. The electrolyzed water in this embodiment can be generated using an existing electrolyzed water generator.

[Cellulase]
Cellulases are enzymes that hydrolyze the glycosidic bonds of β-1,4-glucans such as cellulose. Cellulase is classified according to its mode of action, and includes endoglucanase that degrades β-1,4-glucan from the inside and exoglucanase that degrades β-1,4-glucan from the end. Either cellulase can be used without limitation.

In the present embodiment, cellulase originating from the genus Aspergillus and cellulase originating from the genus Trichoderma are used as the cellulase. Moreover, the form of cellulase is not limited, and it may be in the form of powder or may be in the form of liquid in which cellulase is dissolved in a solvent. Examples of these cellulases include cellulase XL-531 (originating from Aspergillus) (manufactured by Nagase ChemteX Corporation) and cellulase SS (originating from Trichoderma) (manufactured by Nagase ChemteX Corporation).

By enzymatically treating the plant material, which is the object of protein extraction, using cellulase, the cellulose that constitutes the cell wall can be decomposed, and components such as proteins contained in the object of protein extraction can be efficiently extracted. .

The amount of cellulase to be used can be appropriately set in consideration of the cellulose content in the protein extraction target and the titer of the cellulase to be used. For example, in the first mixing step, the amount of Aspergillus-derived cellulase to be mixed can be 0.01% by mass to 2% by mass with respect to the electrolyzed water. The amount of cellulase originating from the genus Trichoderma can be 0.01% by mass to 2% by mass with respect to the electrolyzed water. For example, when using cellulase XL-531 or cellulase SS, each of them can be mixed in 0.5% to 2.0% by volume with respect to electrolyzed water.

[pH adjuster]
A pH adjuster adjusts the hydrogen ion concentration, and examples thereof include acids, bases, and salts. Since each enzyme such as cellulase has a pH at which it is most active, it is necessary to maintain the pH of the reaction mixture containing the enzyme and enzyme substrate within a specific range in the enzymatic reaction. In order to maintain the pH within a specific range, it is common to use a buffer containing a pH adjuster with a buffering action as a solvent for the enzyme mixture, or add a pH adjuster such as an acid or base during the enzyme reaction. Adding is done. However, in this embodiment, no pH adjuster is used for the purpose of adjusting the pH of the reaction mixture.

[First mixing step]
In the first mixing step, electrolyzed water having a temperature of 30° C. to 70° C. and a pH of 4 to 5, cellulase originating from the genus Aspergillus, and cellulase originating from the genus Trichoderma are mixed to obtain an enzyme mixture. It is a process.

Specifically, electrolyzed water having a pH of 4 to 5 is generated in advance by an electrolyzed water generator, and the temperature is adjusted to 30°C to 70°C. The temperature can be adjusted by heating a container containing electrolyzed water from the outside with a heater or the like, or by bringing high humidity hot air containing electrolyzed water of pH 4 to 5 into contact with the electrolyzed water in the container. be done.

The high-humidity hot air in the present embodiment means hot air humidified to high humidity by spraying electrolyzed water generated by an electrolyzed water generator toward a burner or the like in the form of a mist, as shown in Patent Document 2, for example. and Also, the high-humidity hot air is in the form of mist whose temperature and humidity can be changed. This high-humidity hot air contains high-density steam with high thermal conductivity, and the temperature can be adjusted without greatly increasing the amount of liquid.

The above-described electrolyzed water at a temperature of 30° C. to 70° C. and a pH of 4 to 5 is mixed with cellulase originating from the genus Aspergillus and cellulase originating from the genus Trichoderma to obtain an enzyme mixture. . At this time, pH adjusters such as acids, bases and salts are not added.

[Second mixing step]
The second mixing step is a step of mixing the enzyme mixture obtained in the first mixing step and the protein extraction target to obtain a reaction mixture. The mixing ratio of the enzyme mixture and the protein extraction target can be appropriately set in consideration of the cellulose content and the cellulase titer. It can be about 50L. In the second mixing step, in order to suppress the temperature drop due to mixing the protein extraction target, it is heated from the outside or contacted with high humidity hot air containing electrolyzed water of pH 4 to 5. good too.

[Extraction process]
The extraction step is a step of extracting protein from the protein extraction target by maintaining the reaction mixture obtained in the second mixing step at a temperature of 30° C. to 70° C. and a pH of 4 to 5. The temperature and pH conditions of the reaction mixture were adjusted to the optimum conditions for cellulase in consideration of the reaction efficiency of cellulase. Further, in order to increase the reaction efficiency between the protein extraction target and cellulase, the protein extraction target may be pulverized in the extraction step.

