JP2004313170A - Method for producing soybean whey-fractionated product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To fractionate soybean whey to soybean trypsin inhibitor concentrates (preferably, fractions mainly comprising BBI type and Kunitz type), respectively, and a soybean β-amylase concentrate and a soybean oligosaccharide concentrate without adding salt. <P>SOLUTION: The method for producing soybean whey-fractionated product comprises (a) a process for concentrating the soybean whey and collect aggregated precipitation precipitated under an acidic condition and (b) a process for fractionating the supernatant to a higher molecular fraction and a lower molecular fraction. By the method, the soybean whey can be fractionated into 3 fractions, the soybean trypsin inhibitor concentrate, the soybean β-amylase concentrate and/or the soybean oligosaccharide concentrate. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for easily producing useful fractions such as a trypsin inhibitor having a physiological activity and a concentrate of β-amylase which is a saccharifying enzyme of starch contained in soybean.

  As a trypsin inhibitor which is one of the physiologically active substances contained in soybean, a Kunitz type having a molecular weight of about 20,000 and an isoelectric point of 4.5 and a BBI type having a molecular weight of about 8,000 and an isoelectric point of 4.2 are known. A disposable absorbent product having a skin care composition containing a cancerous pleural and ascites retention inhibitor (Patent Document 1), an inflammatory edema enhancement inhibitor (Patent Document 2), and an enzyme inhibitor (Patent Document 3) ) Etc. have been reported.

  Soybean β-amylase is an enzyme that hydrolyzes amylose of starch from its non-reducing end in the form of maltose and has a molecular weight of 57,000 and an isoelectric point of 5.5, and is used for producing starch syrup and maltose. It is directly used in bread and Japanese confectionery as an anti-aging agent. In addition, soybeans have extremely low α-amylase activity, and soybean β-amylase has higher heat resistance than other plant-derived β-amylases, indicating superiority in the production of high-purity maltose and antiaging of foods. ing.

  Oligosaccharides contained in soybeans are high in carbohydrates having a physiological activity, and include stachyose, raffinose, verbascose, pinitol, chiro-inositol, myo-inositol, sucrose, etc. The carbohydrates possessed are stachyose, raffinose and verbascose, and their use as bifidobacteria has been reported in Patent Documents 4 and 5 and the like.

  At the laboratory level, various purification methods have been proposed as a fractionation method of soybean trypsin inhibitor by a combination of isoelectric point precipitation, salting out, ion exchange chromatography, gel filtration and the like. Industrial methods include Patent Document 6 combining an ultrafiltration membrane and ion exchange chromatography, and Patent Document 7 combining a trypsin inhibitor extraction method and ion exchange chromatography.

  There are Patent Documents 8 and 9 using an ultrafiltration membrane as a fractionation method of soybean β-amylase, and Patent Document 10 using a synthetic aluminum silicate as an adsorbent.

  Patent Document 11 discloses a method for fractionating soybean oligosaccharides in which soybean whey is neutralized with calcium hydroxide in the presence of calcium chloride to reduce protein with precipitation, and after heating soybean whey, the pH is adjusted to 3.0 or less with phosphoric acid. Patent Document 12 and the like in which protein is reduced together with the resulting precipitate.

As described above, soybean trypsin inhibitor, soybean β-amylase, and soybean oligosaccharide are extremely useful substances, and if a simple separation technique leading to industrialization can be established, social contribution will be great.
On the other hand, examples of the conventional methods for fractionating soybean trypsin inhibitor, fractionating soybean β-amylase, and fractionating soybean oligosaccharides have been given. Therefore, there is no technical proposal to comprehensively use soy whey containing various useful components.

Japanese Patent Publication No. 6-23113 JP-A-7-10772 JP 2002-505916 A JP-A-3-22971 JP-A-60-066978 JP-A-6-145198 JP-A-10-066572 Japanese Patent Publication No. 57-11636 JP-A-7-107974 JP-B-57-52386 JP-A-60-66978 Japanese Patent No. 2635210

The method of recovering protein by adding salt to soy whey is used at the experimental level, but it is not practical because it requires a large amount of salt, and multiple components are directly mixed into ion exchange chromatography to recover specific components. It is also not realistic to serve soy whey that is.
Although the method of fractionating proteins with an ultrafiltration membrane is effective, soybean whey gradually causes precipitation of trypsin inhibitor as a main component and causes clogging of the ultrafiltration membrane, leading to a decrease in flux. Therefore, it is difficult to concentrate the concentrated soybean whey at a high concentration.
Also, the difference in molecular weight between soybean trypsin inhibitor and soybean β-amylase is small, and it is unsuitable when soybean whey is directly treated with an ultrafiltration membrane and separated from each other. Furthermore, it is almost impossible to fractionate the BBI-type trypsin inhibitor fraction, the Kunitz-type trypsin inhibitor fraction, and the β-amylase fraction from dilute soybean whey with an ultrafiltration membrane.
In view of the above, the present invention provides a method for fractionating soybean whey into soybean trypsin inhibitor concentrates (preferably, fractions mainly composed of BBI type and Kunitz type) without adding salt, soybean β-amylase concentrate and It was intended to be fractionated into soy oligosaccharide concentrate.

