JP2011234694A - Method for producing soybean beta-amylase preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high-titer soybean beta-amylase preparation applicable to the food industry, etc.SOLUTION: Soybean extract liquid or soybean whey is filtered through a microfiltration membrane whose pore diameter is 0.1-0.45 μm, and the filtered liquid is concentrated till the beta-amylase titer is 10,000 units/g or larger by using an ultrafiltration membrane under the condition of liquid temperature of 15-60 °C, and dried to obtain the soybean beta-amylase preparation. Preferably, pH of the soybean extract liquid or soybean whey when filtering through the microfiltration membrane is adjusted to 4.0-5.0, and pH of the concentrated liquid before dried is adjusted to 4.2-4.8. In this way, the high-titer soybean beta-amylase preparation applicable to the food industry, etc., can be efficiently produced while avoiding problems concerning the waste liquid treatment or introduction of additional production steps or equipment.

Description

  The present invention relates to a method for producing a soybean β-amylase preparation that can be used for food industry applications and the like.

β-Amylase is used in the field of food industry and the like as an enzyme agent used in the production of maltose and syrup and anti-aging preparations such as candy and candy. For example, soybean-derived β-amylase is an enzyme extracted from plant seeds with eating habits, and has high heat resistance and stability compared to β-amylase derived from other plants (wheat, sweet potato, etc.). Therefore, it is widely used. Such a β-amylase preparation is required to be a high-titer preparation that can achieve the desired effect with a small amount of addition and can be easily prepared to a desired titer. It is advantageous to use a high-potency soybean β-amylase preparation of about 10,000 units / g or more.
Since a high-titer β-amylase preparation can achieve the desired effect with a smaller amount of use, it has the advantage that it does not affect the color and flavor of the food used. In addition, when producing a high-titer β-amylase preparation, when the impurity content in the preparation is reduced and the purity of β-amylase is increased, the color tone of the obtained powder β-amylase preparation becomes whiter. Thus, the transparency of the obtained liquid β-amylase preparation is expected to be further increased. Also from such a viewpoint, a high-titer β-amylase preparation is demanded particularly for foods that require high transparency such as chickenpox and foods that require high whiteness such as strawberries.

  As an industrial production method of soybean β-amylase preparation, general vacuum evaporation concentration, freeze-drying, spray-drying treatment, etc. can be considered, but in any case, the titer of the obtained β-amylase preparation is sufficient. is not. In addition, a method of purification using a column packed with an aluminum silicate or the like as an adsorbent is also known (for example, see Patent Document 1). The titer of the soybean β-amylase preparation obtained by this method is also known. Only low-potential preparations up to 2,000 units / g can be produced.

  As another method, a salting out method in which mirabilite (sodium sulfate) or the like is added is known. For example, by directly salting out a soybean raw material containing β-amylase, or by salting out a crude enzyme solution that has been concentrated to a certain titer using the above-mentioned various methods, etc., 50,000 units / g A soybean β-amylase preparation having a higher titer than powder can be obtained (for example, see Patent Document 2). However, the salting-out method requires a step of pulverizing the lump-like dried solids in order to obtain a uniform powder because the recovered product is a clay-like lump, and a large amount of high-concentration salt solution is required. It also has the problem of waste disposal as a by-product.

  Also known is a method of obtaining a β-amylase preparation by adding a metal salt or hydroxide to a crude enzyme solution containing β-amylase and using an ultrafiltration membrane (UF membrane) or a reverse osmosis membrane. (For example, refer to Patent Document 3). However, even if concentration is simply performed using a UF membrane, it is still possible to produce only liquid products having a titer of about 2,000 units / g. The problem in the manufacturing method using these membranes is clogging of the membranes due to precipitates, viscous substances, and the like that are generated in large amounts during UF membrane concentration.

  Therefore, as a method for improving the above-mentioned problems, UF membrane concentration is performed at about 50 ° C., and the concentrated solution is collected once before clogging of the membrane and allowed to stand at 10 ° C. and then decanted or centrifuged. A method has been proposed in which the precipitate is removed or the pH of the concentrated solution is adjusted to 5.0 to 7.0 to partially redissolve the precipitate that causes clogging (for example, see Patent Document 3). . It is possible to produce high-potency liquid soybean β-amylase exceeding 10,000 units / g by introducing such precipitation removal / re-dissolution treatment one or more times during the UF membrane concentration. It is.

