JP4165329B2 - Production method of soybean whey fraction - Google Patents

Description

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

  The trypsin inhibitor which is one of the physiologically active substances contained in soybean is known to be 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. Disposable absorbent product having skin care composition containing cancerous chest / ascites retention inhibitor (Patent Document 1), inflammatory edema enhancement inhibitor (Patent Document 2) and enzyme inhibitor as physiological functions (Patent Document 3) ) Etc. have been reported.

  Soybean β-amylase is an enzyme that hydrolyzes starch amylose in the form of maltose from the non-reducing end, has a molecular weight of 57,000, has an isoelectric point of 5.5, and is used for the production of chickenpox and maltose. It is used directly in bread and Japanese confectionery as an anti-aging agent. In addition, soybean has very little α-amylase activity, and soybean β-amylase has higher heat resistance than β-amylase derived from other plants, so it has an advantage in high-purity maltose production and food aging prevention applications. ing.

  Many oligosaccharides contained in soybeans have physiologically active sugars such as stachyose, raffinose, verbose course, pinitol, chiro-inositol, myo-inositol, sucrose, etc. The carbohydrates possessed are stachyose, raffinose, and verbus course, and their use as bifido factors has been reported in Patent Document 4, Patent Document 5, and the like.

  As a method for fractionating soybean trypsin inhibitor, various purification methods have been proposed at the laboratory level by a combination of isoelectric precipitation, salting out, ion exchange chromatography, gel filtration and the like. As an industrial method, there are Patent Document 6 in which an ultrafiltration membrane and ion exchange chromatography are combined, and Patent Document 7 in which a trypsin inhibitor extraction method and ion exchange chromatography are combined.

  As a method for fractionating soybean β-amylase, there are Patent Document 8 and Patent Document 9 using an ultrafiltration membrane, Patent Document 10 using synthetic aluminum silicate as an adsorbent, and the like.

  As a method for fractionating soy oligosaccharides, Patent Document 11 which reduces protein along with precipitates formed by neutralizing soy whey with calcium hydroxide in the presence of calcium chloride, pH 3.0 or less with phosphoric acid after heating soy whey Patent Document 12 that reduces the protein together with the precipitate produced after the adjustment.

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 fractionation method of soybean trypsin inhibitor, the fractionation method of soybean β-amylase, and the fractionation method of soybean oligosaccharide have been mentioned, but all the methods specialized for the target substance are discussed. There is no technical proposal to comprehensively use soy whey containing various useful ingredients.

Japanese Patent Publication No. 6-23113 Japanese Patent Laid-Open No. 7-10772 JP-T-2002-505916 JP-A-3-22971 Japanese Patent Laid-Open No. 60-066978 JP-A-6-145198 Japanese Patent Application Laid-Open No. 10-066572 Japanese Patent Publication No.57-11636 JP-A-7-107974 Japanese Patent Publication No.57-52386 JP 60-66978 A 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 mixed directly into ion-exchange chromatography to recover specific components. Serving soy whey is also not realistic.
Although the method of fractionating protein with an ultrafiltration membrane is effective, soybean whey gradually develops starch mainly composed of trypsin inhibitor, which causes clogging of the ultrafiltration membrane and causes a decrease in flux. Therefore, it is difficult to concentrate dilute soybean whey at a high concentration.
Also, soybean trypsin inhibitor and soybean β-amylase have a small difference in molecular weight, and are not suitable 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 using an ultrafiltration membrane.
In view of the above, the present invention fractionates soybean whey into a soybean trypsin inhibitor concentrate (preferably a fraction mainly composed of BBI type and Kunitz type) without the need to add a salt, and a soybean β-amylase concentrate and The purpose was to fractionate into soy oligosaccharide concentrate.