The inventors of the present invention can collect 80% or more of the activity of trypsin inhibitor present in soy whey as a precipitate by concentrating soy whey by-produced in the production process of isolated soy protein and then adjusting the pH to a specific range. Obtained knowledge.
Next, when a polar solvent was added to the supernatant separated in the above step to perform fractionation, it was found that 85% or more of the activity of β-amylase present in soybean whey could be recovered as a precipitate.
On the other hand, it has been found that by hydrolyzing and partially dissolving the recovered precipitate of trypsin inhibitor in a specific pH range, it can be fractionated into a fraction mainly composed of BBI type and Kunitz type.
Then, when the supernatant separated in the above step was attempted to concentrate β-amylase using an ultrafiltration membrane, the precipitate generated in advance was removed as a trypsin inhibitor fraction, so that the flux was reduced. There was almost no discovery that 85% or more of the β-amylase titer present in soy whey could be recovered as a concentrate.

The present invention is the following method completed based on the above findings.
1. A method for producing a soy whey fraction, comprising the following steps.
(A) a step of concentrating soy whey and collecting coagulated sediment precipitated under acidic conditions;
(B) a step of fractionating the obtained supernatant into a high molecular fraction and a low molecular fraction;
2. The coagulated precipitate recovered in step (a) is a soybean trypsin inhibitor concentrate, the high molecular fraction obtained in step (b) is a soybean β-amylase concentrate, and the low molecular fraction is soybean oligosaccharide. Manufacturing method.
3. The concentration of the soy whey in the step (a) is performed at pH 3.5 to 8.5, and the solid content is concentrated to the range of 30% by weight to 50% by weight. Manufacturing method.
4. Concentrating the soy whey in step (a) at a product temperature of 20 ° C to 80 ° C. Manufacturing method.
5. The step (a) of depositing the aggregated precipitate is performed at pH 1.7 to 5.2. Manufacturing method.
6. Water is added to the precipitate obtained in the step (a) and partially redissolved in the range of pH 2.0 to 4.8, and this is subjected to solid-liquid fractionation. Manufacturing method.
7. 5. The liquid part obtained by the solid-liquid fractionation is a BBI-type trypsin inhibitor concentrate, and the solid part is a Kunitz-type trypsin inhibitor concentrate. Manufacturing method.
8. In the step (b), a polar solvent is added to the obtained supernatant, and a fractionation step (b-1) in which the supernatant is fractionated into a new supernatant and a precipitated precipitate, or the obtained supernatant is applied to an ultrafiltration membrane. (B-2) for separating the concentrate into a concentrate and a permeate, or a combination thereof. Manufacturing method.
9. In the step (b-1), the polar solvent is ethanol, the ethanol concentration is 30% by weight to 85% by weight based on the total of water and ethanol in the supernatant to which the polar solvent is added, and the temperature is -5 ° C to 15 ° C. 7. Implement within the range of Manufacturing method.
10. 7. In the step (b-2), the molecular weight cut off of the ultrafiltration membrane is in the range of 5,000 to 250,000. Manufacturing method.
11. 7. Step (b-2) is performed at a temperature of 65 ° C. or less, a pH of 4.0 to 7.0, and a solid content of the supernatant of 50% by weight or less. Manufacturing method.
12. 7. Concentrate the fresh supernatant obtained in step (b-1) or the permeate obtained in step (b-2) to a solid content of 50% to 85% by weight. Manufacturing method.

According to the present invention, it has become possible to fractionate soy whey into three fractions: soybean trypsin inhibitor concentrate, soybean β-amylase concentrate and / or soybean oligosaccharide concentrate.
Furthermore, fractionation of the BBI-type trypsin inhibitor and the Kunitz-type trypsin inhibitor from the soybean trypsin inhibitor concentrate became possible, and the diafiltration of soybean β-amylase with a UF membrane enabled high concentration.
These fractions can be used as intermediate raw materials for soybean trypsin inhibitor, soybean β-amylase, and soybean oligosaccharides.
By using the process of the present invention, soybean trypsin inhibitor, soybean β-amylase, and soybean oligosaccharide can be efficiently separated from soybean whey in a series of steps, and soybean whey can be comprehensively used. It is now possible.