The temperature and pH may be maintained, for example, by continuously or intermittently contacting the reaction mixture with hot, humid air generated from electrolyzed water of appropriate pH. The electrolyzed water with an appropriate pH for maintaining the pH refers to more alkaline electrolyzed water, for example, when the reaction mixture is more acidic than pH 4-5, and the reaction mixture has a pH of 4-5. When it becomes more alkaline, it refers to more acidic electrolyzed water. In addition, as a method of bringing the high-humidity hot air into contact with the reaction mixture, for example, there is a method of supplying high-humidity hot air toward the liquid surface of the reaction mixture, or a method of providing a high-humidity hot air outlet in the reaction mixture. A method of installing and supplying high-humidity hot air into the reaction mixture is exemplified. Further, the contact with the high-humidity hot air may be continued throughout the extraction process, or may be intermittently stopped while the temperature and pH are maintained. Since the high-humidity hot air contains high-density steam with high thermal conductivity, the liquid volume of the reaction mixture is greatly increased by maintaining the temperature and pH using the method of contacting the high-humidity hot air of electrolyzed water. temperature and pH conditions can be maintained without That is, even if the high-humidity hot air is brought into contact, the liquid volume of the reaction mixture hardly increases, and does not increase by 0.5% by mass. A decrease in cellulase reaction efficiency can be suppressed.

The temperature and pH of the reaction mixture in the extraction step may be maintained by simply adding electrolyzed water having an appropriate pH to the reaction mixture or heating the reaction mixture with a heater or the like. .

The maintenance time (enzyme reaction time) in the extraction step can be appropriately set according to the amount and properties of the protein extraction target, and the temperature of the reaction mixture is maintained at 30 ° C. to 70 ° C. for 30 minutes to 90 minutes. And by maintaining the pH at 4-5, the protein can be efficiently extracted. For example, when the protein extraction target is tea leaves, it can be about 60 to 70 minutes, and when the protein extraction target is tea leaves, it can be about 45 to 60 minutes.

Addition of a pH adjuster, which is generally used when performing an enzymatic reaction, means addition of a compound such as an acid, a base, or a salt that is not originally contained in a protein extraction target. In the present embodiment, the electrolyzed water having a temperature of 30° C. to 70° C. and a pH of 4 to 5 is used. A protein can be extracted from a protein extraction target by maintaining the pH and temperature of the reaction mixture containing the mixture in an appropriate range. As a result, it becomes possible to effectively utilize proteins contained in protein extraction targets such as tea leaves, which have conventionally been difficult to utilize.

(Production of protein extract)
In the production of the protein extract, a mixture containing electrolyzed water at a temperature of 30 ° C. to 70 ° C. and a pH of 4 to 5, cellulase derived from Aspergillus, and cellulase derived from Trichoderma is added to the reaction. The temperature of the mixed solution is maintained at 30° C. to 70° C. and the pH is maintained at 4 to 5 by applying high-humidity hot air generated using electrolyzed water having a desired pH to act on the protein extraction target. and extracting an extract containing protein from the protein extraction target.

Specifically, the enzyme mixture described in the first mixing step is allowed to act on the protein extraction target to obtain the reaction mixture described in the second mixing step. The temperature of the mixed solution during the reaction is maintained at 30° C. to 70° C. and the pH is maintained at 4 to 5 using electrolyzed water having a desired pH. Then, the protein described in the extraction step is extracted to produce a protein extract.

In addition, the best configuration, method, etc. for carrying out the present invention have been disclosed in the above description, but the present invention is not limited thereto. That is, although the present invention has been described primarily in terms of particular embodiments, it is possible to modify the above-described embodiments in terms of shape, material, quantity, and/or size without departing from the spirit and scope of the invention. Various modifications can be made to other detailed configurations by those skilled in the art. Therefore, the descriptions that limit the shape, material, etc. disclosed above are exemplified to facilitate understanding of the present invention, and do not limit the present invention. The description by the name of the member excluding some or all of the limitations such as , etc. is included in the present invention.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples. However, the present invention is not limited by the following examples. In this example, protein extraction using tea leaves and used tea leaves as a protein extraction target is exemplified.

[Example 1]
(First mixing step)
20 L of pH 4 electrolyzed water was placed in a container with a volume of 40 L, and the high-humidity hot air generated from the pH 4 electrolyzed water was brought into contact with the liquid surface of the electrolyzed water in the container, and the temperature was adjusted to 45°C. At this time, the amount of electrolyzed water was almost unchanged even when it was brought into contact with high-humidity hot air. HOX-60PA (manufactured by Hoshizaki Co., Ltd.) was used as the electrolyzed water generator, and high-humidity hot air was generated by spraying the electrolyzed water generated by the electrolyzed water generator toward the burner.

200 mL of cellulase XL-531 (Nagase ChemteX Co., Ltd.) in the electrolyzed water having a temperature of 45 ° C. and a pH of 4 obtained in this way (1% by volume with respect to the electrolyzed water), Cellulase SS (specific gravity 1.1 g ~1.2 g/mL, Nagase ChemteX Co., Ltd.) was added to 200 mL (1% by volume with respect to electrolyzed water) and stirred to mix. An enzyme mixture was thus obtained.