The present inventors can recover 80% or more of the activity of trypsin inhibitor present in soybean whey as a precipitate by concentrating soy whey produced as a by-product in the production process of isolated soybean protein and then adjusting the pH to a specific range. Obtained knowledge.
Next, a polar solvent was added to the supernatant separated in the above step and fractionation was attempted. As a result, it was found that 85% or more of β-amylase activity present in soybean whey can be recovered as a precipitate.
On the other hand, it was found that the collected trypsin inhibitor precipitate was hydrolyzed within a specific pH range and partially dissolved to fractionate into fractions mainly composed of BBI type and Kunitz type.
Then, when an attempt was made to concentrate the β-amylase from the supernatant separated in the above step using an ultrafiltration membrane, the precipitate generated in advance was removed as a trypsin inhibitor fraction. There was almost no knowledge that 85% or more of the β-amylase titer present in soybean whey could be recovered as a concentrate.

The present invention is the following method completed by the above knowledge.
1. A method for producing a soybean whey fraction comprising the following steps:
(A) The process of concentrating soybean whey and collect | recovering the aggregate deposits which precipitate under acidic conditions.
(B) A step of fractionating the obtained supernatant into a high molecular fraction and a low molecular fraction.
2. The aggregated 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 a soybean oligosaccharide. Manufacturing method.
3. Concentration of the soybean whey in 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 wt% to 50 wt%. Manufacturing method.
4). 1. Concentration of soybean whey in step (a) is carried out at a product temperature of 20 ° C to 80 ° C. Manufacturing method.
5. The step of depositing the aggregated precipitate in step (a) is carried out at a pH of 1.7 to 5.2. Manufacturing method.
6). Water is added to the precipitate obtained in step (a) and partial redissolution is performed in the range of pH 2.0 to 4.8, and this is solid-liquid fractionated. Manufacturing method.
7). The liquid part obtained by the solid-liquid fraction is a BBI type trypsin inhibitor concentrate, and the solid part is a Kunitz type trypsin inhibitor concentrate. Manufacturing method.
8). In step (b), a polar solvent is added to the obtained supernatant, and the fractionation step (b-1) for fractionation into a new supernatant and the precipitated precipitate is performed, or the obtained supernatant is used as an ultrafiltration membrane. Process (b-2) for fractionating into a concentrated solution and a permeate, or a combination thereof. Manufacturing method.
9. In step (b-1), the polar solvent is ethanol, the ethanol concentration with respect to the total of water and ethanol in the supernatant to which the polar solvent is added is 30% to 85% by weight, and the temperature is -5 ° C to 15 ° C. Implement within the range of 8. Manufacturing method.
10. 7. The molecular weight cutoff of the ultrafiltration membrane in the step (b-2) is in the range of 5,000 to 250,000. Manufacturing method.
11. Step (b-2) is performed within a range where the temperature is 65 ° C. or less, the pH is 4.0 to 7.0, and the solid content of the supernatant is 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, soybean whey can be fractionated into three fractions: a soybean trypsin inhibitor concentrate, a soybean β-amylase concentrate, and / or a soybean oligosaccharide concentrate.
Furthermore, BBI type trypsin inhibitor and Kunitz type trypsin inhibitor can be fractionated from the soybean trypsin inhibitor concentrate, and high concentration can be achieved by diafiltration of soybean β-amylase with a UF membrane.
These fractions can be used as intermediate materials for soybean trypsin inhibitor, soybean β-amylase, and soybean oligosaccharide.
If the process of the present invention is used, soybean trypsin inhibitor, soybean β-amylase, and soybean oligosaccharide can be efficiently separated from soybean whey in a series of steps. Soy whey can be used comprehensively. It has become possible.

Step (a): A step of concentrating soybean whey and collecting agglomerated sediment that precipitates under acidic conditions.
As the soybean whey used in the present invention, soybean whey having activity of soybean trypsin inhibitor and soybean β-amylase is used. That is, it is preferable that it does not receive a heat history enough to inactivate soybean trypsin inhibitor or soybean β-amylase in the process in which it occurs.
Soy whey is by-produced in the process of producing separated soy protein or concentrated soy protein from soy. Soy whey with less heat performance is, for example, by coagulating and precipitating soy protein near the isoelectric point (for example, around pH 4.5) in defatted soymilk from which low-denatured defatted soybean is extracted with water in the vicinity of neutrality and then removed. Can be obtained as a removed solution.