  However, in this method, the concentration process is temporarily interrupted, the concentrated solution is taken out from the concentrator and transferred to another container, and additional processes and facilities necessary for pH adjustment, precipitation formation / removal, or centrifugation are required. Introduction is required. In the case where it is necessary to cool the concentrated solution and leave it for a certain period of time for the formation of a precipitate, the time for that is also added to the number of manufacturing days. Moreover, even if it is a necessary treatment individually, the transfer of enzyme solution from container to container and frequent adjustment of pH may cause a decrease in enzyme titer and recovery rate. It can be a cost burden. In particular, in providing enzyme preparations for foods, there is a demand for a method that can be efficiently manufactured while avoiding removal, movement, and adjustment of the concentrated solution as much as possible from the viewpoint of microbial contamination and alteration. .

Japanese Examined Patent Publication No. 63-48517 Japanese Patent Publication No.57-11636 Japanese Patent No. 3513193

  The present invention provides a high-titer soybean β-amylase preparation that can be used for food industry applications, etc., and avoids the problem of disposal and the introduction of additional steps or equipment, It is an object of the present invention to provide a method for producing a soybean β-amylase preparation capable of efficiently producing a soybean β-amylase preparation.

  In order to solve the above-mentioned problems, the present inventor has conducted intensive studies, and a soy extract adjusted to pH 4.0 to 5.0 on a microfiltration membrane (MF membrane) having a pore size of 0.1 to 0.45 μm or UF is passed through soy whey, using a ultrafiltration membrane with a fractional molecular weight of 13,000 or less, and a β-amylase titer of 10,000 units / g or more with a passing liquid at 15-60 ° C. It has been found that high-titer liquid soybean β-amylase preparation can be obtained by membrane concentration. Furthermore, when drying to prepare a powder formulation, adjusting the pH of the β-amylase concentrate to 4.2 to 4.8 is extremely effective in suppressing loss of β-amylase titer. I knew that there was. Then, by drying the obtained concentrated liquid, it was confirmed that a high-potency powdered soybean β-amylase preparation of 30,000 to 50,000 units / g could be produced, and the present invention was completed.

That is, the present invention is as follows.
(1) A soybean extract or soybean whey is passed through an MF membrane having a pore size of 0.1 to 0.45 μm, and the passing solution is used under the condition of a liquid temperature of 15 to 60 ° C. and using a UF membrane having a molecular weight cut off of 13,000 or less. Then, the method for producing a soybean β-amylase preparation is characterized by performing ultrafiltration concentration until the β-amylase titer is in a liquid state of 10,000 units / g or more.
(2) The method for producing a soybean β-amylase preparation according to (1), wherein the pH of the soybean extract or soybean whey when passing through the MF membrane is adjusted to 4.0 to 5.0.
(3) The method for producing a soybean β-amylase preparation according to (1) to (2), wherein the UF membrane concentration step after passing through the MF membrane is continuously and / or aseptically performed.
(4) A method for producing a powdered soybean β-amylase preparation, characterized in that the pH of the soybean β-amylase solution obtained by the method of (1) to (3) is adjusted to 4.2 to 4.8 and dried. .
(5) pH adjustment is performed using one or more diluted acidic or diluted alkaline solutions selected from sodium hydroxide, sodium bicarbonate, sodium carbonate, sodium acetate, hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, and phosphate. (2 ) And (4), a method for producing a soybean β-amylase preparation.
(6) A soybean β-amylase preparation obtained by the method for producing a soybean β-amylase preparation according to (1) to (5).

  According to the present invention, it is possible to reduce the burden of high salt concentration waste liquid treatment during the conventional salting-out recovery, and to perform the membrane separation and concentration step by step using two different membranes. A high-potency soybean β-amylase preparation that can be used for food industry applications and the like can be produced without introducing steps such as removal and re-dissolution of the precipitate.

(Soybean β-amylase)
The “soybean β-amylase” of the present invention is an enzyme that cleaves from starch or the like with α-1,4-linked glucose in maltose units. The soybean β-amylase titer of the present invention was measured by the Somoji method for the amount of reduction produced by reacting in 1% purified potato starch solution as a substrate in 83 mM sodium acetate buffer (pH 5.5) at 40 ° C. for 20 minutes. The amount of enzyme that produces a reduced amount corresponding to 1 mg of glucose per 10 minutes is defined as 1 unit.