Step (a): a step of concentrating soy whey and collecting coagulated sediment precipitated under acidic conditions.
As the soybean whey used in the present invention, a soybean whey in which soybean trypsin inhibitor or soybean β-amylase has activity is used. That is, it is preferable that the soybean trypsin inhibitor and the soybean β-amylase do not have such a heat history as to deactivate them in the process of occurrence.
Soy whey is a by-product in the process of producing isolated soy protein or soy protein concentrate from soy. Soy whey with a small amount of heat can be obtained by, for example, extracting low-denatured defatted soybeans near neutral with water and removing the okara component, and coagulating and precipitating soybean proteins near the isoelectric point (for example, near pH 4.5). It can be obtained as a solution that has been removed.

  Soy whey, which is a by-product of industrially producing isolated soy protein, is advantageous because it can be used in large quantities as a stable raw material, and this whey typically has a solids content of about About 3%, its solid content composition (hereinafter also referred to as “dry%”) is composed of about 20% crude protein, about 20% ash, and about 60% sugar, and the water extraction ratio from defatted soybeans changes. There is also a tendency that the solid content composition of soy whey hardly fluctuates. When the solid content is about 3%, the activity of soybean trypsin inhibitor is about 5 unit / ml, and the activity of soybean β-amylase is about 100 unit / ml, which can be suitably used in the present invention.

  The method for measuring soybean trypsin inhibitor activity herein is described in O. C. S. The measurement of the soybean β-amylase activity was carried out by using the potato starch as a substrate and reacting with whey (amylase solution) to determine the reducing sugar produced by the Somogi-Nelson method. The amount of enzyme required to produce a sugar equivalent to 1 mg of glucose in 10 minutes at 10 ° C. was defined as 1 unit.

  The concentration of soy whey in the step (a) is performed at pH 3.5 to 8.5, preferably pH 4.5 to 7.0, and at a solution temperature (product temperature) of 20 ° C to 80 ° C, preferably 20 ° C to 65 ° C, It is appropriate to concentrate the solids to a range of 30% to 50%, preferably 35% to 45% by weight.

  If the pH is less than 3.5, β-amylase tends to be unstable, and if the pH exceeds 8.5, the trypsin inhibitor tends to be unstable. If the solution temperature is lower than 20 ° C., there may be a mechanical limit in the decompression device, and if it exceeds 65 ° C., the activity of β-amylase tends to decrease, but 60% activity remains at 70 ° C. . That is, when the pH, temperature and concentration are out of the above ranges, the recovery of soybean trypsin inhibitor activity tends to decrease, and the activity of β-amylase recovered in step (b) also tends to decrease.

  The Kunitz type trypsin inhibitor has an isoelectric point of pH 4.5, the BBI type has an isoelectric point of pH 4.2, and the β-amylase has an isoelectric point of pH 5.5, but soy whey is used as it is. Even if the isoelectric point precipitation is attempted by adjusting the pH at the isoelectric point, coagulation and precipitation of the target substance do not occur, or the amount is extremely small.

By adjusting the solid content to the above range, the ash concentration in the whey is relatively increased, and the same effect as salting out is obtained, so that the trypsin inhibitor fraction can be specifically fractionated.
Conventionally, there is a salting-out method as a method for recovering protein from soybean whey. For example, when an attempt is made to precipitate soybean trypsin inhibitor with salt, a large amount of salt as much as 14% is added to soybean whey having a solid content of 3%. It was not realistic because it was necessary.

  A known means can be used as the concentrating means. Concentration under reduced pressure can prevent the temperature from rising and can be concentrated efficiently.

The soy whey concentrated as described above is subjected to acidic conditions, preferably to a pH of 1.7 to 5.2, and more preferably to a pH in the range of 2.7 to 4.8. Fractionate. This aggregate precipitate is enriched in soybean trypsin inhibitor.
Even when the pH is out of the range of 1.7 to 5.2, the temperature can be reduced to 20 ° C. or less with sufficient care for the inactivation of β-amylase due to low pH.

  The coagulated precipitate thus obtained is a soybean trypsin inhibitor concentrate, which is partially dissolved in a range of pH 2.0 to 4.8, preferably pH 3.0 to pH 4.4, and further subjected to solid-liquid fractionation. Can be. The liquid part obtained by the solid-liquid fractionation can be a BBI-type trypsin inhibitor concentrate, and the solid part can be a Kunitz-type trypsin inhibitor concentrate. Since the salt concentration becomes lower than that of concentrated whey due to the addition of water, the BBI-type trypsin inhibitor re-dissolves, whereas the Kunitz-type trypsin inhibitor does not dissolve, and both are separated into a supernatant and a precipitate. You can do it.

  The degree of water addition is 1.5 times or more, preferably 2 to 10 times, and most preferably 2.5 to 4 times the amount of the aggregated precipitate (hydrated product).

  As a means for this fractionation (solid-liquid separation), a known solid-liquid separator, for example, a centrifugal separator, a membrane separator or the like can be used.