  Soy whey produced as a by-product in the process 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 solid content of about The solid content composition (hereinafter also referred to as “dry%”) is about 3%, and is composed of about 20% crude protein, 20% ash, and about 60% sugar, and the water extraction ratio from defatted soybeans has changed. However, there is almost no change in the solid content composition of soybean whey. If the solid content is about 3%, soybean trypsin inhibitor is contained in an activity of about 5 units / ml, and soybean β-amylase is contained in an activity of about 100 units / ml, and can be suitably used in the present invention.

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

  The concentration of soybean whey in the step (a) is carried out at pH 3.5 to 8.5, preferably pH 4.5 to 7.0, and the solution temperature (article temperature) is 20 ° C. to 80 ° C., preferably 20 ° C. to 65 ° C., It is appropriate to concentrate the solid content to a range of 30 wt% to 50 wt%, preferably 35 wt% to 45 wt%.

  Below pH 3.5, β-amylase tends to be unstable, and above pH 8.5, trypsin inhibitor tends to become unstable. Further, when the solution temperature is less than 20 ° C., the pressure reducing device may have a mechanical limit, and when it exceeds 65 ° C., the activity of β-amylase tends to decrease, but 60% activity remains at 70 ° C. . That is, outside the above pH, temperature and concentration ranges, the recovery rate 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 of trypsin inhibitor has an isoelectric point of pH 4.5, the BBI type has an isoelectric point of pH 4.2, and β-amylase has an isoelectric point of pH 5.5. Even if the isoelectric point precipitation is attempted by adjusting the pH at the isoelectric point, no aggregation or precipitation of the target substance occurs or the amount is extremely small.

By adjusting the solid content to the above range, the ash concentration in the whey is relatively increased, the same effect as salting out can be obtained, and 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 soy trypsin inhibitor is precipitated with salt, a large amount of 14% salt is added to 3% soybean whey. It was not realistic because it was necessary.

  A known means can be used as the concentrating means. However, concentration under reduced pressure can prevent temperature rise and can concentrate efficiently.

The soy whey concentrated as described above is obtained by adjusting the aggregated precipitate and the supernatant which are precipitated by adjusting the pH to 1.7 to 5.2, more preferably 2.7 to 4.8 under acidic conditions. Fractionation. This aggregated precipitate is enriched with soybean trypsin inhibitor.
Even if it is outside the range of pH 1.7 to 5.2, it is possible if the temperature is kept at 20 ° C. or less, paying sufficient attention to inactivation of β-amylase due to low pH.

  The aggregated precipitate obtained in this way is a soybean trypsin inhibitor concentrate, which is hydrolyzed and partially dissolved in the range of pH 2.0 to 4.8, preferably pH 3.0 to pH 4.4, and further solid-liquid fractionated. Can do. The liquid part obtained by this solid-liquid fraction can be a BBI type trypsin inhibitor concentrate, and the solid part can be a Kunitz type trypsin inhibitor concentrate. This is because the salt concentration becomes lower than that of concentrated whey due to the addition of water, so that the BBI type trypsin inhibitor is redissolved, whereas the Kunitz type trypsin inhibitor does not dissolve, and both are separated into a supernatant and a precipitate. It can be done.

  The degree of hydration is 1.5 times or more, preferably 2 to 10 times, most preferably 2.5 to 4 times that of the aggregated precipitate (hydrous material).

  As a means for this fractionation (solid-liquid separation), a known solid-liquid separation device such as a centrifugal separation device or a membrane separation device 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.
In this fractionation, a polar solvent is added to the supernatant obtained in the step (a), and the fraction is separated into an aggregate and a new supernatant (b-1), or the supernatant is ultrafiltered. It can carry out by the process (b-2) which processes with a film | membrane, and fractionates into a concentrate and a permeate, or its combination.