(Soybean β-amylase preparation)
The “soybean β-amylase preparation” of the present invention refers to liquid (including aqueous solution, suspension, slurry, etc.) and powder preparations. Also included are powdered preparations molded into suitable shapes.
The enzyme titer of the soybean β-amylase preparation of the present invention is not particularly limited as long as it is high enough to exhibit the effect required according to the purpose of use, but per unit weight practical for food industry applications and the like. In general, the titer is preferably 10,000 units / g or more in the case of a liquid enzyme preparation and 30,000 to 50,000 units / g or more in the case of a powdery enzyme preparation. If a preparation with a higher titer can be obtained, a preparation with a desired titer can be freely prepared, which is more preferable.

(Soybean extract, soybean whey)
The “soybean extract” as used in the present invention refers to an extract from soybean that is a raw material for recovering soybean β-amylase. Soybean is extracted by an arbitrary method, and may include various crudely purified soybean β-amylase compositions, solutions and suspensions of roughly purified soybean β-amylase preparations, and the like. The term “soy whey” as used in the present invention is a general term for ingredients produced as a by-product in the process of producing separated soy protein from defatted soybeans and low-temperature defatted soybeans, and generally a soy protein aqueous solution obtained by water extraction of soybeans. From the above, it is the aqueous layer section obtained when the protein is separated by acid precipitation, and its preparation method is well known to those skilled in the art.

  Although the specific manufacturing method of "soybean extract" is not limited at all, For example, finely crushed raw soybeans are suspended in tap water to a concentration of 7 to 10% (w / w), and occasionally stirred. After soaking for 10 to 30 minutes, the mixture is allowed to stand for about 10 minutes to remove crude solids by decantation, centrifugation or filter cloth filtration, and 0.1 to 1.0 N acetic acid or hydrochloric acid is added. The soybean extract of the present invention can be prepared by adjusting the pH to 4.5 ± 0.5 at ˜50 ° C. and removing the generated precipitate.

(MF membrane, UF membrane)
The “MF membrane” of the present invention is a membrane for performing solid-liquid separation, generally having a pore size of about 0.01 to 10 μm, and the substance to be blocked is a certain shape or more. Particles or fungus bodies of the size. Examples of commercially available MF membranes include module types ULP-143 (nominal pore diameter 0.45 μm), USP-143 (nominal pore diameter 0.1 μm), PSP-103 (nominal pore diameter 0.1 μm) manufactured by Asahi Kasei Chemicals Corporation. , PMP-102 (nominal pore diameter 0.25 μm) and the like.
In producing the soybean β-amylase preparation of the present invention, these MF membranes having a pore diameter of 0.01 to 10 μm can be used. In particular, when an MF membrane having a pore size of 0.1 to 0.45 μm is used, β-amylase passes through and at the same time efficiently removes various substances that interfere with the concentration work, such as viscous substances and solids. Can be preferred. In addition, when the MF membrane having the above pore diameter is employed, it is preferable because it can also serve as a sterilization step in which contaminating microorganisms can be efficiently removed at the same time.

  The “UF membrane” of the present invention is a membrane designed by setting various predetermined molecular weight ranges, and prevents polymer substances and colloidal substances exceeding the set size and has a size smaller than that. It is a “molecular sieve” that permeates low molecular weight substances and ions. Examples of commercially available MF membranes include module types ACP3013D (fraction molecular weight 13,000), SLP-2053 (fraction molecular weight 10,000), and AIP3013D (fraction molecular weight 6,000) manufactured by Asahi Kasei Chemicals Corporation. There is. The β-amylase of the present invention has a molecular weight of about 60,000, and in producing the soybean β-amylase preparation of the present invention, a membrane having a size that does not allow β-amylase to pass through is selected and used for efficient concentration. It can be performed. That is, in consideration of the fractional molecular weight of 60,000 or less and the recovery rate, the concentration can be efficiently performed by using a UF membrane preferably having a fractional molecular weight of 13,000 or less.

(Production of β-amylase preparation by MF membrane passage and UF membrane concentration)
The β-amylase preparation of the present invention is characterized in that the soybean extract and soybean whey are first purified by passing through an MF membrane and then concentrated using a UF membrane. Thus, by using two types of membranes step by step, the clogging phenomenon that has occurred during the conventional UF concentration can be suppressed, and high-magnification concentration can be easily realized.