Step (b): a step of fractionating the supernatant obtained in step (a) into a high molecular fraction and a low molecular fraction.
This fractionation is carried out by adding a polar solvent to the supernatant obtained in the step (a), and then separating the aggregate into a new supernatant with the aggregated substance (b-1), or ultrafiltration of the supernatant It can be performed by the step (b-2) of treating with a membrane and fractionating into a concentrated solution and a permeated solution, or a combination thereof.

First, the step (b-1) will be described.
A polar solvent is added to the supernatant obtained in the step (a) to fractionate into aggregates and supernatant which precipitate. This aggregate is useful as a soybean β-amylase concentrate, and the newly generated supernatant can be used as a raw material for oligosaccharides. The newly generated supernatant is preferably concentrated to a solid content of 50% or more.

The polar solvent used here is preferably ethanol for food use, and the concentration of ethanol is adjusted to 30% to 85% by weight, preferably 50% to 80% by weight based on the sum of water and ethanol in the supernatant to which ethanol has been added. However, the temperature is preferably set in a range of -5C to 15C, preferably 0C to 10C.
If the ethanol concentration is low, β-amylase is dissolved in a low-concentration ethanol solution, and if it is high, saccharides are also precipitated. From the viewpoint of the bactericidal effect, it is more appropriate to carry out the ethanol concentration at 65% by weight to 75% by weight.
If the temperature is higher than the above temperature, the activity of the β-amylase fraction tends to decrease.

  Since the supernatant obtained in the step (a) has a high solid content of about 30% to 50% by weight, the oligosaccharides in the mother liquor should be distributed in the step (b-1) in a large amount to the precipitate. Alternatively, the supernatant in step (a) can be appropriately diluted to dissolve oligosaccharides in the supernatant during ethanol precipitation to increase the recovery rate.

The fresh supernatant obtained in the step (b-1) is preferably prepared by concentrating the oligosaccharide fraction to 50% by weight or more to reduce the volume for storage and transportation and to prevent spoilage. The raw material for the oligosaccharide preferably has a concentration range that is fluid and has a very low level of spoilage. The solid content is usually 50% to 85% by weight, preferably 55% to 80% by weight.
A purified oligosaccharide can be obtained by subjecting the supernatant concentrate to a purification treatment such as desalting.

Next, the step (b-2) of the present invention will be described.
The supernatant obtained in the step (a) is fractionated into a concentrate and a permeate using an ultrafiltration membrane.
This concentrate is useful as a soybean β-amylase concentrate, and the permeate can be used as a raw material for soybean oligosaccharides.

In the ultrafiltration performed here, it is preferable to use an ultrafiltration membrane having a cut-off molecular weight of 5,000 to 250,000, preferably a cut-off molecular weight of 10,000 to 100,000.
The supernatant obtained in step (a) can be used without dilution in the ultrafiltration step, but as the concentration proceeds, the solid content of the concentrate increases and the flux (permeate flux) decreases. Therefore, a diafiltration method in which water is appropriately added and ultrafiltration is performed to maintain the solid content of the concentrated liquid at 50% by weight or less, preferably 45% by weight or less can be adopted.
The higher the temperature of the solution passed through the ultrafiltration membrane, the higher the flux, but the temperature is high enough to not deactivate β-amylase, 65 ° C., preferably at 55 ° C. or lower, and the pH at this time is also preferable. β-Amylase is preferably carried out in the range of pH 4.0 to pH 7.0, preferably pH 4.5 to pH 6.0, and β-amylase is stable even at pH 6.0 or more, but is acidic at pH 6.0 or more. It is not preferable because a precipitate is generated due to insolubilization of phytic acid dissolved on the side.

  The permeate obtained in step (b-2) contains 50% by weight or more of oligosaccharides, but contains 20% by weight or more of ash, so that it can be desalted and provided as a food material.

On the other hand, it is preferable that the oligosaccharide fraction of the permeate obtained in the step (b-2) is concentrated to 50% by weight or more to reduce the volume for storage and transportation and to prevent spoilage.
For example, the permeated liquid is preferably a flowable soybean oligosaccharide raw material and has a very low concentration of spoilage, and usually has a solid content of 50% to 85% by weight, preferably 55% to 80% by weight. Is appropriate.
A purified oligosaccharide can be obtained by subjecting the permeate concentrate to a purification treatment such as desalting.

  Through the above steps, soybean trypsin inhibitor, β-amylase and soybean oligosaccharide can be efficiently separated from soybean whey in a series of steps.

  Examples will be described below to describe embodiments of the present invention.