First, the step (b-1) will be described.
A polar solvent is added to the supernatant obtained in the step (a) to separate into an aggregate and a supernatant that are precipitated. This aggregate is useful as a soybean β-amylase concentrate, and the newly produced supernatant can be used as a raw material for oligosaccharides. The newly produced 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 ethanol concentration with respect to the total of water and ethanol in the supernatant added with ethanol is adjusted to 30% to 85% by weight, preferably 50% to 80% by weight. However, it is good to carry out by putting it in the range of -5 degreeC-15 degreeC, preferably 0 degreeC-10 degreeC.
When the ethanol concentration is low, β-amylase is dissolved in a low concentration ethanol solution, and when it is high, carbohydrates are also precipitated. From the viewpoint of the bactericidal effect, the ethanol concentration is more preferably 65% by weight to 75% by weight.
When the temperature is higher than the above temperature, the activity of the β-amylase fraction is likely to decrease.

  In addition, since the solid content of the supernatant obtained in step (a) is as high as about 30% to 50% by weight, in order to avoid a large amount of oligosaccharide in the mother liquor being distributed to the precipitate in step (b-1). It is also possible to appropriately dilute the supernatant of step (a) and dissolve oligosaccharides in the supernatant during ethanol precipitation to increase the recovery rate.

The new supernatant obtained in the step (b-1) is preferably reduced in volume for storage and transportation and prevented from decay by concentrating the oligosaccharide fraction to 50% by weight or more. Further, a concentration range that is fluid and has very little spoilage as a raw material for the oligosaccharide is preferable, and it is appropriate to set the solid content to 50 to 85% by weight, preferably 55 to 80% by weight.
The supernatant concentrate can be subjected to a purification treatment such as desalting to obtain a purified oligosaccharide.

Next, the process (b-2) of this invention is demonstrated.
The supernatant obtained in step (a) is fractionated into a concentrated solution and a permeated solution 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 molecular weight cut-off of 5,000 to 250,000, preferably 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 (permeation flux) decreases. For this reason, a diafiltration method in which ultrafiltration is performed with appropriate addition of water in order to maintain the solid content of the concentrated liquid at 50% by weight or less, preferably 45% by weight or less can be employed.
The higher the temperature of the solution that passes through the ultrafiltration membrane, the higher the flux, but it is desirable that the temperature be such that the β-amylase is not inactivated at 65 ° C., preferably 55 ° C. or less. It is desirable that β-amylase is stable at pH 4.0 to pH 7.0, preferably pH 4.5 to pH 6.0. β-amylase is stable at pH 6.0 or higher, but acidic at pH 6.0 or higher. This is not preferable because starch is generated due to insolubilization of phytic acid dissolved on the side.

  The permeate obtained in the 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 to reduce the volume and prevent spoilage for storage and transportation by concentrating the oligosaccharide fraction of the permeate obtained in step (b-2) to 50% by weight or more.
For example, this permeate has a fluidity as a raw material for soybean oligosaccharides and preferably has a concentration range that is extremely low in 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.

  The following examples illustrate embodiments of the present invention.

[Example 1]
(A) Process:
70 kg of soybean whey obtained in the isolated soybean protein production process (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. 1 dry%, ash 19.7 dry%, carbohydrate 60.2 dry%) was concentrated under reduced pressure using an evaporator (CEP-L type) manufactured by Okawara Seisakusho under the conditions of a soybean whey evaporation temperature of 50 ° C. (heating temperature 80 ° C.). As a result, 4,600 g of concentrated whey having a total content of 41.3 wt%, a total trypsin inhibitor activity of 333,000 units and a total β-amylase activity of 8,530,000 units (about 300 g loss due to adhesion, etc.) was obtained.
The concentrated whey was cooled to 15 ° C., adjusted to pH 4.0 with phosphoric acid, allowed to stand for 3 hours, and then centrifuged (1,500 G × 10 minutes) to separate into a supernatant and a precipitate.
4,297 g of the separated supernatant (step (a) step 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 precipitate was precipitated. 324 g of ((a) step precipitate) 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 partition rates of both enzyme activities were 11.6% for the trypsin inhibitor, 88.4% for the precipitate, 92.1% for the β-amylase, and 7.9% for the precipitate. .