  In order to produce the soybean β-amylase preparation of the present invention, first, a soybean extract or soybean whey containing soybean β-amylase is subjected to an MF membrane passage step. In the present invention, it is important to perform MF membrane passage treatment prior to UF membrane concentration. Due to this operation, some of the viscous substances that are present in the soybean extract and soybean whey and interfere with the concentration of soybean β-amylase and the cause of lowering the titer per unit weight of soybean β-amylase preparation A part of the contaminating substance can be efficiently removed prior to UF membrane concentration. As a result, the titer content of β-amylase is relatively increased, the production of concentrated precipitates and viscous substances in the UF membrane concentration step can be dramatically reduced, and a high-titer preparation can be prepared.

  Next, the MF membrane passage liquid is subjected to a UF membrane concentration step. There are a method in which both steps are performed continuously and a batch method in which the UF membrane concentration step is performed after all the MF membrane passage steps are completed. Either method can be used, and which method should be used should be selected based on multifaceted judgments with emphasis on overall efficiency, but it can be performed quickly and reduces the risk of microbial contamination. From the viewpoint of easy reduction, both steps are performed continuously, more preferably under aseptic conditions. That is, an MF membrane having a pore size of 0.01 to 0.45 μm, preferably 0.1 to 0.45 μm is adopted in the MF membrane treatment performed earlier, and the UF membrane concentration step after passing through the MF membrane is performed under aseptic conditions. If it carries out by, manufacture of the soybean beta-amylase formulation which reduced the risk of microbial contamination can be implement | achieved easily.

  When concentrating the UF membrane, a washing (diafiltration) step can be appropriately added. Specifically, when transferring the MF membrane permeate into the UF device and concentrating it, sanitized or sterilized tap water, distilled water, ion-exchanged water, an arbitrary buffer, etc. are added as necessary. UF concentration can be performed. This step is effective for eliminating the substances having a molecular weight equal to or lower than the molecular weight of the UF membrane from the UF membrane concentrate to increase the purity of soybean β-amylase.

(Concentration ratio)
The concentration factor in the UF membrane is not particularly limited, but as a guideline, it is preferably used for food industry applications, etc. by performing concentration so that the β-amylase titer in the concentrate is 10,000 units / g or more. A possible high-titer soybean β-amylase preparation can be obtained. The concentration ratio required for this depends on the specific activity of soybean β-amylase contained in the raw material. For example, according to the method of the present invention, the amount of the first raw material liquid (soy extract, soybean whey) On the other hand, concentration 50 times or more, preferably 100 times or more can be performed smoothly.

(PH during treatment)
In the present invention, setting the pH of the raw material liquid (soybean extract or soybean whey and concentrated solution) to a predetermined range when passing through the MF membrane and performing UF membrane concentration and washing is β-amylase. This is important for preventing loss of activity and improving the yield of the resulting amylase preparation.
Specifically, the soybean extract or soybean whey during passage through the MF membrane is adjusted to pH 3.9 to 5.1, preferably pH 4.0 to 5.0, more preferably pH 4.3 to 5.0. That is important. If the raw material liquid adjusted to such a pH range is processed, high-magnification soybean β- can be obtained smoothly when high-concentration concentration is performed when MF membrane passage and subsequent UF concentration and washing are performed. Amylase preparations can be obtained with high recovery.

  In addition, it is important to adjust the pH of the ultrafiltration membrane concentrate when performing a drying process such as spray drying to obtain a powder formulation. The concentrate is adjusted to pH 3.9 to 5.1, preferably pH 4.2 to 4.8. In the production process of the present invention, the pH of the concentrated solution that has been subjected to the two-stage membrane treatment of the MF membrane and the UF membrane is usually lowered to 4.0 or lower, but the pH of the concentrated solution when performing a drying treatment such as spray drying treatment By adjusting to within the above range, a soybean β-amylase preparation can be obtained with a higher recovery rate.