[Example 1]
(A) Step:
70 kg of soy whey (solid content: 2.9% by weight, pH 4.6, total trypsin inhibitor activity: 357,000 units, total β-amylase activity: 9,120,000 units, crude protein content: 20 kg) 1 dry%, ash content 19.7 dry%, carbohydrate 60.2 dry%) were concentrated under reduced pressure at 50 ° C. (heating temperature 80 ° C.) of soybean whey using an evapor (CEP-L type) manufactured by Okawara Seisakusho and solidified. 4,600 g of concentrated whey having a total trypsin inhibitor activity of 333,000 units and a total β-amylase activity of 8,530,000 units were obtained in an amount of 41.3% by weight (about 300 g loss due to adhesion or the like).
The concentrated whey was cooled to 15 ° C., adjusted to pH 4.0 with phosphoric acid, allowed to stand for 3 hours, centrifuged (1,500 G × 10 minutes), and separated into a supernatant and a precipitate.
4,297 g of the separated supernatant (step (a) supernatant) was obtained, the solid content was 40.0% by weight, the total trypsin inhibitor activity was 40,000 units, the total β-amylase activity was 8,200,000 units, and the 324 g of ((a) step precipitation) was obtained, the solid content was 55.0% by weight, the total trypsin inhibitor activity was 305,000 units, and the total β-amylase activity was 700,000 units.
Therefore, the distribution ratio of both enzyme activities was 11.6% for the trypsin inhibitor in the supernatant, 88.4% for the precipitate, 92.1% for β-amylase in the supernatant, and 7.9% for the precipitate. .

(B-1) Step:
(A) Process The supernatant of 4,200 g was cooled to 8 ° C. and 4,000 g of primary ethanol (purity: 99.5%) (ethanol concentration: 61.3% by weight) was softly added with gentle stirring with propeller stirring. Although it was possible to form aggregates like rice cakes and separate them as they were, they were separated into supernatant and precipitate by centrifugation (1,500 G × 10 minutes) after 15 minutes.
2,028 g of the separated precipitate (step (b-1) precipitate) was obtained, the solid content was 55.2% by weight, the total β-amylase activity was 7,970,000 units, and the total trypsin inhibitor activity was 9,800 units. .
The supernatant was subjected to simple distillation (heating temperature: 65 ° C.) using a rotary evaporator, and ethanol was removed to obtain 2,530 g of a solution having a solid content of 29.8% by weight ((b-1) process supernatant).

(B-1) Step The precipitation can be almost completely dissolved by adding about 3 times the amount of water and stirring and dissolving with a homomixer (3,000 rpm), so that the β-amylase can be highly purified using this precipitation as a raw material. .
(B-1) Process The supernatant (2,500 g) was further concentrated under reduced pressure at a vaporization temperature of 60 ° C. and a heating temperature of 90 ° C. using a thin-film flash evaporator (MF-10A manufactured by Tokyo Rika) to concentrate 60.1 wt% solids. The product was obtained in an amount of 1,120 g.
This concentrate contained 76.3% by weight of saccharide per solid weight, of which 32.0% was oligosaccharides of 3 or more saccharides.

[Example 2]
The same soybean whey as in Example 1 was concentrated under reduced pressure in the same manner as described in Example 1 and concentrated to a solid content of 51.5%. 300 g of concentrated whey (total trypsin inhibitor activity 26,000 units, total β-amylase activity 698,000 units). Was cooled to 15 ° C., adjusted to pH 4.0, allowed to stand for 3 hours, and then separated into a supernatant and a precipitate by centrifugation (1,500 G × 10 minutes).

271 g of the separated supernatant was obtained, having a solid content of 50.9% by weight, total trypsin inhibitor activity of 15,000 units and total β-amylase activity of 611,000 units, and 34 g of precipitate were obtained, 48.8% by weight of solid content, and total trypsin inhibitor. The activity was 13,000 units, and the total β-amylase activity was 101,000 units.
When concentrated to a solid content of 51.5% by weight, the degree of selective precipitation of the trypsin inhibitor was inferior to that of Example 1.

[Example 3]
The same soybean whey as in Example 1 was concentrated under reduced pressure in the same manner as described in Example 1, and 300 g of concentrated whey concentrated to a solid content of 28.7% by weight (total trypsin inhibitor activity 15,000 units, total β-amylase activity 361,000 units) ) Was cooled to 15 ° C., adjusted to pH 4.0, allowed to stand for 3 hours, and then separated into a supernatant and a precipitate by centrifugation (1,500 G × 10 minutes).
286 g of the separated supernatant was obtained, the solid content was 27.9% by weight, the total trypsin inhibitor activity was 7,300 units, the total β-amylase activity was 320,000 units, the precipitate was 18 g, and the solid content was 35.2% by weight. Total trypsin inhibitor activity was 8,100 units and total β-amylase activity was 54,000 units.

At a concentration of 28.7% by weight solids, the extent of trypsin inhibitor precipitation was insufficient compared to Example 1.