(B-1) Step:
(A) 4,200 g of the process supernatant is cooled to 8 ° C., and 4,000 g of primary ethanol (purity 99.5%) (ethanol concentration 61.3% by weight) is added gently with stirring with a propeller. It was possible to form agglomerate-like aggregates and separate them as they were, but after 15 minutes, they were centrifuged (1,500 G × 10 minutes) and separated into supernatant and precipitate.
2,028 g of the separated precipitate ((b-1) step 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.) with a rotary evaporator to remove ethanol to obtain 2,530 g of a solution having a solid content of 29.8% by weight ((b-1) step supernatant).

(B-1) Step precipitation is almost completely dissolved by adding about 3 times the amount of water and stirring and dissolving with a homomixer (3,000 rpm). Therefore, highly purified β-amylase can be obtained using this precipitate as a raw material. .
(B-1) 2500 g of the process supernatant was further concentrated under reduced pressure at a vaporization temperature of 60 ° C. and a heating temperature of 90 ° C. with a thin film flash evaporator (manufactured by Tokyo Science and Technology MF-10A type), and concentrated to a solid content of 60.1% by weight. 1,120 g of product was obtained.
This concentrate contained 76.3% by weight of saccharides per weight of solid content, 32.0% of which was oligosaccharides of 3 or more saccharides.

[Example 2]
300 g of concentrated whey concentrated under reduced pressure by the same method and concentrated to a solid content of 51.5% using the same soybean whey as in Example 1 (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, the solid content was 50.9 wt%, the total trypsin inhibitor activity was 15,000 units, the total β-amylase activity was 611,000 units, and 34 g of the precipitate was obtained, the solid content was 48.8 wt%, the 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 trypsin inhibitor was inferior to that of Example 1.

Example 3
300 g of concentrated whey concentrated under reduced pressure using the same soybean whey as in Example 1 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%, the total trypsin inhibitor activity was 7,300 units, the total β-amylase activity was 320,000 units, 18 g of the precipitate was obtained, the solid content was 35.2% by weight, The total trypsin inhibitor activity was 8,100 units, and the total β-amylase activity was 54,000 units.

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

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

[Application Example 1]
Prepared in the same manner as in Example 1 (a) Add 500 g of water to 100 g of the precipitate (step 56.1% solid content, total trypsin inhibitor activity 95,000 units), and stir and dissolve with a homomixer (3,000 rpm × 15 minutes). Then, the supernatant and the precipitate were separated by centrifugation (1,500 G × 10 minutes). As a result, 580 g of the supernatant (solid content 7.9%, total trypsin inhibitor activity 93,000 units) was obtained. 20 g (51.5% solids, total trypsin inhibitor activity 3,500 units) was obtained.
From this result, since the trypsin inhibitor coagulated and precipitated in the step (a) is redissolved by adding water, the trypsin inhibitor can be highly purified from the precipitate in the step (a).

Example 5
(A) Process:
Soybean whey 200 kg (dry matter solid content 3.0 wt%, 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 content 20 0.1 dry%, carbohydrates 59.6 dry%) was concentrated under reduced pressure using an evaporator (CEP-L type) manufactured by Okawara Seisakusho under the conditions of the evaporation temperature of soybean whey at 50 ° C and the heating temperature at 80 ° C. %, 14,000 g (about 200 g loss due to adhesion, etc.) of concentrated whey with 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).
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, and then centrifuged (1,500 G × 10 minutes) to separate into a supernatant and a precipitate.
12,850 g of the separated supernatant (step (a) process supernatant) was obtained, the dry matter solid content was 41.3% by weight, the total trypsin inhibitor titer was 110,500 units (8.6 units / g), and the total β-amylase activity. Is 1,500,000 units (1,595 units / g), 1,150 g of precipitate ((a) step precipitate) is obtained, the dry matter solid content is 52.3% by weight, the total trypsin inhibitor activity is 820,000 units (713 units / 713). g), the total β-amylase titer was 1,530,000 units (1330 units / g).
Therefore, the partition rates of both enzyme activities were 11.9% for the trypsin inhibitor, 88.1% for the precipitate, 93.1% for the β-amylase, and 6.9% for the precipitate. .