  One of the most important things in the production of an enzyme agent is to recover the enzyme without compromising its potency. Therefore, in general, the handling operation is usually performed under conditions according to the stable pH range of the enzyme to be recovered. In the case of soybean β-amylase, the isoelectric point is pH 5.5 and the stable pH range is pH 5-6. However, in the method of the present invention, unexpectedly, MF membrane passage and UF membrane concentration and washing are performed in a pH range in the acidic range rather than the stable pH range of soybean β-amylase. The inventor has also found that the titer recovery rate can be increased by pulverization in the acidic pH range rather than the stable pH range of soybean β-amylase in the drying treatment for obtaining. Although the detailed cause is unknown, in the presence of various components in the physical operation of membrane concentration, the isoelectric point and solubility of each component, the passing material and the properties of the material remaining inside the membrane It is speculated that setting the pH of the concentrated liquid in such a pH range due to changes, quantitative balance changes, and the like resulted in an increase in soybean β-amylase activity recovery rate.

(PH adjustment)
The pH in the production method of the present invention is adjusted by a substance that does not impair the β-amylase titer, for example, sodium hydroxide, sodium bicarbonate, sodium carbonate, sodium acetate at a low concentration (about 0.1 to 1 molar concentration). , Hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, phosphate and the like can be added. The pH adjustment may be performed before each step depending on the pH of the raw material, or may be additionally performed in the middle of the film treatment as necessary. If it is not necessary to perform additional pH adjustment depending on the raw material used or the lot of processing, the film may be subjected to film processing without performing special adjustment processing.
In the method of the present invention, the precipitation re-dissolution step by pH adjustment during concentration, which is necessary in the conventional UF membrane concentration method, is not necessary, and the production can be completed with minimum pH adjustment.

(Temperature conditions)
The temperature during operation in the production method of the present invention can be selected and set from a wide range, and can be arbitrarily selected at 15 to 60 ° C., preferably 40 ° C. or less.
In general, it is not preferable to perform membrane treatment of an enzyme for a long time at 25 to 40 ° C. where microorganisms are likely to grow. Therefore, avoiding these temperature ranges, a temperature as low as 15 ° C. or lower, or exceeding 45 ° C. The treatment is often performed at a high temperature. However, in the production method of the present invention, the membrane concentration can be completed quickly and efficiently by carrying out a two-stage membrane treatment under a predetermined pH condition. The risk is greatly reduced. From this point of view, the method of the present invention has the advantage that there are few restrictions on temperature conditions during production, and production can be carried out near room temperature without heating and cooling costs and labor.
In the method of the present invention, the step of heating and cooling the concentrated solution, which was necessary in the conventional UF membrane concentration method (concentration by heating to 50 ° C., cooling to 10 ° C. or less to form a precipitate) (Removing, reheating and continuing the concentration) becomes unnecessary, and the main purification / recovery steps in production, ie, MF membrane passage and UF membrane concentration, can be performed without the need for temperature adjustment.

(Further purification operation and added components)
In producing the soybean β-amylase preparation of the present invention, any additional purification step may be added as necessary. Moreover, you may add the stabilizer for hold | maintaining an enzyme activity, and the various additive components which assist the other process. The addition of these additional components and the introduction of the additional step can be performed at any stage before and after passing through the MF membrane and the UF membrane concentration step.

(Dry)
“Drying” in the present invention refers to any known drying method, and includes, for example, spray drying, freeze drying, and vacuum drying. In particular, spray drying is practically preferable because a pulverization step after drying is unnecessary.
Although the conditions of spray drying are not limited, in general, the sample liquid is sprayed or scattered in hot air at 100 to 200 ° C. to dry and powderize. Various types of spray-drying devices are commercially available. For example, a MM2000 model manufactured by Niro Co., Ltd. is used, and hot air temperature of 116 to 125 ° C., exhaust air temperature of 63 to 66 ° C., and the amount of sample supplied into the device are changed. The β-amylase of the present invention can be powdered under the condition of 14.5 to 15.0 ml per minute.
In lyophilization, a frozen sample solution is generally dried under vacuum, and the resulting dried product is ground and powdered with a mortar or the like. For example, using a RLE-103 model manufactured by KYOWAC, a sample solution frozen at −50 to 40 ° C. is placed under vacuum and dried. As a method for pulverizing the dried product, a KR-3 type small pulverizer manufactured by Kyoritsu Riko Co., Ltd. can be used in addition to a mortar. For example, after operating for 10 seconds at 9000 to 15,000 rpm, the powder can be pulverized while preventing temperature rise by grinding for 1 to 5 times after standing for 30 seconds.