[Example 4]
Step (a) was carried out in the same manner as in Example 1, and 300 g (total β-amylase activity: 625,000 units) of the supernatant of the step (a) having a solid content of 42.3% by weight was added to ethanol at room temperature (25 ° C.). 270 g was stirred and gently added (alcohol concentration: 60.6%), and then separated into a supernatant and a precipitate by centrifugation (1,500 G × 10 minutes). 145 g of a precipitate was obtained, the solid content was 56.4%, and the total β-amylase activity was 313,000 units.
The yield of solids from the supernatant of the step (a) in the precipitation of the step (b-1) of Example 1 was 66.6%, whereas the yield of solids from Example 4 was 64.4%, which was almost 64.4%. It was equivalent.
However, the recovery rate of β-amylase activity in Example 1 was 97%, which was almost the entire amount, whereas that in Example 4 was 50%, and it is considered that there was inactivation due to temperature.

[Application Example 1]
Step (a) prepared in the same manner as in Example 1 To 100 g of precipitate (solid content: 56.1%, total trypsin inhibitor activity: 95,000 units), 500 g of water was added, and the mixture was stirred and dissolved with a homomixer (3,000 rpm × 15 minutes). After centrifugation (1,500 G × 10 minutes), the supernatant was separated into a precipitate, and as a result, 580 g (solid content: 7.9%, total trypsin inhibitor activity: 93,000 units) of the supernatant was obtained. 20 g (solid content 51.5%, total trypsin inhibitor activity 3,500 units) were obtained.
From these results, the trypsin inhibitor aggregated and precipitated in the step (a) is redissolved by adding water, so that the trypsin inhibitor can be highly purified from the precipitation in the step (a).

[Example 5]
(A) Step:
200 kg of soy whey (dry matter solid content 3.0% by weight, pH 4.6, total trypsin inhibitor titer 960,000 units, total β-amylase titer 22,400,000 units, crude protein content 20.3 dry%, ash 20 .1dry%, carbohydrate 59.6dry%) was concentrated under reduced pressure at a temperature of 50 ° C. and a heating temperature of 80 ° C. of soybean whey using an evapor (CEP-L type) manufactured by Okawara Seisakusho Co., Ltd., and 42.2 wt. Thus, 14,000 g of concentrated whey having a total trypsin inhibitor titer of 930,000 units (66 units / g) and a total β-amylase titer of 22,010,000 units (1572 units / g) were obtained (about 200 g loss due to adhesion or the like).
The concentrated whey was cooled to 15 ° C., adjusted to pH 4.0 with phosphoric acid, allowed to stand at 10 ° C. for 3 hours, centrifuged (1,500 G × 10 minutes), and separated into a supernatant and a precipitate.
The separated supernatant (the supernatant in the step (a)) was obtained in an amount of 12,850 g, the dry matter solid content was 41.3%, the total trypsin inhibitor titer was 110,500 units (8.6 units / g), the total β-amylase activity. Is 20,500,000 units (1,595 units / g), 1,150 g of precipitate (step (a) precipitation) is obtained, the dry matter solid content is 52.3% by weight, the total trypsin inhibitor activity is 820,000 units (713 units / g). g), the total β-amylase titer was 1,530,000 units (1330 units / g).
Therefore, the distribution ratio of both enzyme activities was 11.9% for the trypsin inhibitor in the supernatant, 88.1% for the precipitate, 93.1% for β-amylase in the supernatant, and 6.9% for the precipitate. .

(A) Redissolving the process precipitate:
(A) Step While maintaining the pH of 4.0 g by adding 2,000 g of water to 1,000 g of the precipitate, the mixture was stirred and dissolved using a homomixer (4,000 rpm × 15 minutes) and then centrifuged (5, 5). 000 G x 10 minutes) to separate into a supernatant and a precipitate.
2,767 g of the supernatant was obtained, having a solid content of 14.2% and a total trypsin inhibitor titer of 595,000 units (215 units / g), and 233 g of precipitate was obtained, having a solid content of 55.8% and a total trypsin inhibitor titer of 221,000 units ( 948 units / g).
The supernatant and the protein of the precipitate were analyzed by SDS electrophoresis.

The protein of the (a) step supernatant, the (a) step precipitate, the lectin, the (a) step precipitate re-dissolved precipitate, and the protein of the (a) step precipitate re-dissolved supernatant are shown in FIG. 1 by the SDS electrophoresis pattern.
FIG. 1 shows the behavior of a protein having a molecular weight of 42 kDa or less, but the protein is distributed to lane (2), in which almost no trypsin inhibitor is present in lane (1) (some BBI type remains).
Kunitz-type trypsin inhibitor was distributed on the precipitation side of lane (3) in which lane (2) was hydrolyzed and redissolved, and BBI-type trypsin inhibitor was distributed on the supernatant side of lane (4).
As described above, it was found from the precipitation in the step (a) that the Kunitz-type trypsin inhibitor and the BBI-type trypsin inhibitor could be roughly fractionated due to the difference in solubility.