(A) Re-dissolution of process precipitate:
(A) Step precipitation 2,000 g was added to 1,000 g and the precipitate was maintained at pH 4.0 while stirring and dissolved using a homomixer (4,000 rpm × 15 minutes), followed by centrifugation (5, 000 G × 10 minutes) and separated into supernatant and precipitate.
2,767 g of the supernatant was obtained with a solid content of 14.2%, a total trypsin inhibitor titer of 595,000 units (215 units / g), and 233 g of a precipitate with a solid content of 55.8% total trypsin inhibitor titer of 221,000 units ( 948 unit / g).
The supernatant and precipitated protein were analyzed by SDS electrophoresis.

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

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

(Table 1)

(A) The process supernatant was adjusted to 40 Brix and 12.8 kg was processed.
In order to maintain the concentration of the UF membrane concentrate at about 40 Brix, water was added at 60 minutes, 90 minutes, 110 minutes, and 120 minutes, respectively.
As the concentration progresses, the flux decreases as the concentration increases, but it is thought that clogging is not caused because the flux recovers by adjusting the water concentration, and the operation was completed in 130 minutes, but the flux was remarkable. Since there is no decrease, further concentration is possible.
The average flux was 4.1 kg / m2 / hr, and the obtained concentrated liquid had a dry matter solid content of 38.0% and a β-amylase titer of 13,500 U / g (35,500 U / g in terms of dry matter). The composition of the UF membrane permeate is shown in Table 2.

(Table 2)

  Moreover, as a result of analyzing carbohydrates using water as a mobile phase in a high performance liquid chromatograph (detector / L-6200, manufactured by Hitachi, Ltd./pump/L3350, column / Shodex Sugar SC-1011), dry matter solid content A soybean oligosaccharide concentrate containing 27.8% oligosaccharide (raffinose + stachyose), 7.3% pinitol, 1.0% chiro-inositol, 2.6% myo-inositol, etc. there were.

Example 6
The UF membrane permeate was hydrated to adjust the dry matter solid content to 20% (ash content: 4.5%), and desalted with an electrodialyzer (Asahi Kasei Micro Acylizer S3, Cartridge / AC-220-550). As a result, when the electrical conductivity was reduced to about 1.0 mS / cm (ash content 0.2%, dry matter solid content 16.5%), a soy oligosaccharide concentrate with good flavor was obtained.

[Example 7] (Example combining UF membrane (ultrafiltration membrane) concentration and ethanol precipitation)
(A) The concentration of the process supernatant was adjusted by adding water to 40 Brix (solid content was about 38%, β-amylase titer 1,470 U / g), manufactured by Daizen Membrane Co., Ltd. ultrafiltration membrane (fractionated molecular weight 30) , 000, membrane area 1.4 m 2, hollow fiber module), while maintaining the concentration on the concentration side at 40 Brix, while appropriately adding water, flow rate 1 m 3 / hr, back pressure 3 kg / cm 2, liquid temperature 45 ° C., pH 4.5 The dry matter solid content was 38.0%, and the β-amylase titer was 13,500 U / g (the dry matter equivalent was 35,500 U / g). 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 allowed to stand for 10 minutes, followed by centrifugation to obtain a precipitate.
The precipitate had a dry matter solid content of 59.2%, a β-amylase titer of 23,400 U / g (39,500 U / g in terms of dry matter), and β-amylase was further concentrated.
Moreover, there was no β-amylase activity in the ethanol precipitation supernatant.

Example 8
Concentrated whey 450 g (total trypsin inhibitor titer 30,500 units) obtained by concentrating soybean whey under reduced pressure by the same method as in Example 1 to a solid content of 40.6% by weight was added to 50 g (total trypsin inhibitor) in nine 100 ml beakers. Titer (3,300 units) and cooled to 15 ° C., and then pH 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, After adjusting to 5.5 with hydrochloric acid or sodium hydroxide and allowing to stand for 8 hours, the supernatant was 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 is pH 1.5 / 2,500 units, pH 2.0 / 1, 650 units, pH 2.5 / 1, 330 units, pH 3.0 / 820 units, pH 3.5 / 540 units, 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. That's right.