(Characteristic evaluation of the obtained soybean β-amylase preparation)
The obtained soybean β-amylase preparation can be evaluated from various properties.
For example, since the soybean β-amylase preparation of the present invention is produced using a characteristic production method of two-stage membrane treatment, a specific size, property viscous substance, etc. that passes through the MF membrane at a specific pH. Is effectively removed.
Specifically, the soybean β-amylase preparation of the present invention is characterized in that polysaccharides, viscous substances, gel-like substances and the like having a particle size of about 0.1 μm or more are reduced to below a certain level. Therefore, the soybean β-amylase preparation of the present invention has a high purity and a high titer product as compared with an enzyme preparation produced only by UF membrane concentration without passing through an MF membrane. As a result, there is an advantage that the same effect can be obtained by using a small amount of the preparation.

  In addition, since the soybean β-amylase preparation of the present invention has a high purity, it has an advantage that it can be used more favorably when used for a light-colored product than the conventional ones. . The soybean β-amylase preparation of the present invention has higher transparency in a liquid state, and can be more suitably used when used for a product for which high transparency is desired. That is, the β-amylase preparation of the present invention has a great practical advantage that the degree of influence on the color and flavor of the food used is small.

  Furthermore, since the soybean β-amylase preparation of the present invention is produced without salting out, it has a feature that it does not contain an unnecessarily large amount of salts. Specifically, the soybean β-amylase preparation of the present invention is characterized by very little salts such as sodium sulfate, ammonium sulfate, potassium sulfate, magnesium sulfate, calcium sulfate, aluminum sulfate, sodium chloride, potassium chloride, and phosphate. Have Therefore, there is an advantage that it is easy to use in applications where it is required not to contain salts without requiring a desalting step.

In addition, the soybean β-amylase preparation of the present invention has a size, a characteristic viscous substance, and the like that pass through an MF membrane having a pore size of 0.1 to 0.45 μm at a specific pH, and is specified at a specific pH. At the same time, substances of size and properties that pass through a UF membrane having a molecular weight cut off of less than a certain amount have been removed, and at the same pH, a specific substance that does not pass through the UF membrane and remains inside is concentrated. It has the characteristic of being included. Specifically, the soybean β-amylase preparation of the present invention has components other than soybean β-amylase having a particle size of less than 0.1 μm and a molecular weight of 13,000 or more, such as trypsin inhibitor, lipoxygenase, lectin, urease, It has the feature of containing a certain amount of protease or the like. Furthermore, among soybean-derived components that are substances having a molecular weight of 13,000 or less, certain amounts of components such as pectin, galactomannan, water-soluble soybean polysaccharide, isoflavone, sucrose, saponin, and phytin are removed by membrane treatment. As a result, it has the characteristic that it becomes the content below the component composition contained in raw material soybean.
As described above, the soybean β-amylase preparation of the present invention having a novel composition can be used as an amylase preparation for the same use as that of a conventional amylase preparation, and has a new use in which the novel composition works advantageously. It can also be expected to provide.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

(Manufacture of liquid soybean β-amylase preparation from soybean whey)
To 20 kg soybean whey (titer 175 units / g), 0.5N aqueous sodium hydroxide solution or 0.5N aqueous acetic acid solution was added, and the pH at 20-24 ° C. was 3.7, 3.9, It adjusted so that it might become 4.1, 4.4, 4.6, 4.8, 5.1, 5.6, 6.1. Subsequently, the pH-adjusted soybean whey was permeated through an MF membrane (USP-143 manufactured by Asahi Kasei Chemicals Corporation). In permeation of the MF membrane, the permeation treatment is performed until the non-permeability section reaches about 1.0 kg, and tap water (drinking water) is added to the non-permeation section to dilute to about 3.0 kg, and then the MF membrane. The permeate was collected while washing out. The MF membrane passage liquid was continuously subjected to ultrafiltration concentration using a UF membrane (manufactured by Asahi Kasei Chemicals Co., Ltd., SLP-2053), and concentrated to about 1 kg.

After the UF membrane concentrate is diluted by adding 3 times the amount of tap water, and then concentrated again to the original concentrate volume (diafiltration), the final limit is The liquid amount in the outer filtration apparatus was concentrated to about 0.3 Kg, and the concentrated liquid was recovered using about 0.9 to 1.2 Kg of tap water as a washing liquid from the apparatus.
The soy β-amylase titer before treatment, the titer of the finally obtained UF membrane concentrate, and the titer of the non-permeated section liquid of the MF membrane (the non-permeated section liquid is about 1.0 kg of tap water) Table 1 shows the activity recovery rate of soybean β-amylase based on recovery by number. The value obtained by subtracting “final titer recovery (%)” and “MF membrane non-permeation titer (%)” from the soy β-amylase titer before treatment is 100 (%). ) ”As an indicator of titer loss.