(B-2) Step:
(A) Step The concentration of the supernatant was adjusted by adding water to 40 Brix (solid content: about 38%, β-amylase titer: 1,470 U / g), and an ultrafiltration membrane (molecular weight cut off: 30) manufactured by Daisen Membrane Co., Ltd. 2,000, a membrane area of 1.4 m 2, a hollow fiber module), a flow rate of 1 m 3 / hr, a back pressure of 3 kg / cm 2, a liquid temperature of 45 ° C., and a pH of 4.5 while appropriately adding water to maintain the concentration on the concentration side at 40 Brix. Table 1 shows the results of the operation under the conditions described above until the β-amylase titer was concentrated about 10-fold.

(Table 1)

(A) Process The supernatant was adjusted to 40 Brix and 12.8 kg was treated.
To maintain the concentration of the UF membrane concentrated solution at about 40 Brix, water was added for 60 minutes, 90 minutes, 110 minutes, and 120 minutes, respectively.
As the concentration increases, the flux decreases as the concentration increases.However, it is considered that clogging does not occur because the flux recovers by adjusting the water concentration, and the operation was completed in 130 minutes, but the flux was remarkable. Further concentration is possible because there is no decrease.
The average flux was 4.1 kg / m2 / hr, and the obtained concentrate had a solid content of 38.0% of dry matter and a β-amylase titer of 13,500 U / g (35,500 U / g in terms of dry matter). Table 2 shows the composition of the UF membrane permeate.

(Table 2)

  In addition, as a result of performing saccharide analysis using water as a mobile phase with a high performance liquid chromatograph (detector / L-6200 manufactured by Hitachi, pump / L3350, column / Shodex Sugar SC-1011), dry matter solid content was determined. A soy oligosaccharide concentrate containing 27.8% of oligosaccharides (raffinose + stachyose), 7.3% of pinitol, 1.0% of chiro-inositol, 2.6% of myo-inositol, etc. there were.

[Example 6]
The UF membrane permeate was adjusted to a dry matter solid content of 20% (ash content: 4.5%) by adding water, and desalting was performed by an electrodialysis device (Asahi Kasei Micro Acylyzer S3, cartridge / AC-220-550). As a result, when the electric conductivity was reduced to about 1.0 mS / cm (ash content 0.2%, dry matter solid content 16.5%), a soybean oligosaccharide concentrate having a good flavor was obtained.

[Example 7] (Example combining UF membrane (ultrafiltration membrane) concentration and ethanol precipitation)
(A) Process The concentration of the supernatant was adjusted by adding water to 40 Brix (solid content: about 38%, β-amylase titer: 1,470 U / g), and an ultrafiltration membrane manufactured by Daisen Membrane Co., Ltd. 2,000, a membrane area of 1.4 m 2, a hollow fiber module), a flow rate of 1 m 3 / hr, a back pressure of 3 kg / cm 2, a liquid temperature of 45 ° C., and a pH of 4.5 while appropriately adding water to maintain the concentration on the concentration side at 40 Brix. The operation was carried out under the conditions described above until the β-amylase titer was concentrated about 10-fold. The dry matter solid content was 38.0%, and the β-amylase titer was 13,500 U / g (35,500 U / g in terms of dry matter). 100 g of the concentrated solution of g) was adjusted to a liquid temperature of 8 ° C., ethanol was also adjusted to 8 ° C., 150 g was added to the concentrated solution, and the mixture was allowed to stand for 10 minutes and centrifuged to obtain a precipitate.
The precipitate had a dry matter solid content of 59.2% and a β-amylase titer of 23,400 U / g (39,500 U / g in terms of dry matter), and the β-amylase was further concentrated.
The ethanol precipitation supernatant did not have β-amylase activity.

Example 8
In the same manner as in Example 1, soy whey was concentrated under reduced pressure to a solid content of 40.6% by weight, and 450 g of concentrated whey (total trypsin inhibitor titer: 30,500 units) was transferred to nine 100 ml beakers in 50 g (total trypsin inhibitor). After titration at 3,300 units) and cooling to 15 ° C., each was adjusted to pH 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, The mixture was adjusted to 5.5 with hydrochloric acid or sodium hydroxide, allowed to stand for 8 hours, separated into a supernatant and a precipitate by centrifugation (1,500 G × 10 minutes), and the total trypsin inhibitor activity of the supernatant was measured.
The total trypsin inhibitor titer of each supernatant was pH 1.5 / 2,500 unit, pH 2.0 / 1,650 unit, pH 2.5 / 1,330 unit, pH 3.0 / 820 unit, pH 3.5 / 540 unit, pH 4.0. / 350 unit, pH 4.5 / 980 unit, pH 5.0 / 1, 480 unit, pH 5.5 / 2, 120 unit, and more than 70% of the total trypsin inhibitor titer is distributed to the precipitation side in the range of pH 3.0 to pH 4.5. It will be.