Example 9
50 g of the aggregated precipitate (trypsin inhibitor fraction) prepared in the same manner as in Example 1 was added with sodium hydroxide or hydrochloric acid to pH 4.0, pH 4.4, pH 4.8, pH 5.2, pH 5.6, pH 6.0. While maintaining the pH of each, 150 g of water was added and stirred with a homomixer (4000 rpm × 20 minutes, 20 ° C.) to promote redissolution.
FIG. 2 shows an SDS-electrophoresis pattern (fraction of trypsin inhibitor) of the supernatant protein which was centrifuged and dissolved in a high-speed centrifuge manufactured by Hitachi Koki at 5,000 G × 20 minutes.
According to FIG. 2, KSTI is not distributed to the supernatant at pH 4.4 in lane (4), but at pH 4.8 and higher in lane (5), KSTI is mixed in the supernatant and cannot be fractionated into BBI and KSTI. .
In addition, at the lower limit of the fractional pH of KSTI and BBI, pH 4.0, pH 3.0, pH 2.0, and pH 1.0 were examined. Although not shown, KSTI is not distributed to the supernatant at pH 3.0, but pH 2 Below 0.0, KSTI was mixed in the supernatant.

  If the production method of the present invention is used, 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 used comprehensively. Is now possible.

It is the SDS electrophoresis pattern of the protein of (a) process supernatant, (a) process precipitation, (a) process precipitation re-dissolution precipitation, and (a) process precipitation 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 symbols

[Figure 1]
(1) Marker
(2) (a) Step supernatant
(3) (a) Precipitation of process
(4) Precipitation redissolution precipitation in step (a)
(5) Precipitate redissolved supernatant in step (a) [Figure 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 soybean whey fraction comprising the following steps:
(A) The process of concentrating soybean whey and collect | recovering the aggregate deposits which precipitate under acidic conditions.
(B) A step of fractionating the obtained supernatant into a high molecular fraction and a low molecular fraction. The aggregate precipitate collected 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 a soybean oligosaccharide. 1 production method. The process according to claim 1, wherein the concentration of soybean whey in step (a) is carried out at pH 3.5 to 8.5, and the solid content is concentrated to a range of 30 wt% to 50 wt%. The process according to claim 1, wherein the concentration of soybean whey in step (a) is carried out at a product temperature of 20 ° C to 80 ° C. The process according to claim 1, wherein the step of depositing the aggregated precipitate in step (a) is carried out at a pH of 1.7 to 5.2. The process according to claim 1, wherein the precipitate obtained in step (a) is added with water, partially redissolved in the range of pH 2.0 to 4.8, and subjected to solid-liquid fractionation. The process according to claim 6, wherein the liquid part obtained by the solid-liquid fraction is a BBI type trypsin inhibitor concentrate, and the solid part is a Kunitz type trypsin inhibitor concentrate. Step (b) is a fractionation step (b-1) in which a polar solvent is added to the obtained supernatant and fractionated into a new supernatant and a precipitated precipitate, or the obtained supernatant is subjected to an ultrafiltration membrane. The production method according to claim 1, wherein the step (b-2) is a step of fractionating into a concentrate and a permeate, or a combination thereof. In the step (b-1), the polar solvent is ethanol, the ethanol concentration with respect to the total of water and ethanol in the supernatant to which the polar solvent is added is 30% by weight to 85% by weight, and the temperature is −5 ° C. to 15 ° C. The manufacturing method of Claim 8 implemented in the range of these. The process according to claim 8, wherein the molecular weight cut-off of the ultrafiltration membrane is in the range of 5,000 to 250,000 in the step (b-2). The process according to claim 8, wherein the step (b-2) is carried out within a range where the temperature is 65 ° C or lower, the pH is 4.0 to 7.0, and the solid content of the supernatant is 50 wt% or lower. 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 wt% to 85 wt%.