  As shown in Table 1, when subjected to two kinds of continuous membrane treatment of MF membrane and UF membrane, soy whey and concentrated liquid at any pH concentrated about 70 times the amount of the initial soy whey. It was possible to carry out smoothly, and a high-concentration liquid soybean β-amylase preparation could be obtained by the method of the present invention. In particular, when the pH is adjusted in the range of pH 3.9 to 5.1 where the missing titer (%) does not exceed 10%, the activity recovery rate of soybean β-amylase is high, and liquid soybean under more preferable conditions A β-amylase preparation could be produced.

(Production of powdered soybean β-amylase preparation from UF membrane concentrated recovery solution)
The soy whey adjusted to pH 4.5 was subjected to MF membrane passage, UF membrane concentration and washing with tap water according to the method of Example 1, and finally about 120 times the capacity of the first soy whey. Until concentrated. Furthermore, it wash | cleaned by diluting 18.5 times with respect to the amount of concentrated liquids using tap water, and concentrating again, and finally it concentrated and collect | recovered to 20 times the first soybean whey. The pH of the concentrated liquid at this time was 3.9 (22 ° C.), which was significantly different from pH 5-6, which is the stable pH range of soybean β-amylase.
Next, the above-mentioned concentrated recovery liquid is divided into about 300 g portions, and the pH at 23-24 ° C. is 3.7, 3.9 using a 0.5 N aqueous sodium hydroxide solution and a 0.5 N aqueous acetic acid solution. It adjusted to 4.2, 4.5, 4.8, 5.1, 5.6, and it was set as the sample for spray drying. As drying conditions by spray drying, MM2000 model manufactured by Niro Co., Ltd. is used. The hot air temperature of 116 to 125 ° C., the exhaust air temperature of 63 to 66 ° C., and the amount of sample supplied into the apparatus are 14.5 to 15 per minute. 0.0 ml. Under the above conditions, 300 to 320 g of each pH-adjusted sample for spray drying was charged to the spray drying apparatus over about 20 minutes. Table 2 shows the results of the powdered state and the activity recovery rate.
The activity recovery rate was determined by adding the recovered powder and the titer amount of the powder remaining in the apparatus to obtain the total recovery amount as a dry powder. The titer amount of the powder remaining in the apparatus was calculated by dissolving and recovering with about 2 liters of ice-cooled distilled water immediately after completion of drying. The titer amount of the powder remaining in the apparatus was 5 to 6% in all cases. Further, as a reference value, pulverization by freeze-drying, which is generally said to have little loss of titer, was also performed (sample pH is 4.5, 25 ° C., 40 hours).

  As shown in Table 2, when the concentrated solution after the soybean whey was treated with two continuous membranes of MF membrane and UF membrane was spray-dried, the concentrated solution of any pH smoothly powdered. It was possible to obtain a high-titer powdered soybean β-amylase preparation by the method of the present invention. In particular, when the pH is adjusted in the range of pH 3.9 to 5.1 where the titer recovery rate (%) exceeds 80%, the activity recovery rate of soybean β-amylase is high, and the titer recovery rate ( %) Was adjusted to a pH range of 4.2 to 4.8 exceeding 90%, a high-potency powdered soybean β-amylase preparation could be produced under more preferable conditions.

(Production of powdered soybean β-amylase from soybean extract and soybean whey)
Powdered soybean β-amylase was produced according to the methods described in Examples 1 and 2 using soybean extract and soybean whey produced by conventional methods.
When passing through the MF membrane, module types ULP-143, USP-143, PSP-103, and PMP-102 manufactured by Asahi Kasei Chemicals Corporation were used. After passing through until the amount of the non-permeated liquid reached about 1.0 kg, tap water was added to the non-permeated liquid to dilute to about 3.0 kg, and the MF membrane was permeated again.