(Second mixing step)
Next, 1 kg of tea leaves was added to the enzyme mixture and mixed to obtain a reaction mixture. As the tea leaves, fresh leaves immediately after picking from a tea tree were agitated and steamed at 100° C. for 60 seconds to kill the leaves (obtained from JA Enshu Yumesaki Tea Leaf Promotion Center).

(Extraction process)
Next, an extraction step was performed. That is, while stirring the reaction mixture, the high-humidity hot air generated from the electrolyzed water is brought into contact with the liquid surface of the reaction mixture, and the temperature of the reaction mixture is 40 ° C. to 50 ° C. and the pH is 4 to 4. 5 was maintained for 60 minutes. At this time, the liquid volume of the reaction mixture did not change even when it was brought into contact with high-humidity hot air. Protein was extracted from the tea leaves as described above.

(evaluation and results)
The reaction mixture after the extraction step was passed through a mixer and used as a sample. The amount of protein and the amount of cellulose in this sample were measured. The measurement was consigned to the Japan Food Research Laboratories, the protein was measured by the combustion method, and the cellulose was measured by D.I. A. T. It was carried out according to the method of Southgate et al. (J. Sci. Food. Agric., 20, 331 (1969)). Table 1 shows the results.

As described above, Table 1 shows that the reaction mixture obtained by adding and mixing an enzyme mixture (20 L of electrolyzed water, 200 mL of cellulase XL-531, and 200 mL of cellulase SS) to 1 kg of tea leaves was subjected to an extraction step, and then mixed with a mixer. It shows the content of protein and cellulose in 100 g of a sample subjected to scalding. As shown in Table 1, in the sample of Example 1, 0.4 g of tea leaf-derived protein was detected per 100 g of sample by the combustion method, and cellulose was not detected. This indicated that cellulose was degraded and proteins contained in tea leaves were detected. When the specific gravity of cellulase SS is 1.1 g/mL and the specific gravity of cellulase XL-531 is 1.1 g/mL, the reaction mixture subjected to the extraction process is 21.44 kg (electrolyzed water 20 kg, cellulase 0 .44 kg and 1 kg of tea leaves). It was assumed that there was no increase since the liquid volume remained almost unchanged even when it was brought into contact with high-humidity hot air. Therefore, the above results corresponded to extracting approximately 8.6 g of protein from 100 g of tea leaves.

(Example 2)
The procedure was carried out in the same manner as in Example 1, except that used tea leaves were used as the object for protein extraction. As the used tea leaves, sencha was used as a raw material, and the residue after solution extraction was dried in the sun (obtained from JA Enshu Yumesaki Tea Leaf Promotion Center). The solution extraction was carried out using 100° C. hot water in a 200 mL container with respect to 3 g of tea leaves for an extraction time of 180 seconds. Further, the pH and temperature were maintained for 45 minutes in the extraction process. Moreover, in the extraction process, the liquid volume of the reaction mixture was almost unchanged. Table 1 shows the results.

As shown in Table 1, in the sample of Example 2, 0.8 g of protein derived from used tea leaves/100 g of sample was detected by the combustion method, and no cellulose was detected. This indicated that cellulose was degraded and proteins contained in used tea leaves were detected. The above results, calculated in the same manner as in Example 1, corresponded to the extraction of approximately 17.2 g of protein from 100 g of tea leaves.

From the above evaluation results, according to Examples 1 and 2, which are exemplary embodiments of the present invention, the pH and temperature of the reaction mixture containing the enzyme and the protein extraction target tea leaves and tea leaves are adjusted to the appropriate range. It was shown that the protein can be efficiently extracted from the protein extraction target by maintaining the .

Claims (3)

A first mixing step of mixing electrolyzed water having a temperature of 30° C. to 70° C. and a pH of 4 to 5, cellulase originating from the genus Aspergillus, and cellulase originating from the genus Trichoderma to obtain an enzyme mixture;
a second mixing step of obtaining a reaction mixture by mixing the enzyme mixture and a protein extraction target;
an extraction step of extracting protein from the protein extraction target by maintaining the reaction mixture at a temperature of 30° C. to 70° C. and a pH of 4 to 5,
The protein extraction target is any one or more of tea leaves, used tea leaves, coffee beans, coffee leaves, or nuts and seeds,
Protein extraction method. The extraction step is a step of continuously or intermittently contacting the reaction mixture with high-humidity hot air to maintain the reaction mixture at a temperature of 30° C. to 70° C. and a pH of 4 to 5. be,
The protein extraction method according to claim 1. A mixture containing electrolyzed water having a temperature of 30° C. to 70° C. and a pH of 4 to 5, cellulase originating from Aspergillus, and cellulase originating from Trichoderma was heated to 30° C. during the reaction. ° C. ~ 70 ° C., and by applying high humidity hot air generated using electrolyzed water having a desired pH so that the pH is maintained at 4 to 5, the protein extraction target is acted on, and from the protein extraction target. A method for producing a protein extract, comprising extracting an extract containing protein.