[Example 9]
50 g of aggregated precipitate (trypsin inhibitor fraction) prepared in the same manner as in Example 1 was treated with sodium hydroxide or hydrochloric acid at pH 4.0, pH 4.4, pH 4.8, pH 5.2, pH 5.6, pH 6.0. While maintaining the respective pH values, 150 g of water was added, and the mixture was stirred with a homomixer (4000 rpm × 20 minutes / 20 ° C.) to promote re-dissolution.
FIG. 2 shows an SDS-electrophoresis pattern (trypsin inhibitor fractionation) of the supernatant protein that was centrifuged at 5,000 G × 20 minutes using a high-speed centrifuge manufactured by Hitachi Koki after stirring and re-dissolving.
According to FIG. 2, KSTI is not distributed to the supernatant at pH 4.4 in lane (4), but at pH 4.8 or higher in lane (5), KSTI is mixed into the supernatant and cannot be fractionated into BBI and KSTI. .
In addition, the lower limit of the fractionation pH of KSTI and BBI was examined at pH 4.0, pH 3.0, pH 2.0, and pH 1.0, and although not shown in the figure, at pH 3.0, KSTI is not distributed to the supernatant, At a pH of 0.0 or less, KSTI was mixed into the supernatant.

  By using the production method of the present invention, soybean trypsin inhibitor, soybean β-amylase, and soybean oligosaccharide can be efficiently separated from soybean whey in a series of steps. Is made possible.

It is a SDS electrophoresis pattern of protein of (a) process supernatant, (a) process precipitation, (a) process precipitate re-dissolution precipitation, and (a) process precipitate re-dissolution supernatant. It is the SDS electrophoresis pattern of the protein of the supernatant which redissolved the trypsin inhibitor fraction at each pH.

Explanation of reference numerals

[Fig. 1]
(1) Marker
(2) Supernatant of step (a)
(3) Precipitation of step (a)
(4) Redissolved precipitation in step (a)
(5) Precipitated redissolved supernatant in step (a) [Fig. 2]
(1) Marker
(2) Total precipitation
(3) pH 4.0
(4) pH 4.4
(5) pH 4.8
(6) pH 5.2
(7) pH 5.6
(8) pH 6.0

Claims (12)

A method for producing a soy whey fraction, comprising the following steps.
(A) a step of concentrating soy whey and collecting coagulated sediment precipitated under acidic conditions;
(B) a step of fractionating the obtained supernatant into a high molecular fraction and a low molecular fraction; The aggregate precipitate recovered in step (a) is a soybean trypsin inhibitor concentrate, the high molecular fraction obtained in step (b) is a soybean β-amylase concentrate, and the low molecular fraction is soybean oligosaccharide. Manufacturing method of 1. The method according to claim 1, wherein the concentration of the soy whey in the step (a) is carried out at a pH of 3.5 to 8.5, and the solid content is concentrated to a range of 30 to 50% by weight. The method according to claim 1, wherein the concentration of the soybean whey in the step (a) is performed at a product temperature of 20C to 80C. 2. The method according to claim 1, wherein the step (a) of depositing the coagulated precipitate is performed at a pH of 1.7 to 5.2. The production method according to claim 1, wherein the precipitate obtained in step (a) is added with water and partially redissolved in a pH range of 2.0 to 4.8, and then subjected to solid-liquid fractionation. 7. The method according to claim 6, wherein the liquid part obtained by the solid-liquid fractionation is a BBI-type trypsin inhibitor concentrate, and the solid part is a Kunitz-type trypsin inhibitor concentrate. In the step (b), a polar solvent is added to the obtained supernatant, and a fractionation step (b-1) in which the supernatant is fractionated into a new supernatant and a precipitate, or the obtained supernatant is subjected to an ultrafiltration membrane. The method according to claim 1, wherein the method is a step (b-2) of fractionating into a concentrated solution and a permeated solution, or a combination thereof. In the step (b-1), the polar solvent is ethanol, the ethanol concentration is 30% by weight to 85% by weight based on the total of water and ethanol in the supernatant to which the polar solvent is added, and the temperature is -5 ° C to 15 ° C. 9. The production method according to claim 8, wherein the method is carried out within the range. 9. The method according to claim 8, wherein in step (b-2), the molecular weight cut off of the ultrafiltration membrane is in the range of 5,000 to 250,000. 9. The method according to claim 8, wherein step (b-2) is performed at a temperature of 65 ° C. or lower, a pH of 4.0 to 7.0, and a solid content of the supernatant of 50% by weight or less. 9. The method according to claim 8, wherein the fresh supernatant obtained in step (b-1) or the permeate obtained in step (b-2) is concentrated to a solid content of 50 to 85% by weight.