Subsequently, the permeate of the MF membrane was concentrated using a UF membrane (manufactured by Asahi Kasei Chemicals Corporation, module type SLP-2053). Next, a washing operation is performed to add 3 times the amount of tap water to the UF membrane concentrated liquid to concentrate it to the original concentrated liquid volume, and finally the UF membrane concentrated liquid volume is about 0.2 kg (about 14,000). Concentrate in a concentrator to unit / g).
Thereafter, for the purpose of obtaining the titer recovery rate, the ultrafiltration device was sufficiently washed out with tap water, and the recovered liquid amount was set to 1.0 to 1.2 kg. In addition, in order to determine the β-amylase titer present in the non-permeating section liquid of the MF membrane, the non-permeating section of the MF membrane was collected at about 1.0 Kg, and the liquid amount and the titer amount were measured.

  Next, 300 to 340 g of each of the collected concentrated liquids (Category 1 to Category 6 shown in Table 3) was used to adjust the pH to 4.5 ± 0.1 with a 0.5 N aqueous sodium hydroxide solution. Powdering by spray drying was performed by charging a spray dryer (MM2000 type) according to the conditions described in Example 2. The titer of the obtained powder was measured. The results are shown in Table 3.

  As shown in Table 3, in all of Category 1 to Category 6, a high-titer powdered soybean β-amylase preparation having about 40,000 units or more per gram of powder could be obtained.

(Example in which a soybean β-amylase preparation was produced only by UF membrane concentration without performing MF membrane filtration)
As a comparative example, soy whey (157 units / g) shown in categories 2 and 3 of Table 3 was not allowed to pass through the MF membrane, but was directly filtered by ultrafiltration using a UF membrane (manufactured by Asahi Kasei Chemicals Corporation, SLP-2053). The production of soybean β-amylase preparation was attempted. However, in this case, the solid content or turbid substance contained in the whey liquid and the paste-like component that becomes conspicuous when concentrated are obstructed, so that the concentrated liquid of about 10 times is obtained. It took about 3.5 times the time.
Furthermore, about 10 times the obtained concentrated liquid (1,680 units / ml) was subjected to spray drying. However, a thread-like solid was discharged from the spray nozzle of the spray dryer and fixed to the inner surface of the spray drying apparatus. It was difficult to recover and a powder formulation in a good state could not be obtained. That is, when soybean whey is directly subjected to UF membrane concentration, it is difficult to produce a sufficiently concentrated liquid soybean β-amylase preparation, and a high-potency powdered soybean β-amylase preparation is produced. I couldn't.

  In addition, as a result of confirming the practical effect as an anti-aging preparation of sweet confectionery using various powdered soybean β-amylase preparations obtained by the production method of the present invention, as a result of commercially available β-amylase enzyme preparations The practical effect similar to the case was confirmed. That is, the powdered soybean β-amylase preparation of the present invention can be used in the same manner as the conventional β-amylase enzyme agent, and exhibits the desired effect with a small amount of addition, with the advantage of higher titer. And is expected to be useful, such as being able to manufacture products with fewer unnecessary components. Furthermore, the β-amylase preparation of the present invention has a low degree of effect on the color and flavor of the food used, and a whiter color powder β-amylase preparation, a more transparent liquid β-amylase preparation, It is expected that it can be suitably used for various applications including foods that require a white color tone.

Claims (6)

  A soy extract or soy whey is passed through a microfiltration membrane having a pore size of 0.1 to 0.45 μm, and an ultrafiltration membrane having a molecular weight cut off of 13,000 or less is used under conditions of a liquid temperature of 15 to 60 ° C. Then, the method for producing a soybean β-amylase preparation is characterized by performing ultrafiltration concentration until the β-amylase titer is in a liquid state of 10,000 units / g or more.   The method for producing a soybean β-amylase preparation according to claim 1, wherein the pH of the soybean extract or soybean whey when adjusted to pass through a microfiltration membrane is adjusted to 4.0 to 5.0.   The method for producing a soybean β-amylase preparation according to claim 1 or 2, wherein the ultrafiltration membrane concentration step after passing through the microfiltration membrane is performed continuously and / or aseptically.   A method for producing a powdered soybean β-amylase preparation comprising adjusting the pH of a soybean β-amylase solution obtained by the method of claims 1 to 4 to 4.2 to 4.8 and drying.   The pH adjustment is performed using one or more diluted acidic or diluted alkaline solutions selected from sodium hydroxide, sodium hydrogen carbonate, sodium carbonate, sodium acetate, hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, and phosphate. Item 5. A method for producing a soybean β-amylase preparation according to Item 4.   A soybean β-amylase preparation obtained by the method for producing a soybean β-amylase preparation according to claim 1.