JP2000191694A - Fragmentation and preparation of subunit from 11s globulin of soybean, and its product - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for readily and efficiently fractionating and preparing an acidic subunit and a basic subunit, safe in food sanitation without using a reducing agent on 11S globulin. SOLUTION: An acidic subunit fraction and a basic subunit fraction are fractionated by bringing 11S globulin which is a major component of the soybean protein into contact with steam at a temp. of >=150 deg.C (under about 4 kgf/cm2 gage pressure of the steam). The fractionation is carried out under one of conditions that no reducing agent is used at all at the time of the contact with the steam, a centrifugal separation or a filtration is carried out after the contact with the steam, the temperature is kept at a temp. of >=120 deg.C for >=1 min after the contact with the steam, and the contact with the steam is performed at >=10% concentration of the protein, and the like. As the result, the cost of the reducing agent becomes unnecessary, and the method enables the cost for the removing operation, the treating expense, etc., of the remaining sulfurous acid by the conventional technique to be reduced compared to the conventional technique. The method can contribute to mass production, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to 11S globulin (also referred to as glycinin) which is a main component of soybean protein.
Accordingly, the present invention relates to a fractionation method for obtaining acidic subunits and basic subunits as constituents by a simple method without using a reducing agent.

[0002]

2. Description of the Related Art Soybean protein is not only excellent in nutritional properties among vegetable proteins, but has recently been found to have various physiologically active effects, and is a food material that has attracted attention as a physiologically functional agent. Their physiological functions are expressed from a specific site in soy protein, and establishing technology to efficiently fractionate the soy protein component containing that site is necessary to use soy protein not only as a food material but also as a pharmaceutical material Is an important factor in

[0003] It has been reported that each subunit of soy protein is dissociated during denaturation by heating. For example, Wolf WJ and Tamura T. (Cereal Chem.
46, 331, 1969) studied the thermal denaturation of soybean 11S globulin and revealed that 11S globulin is dissociated into an acidic subunit and a basic subunit by heating. For details of the dissociation conditions, see Tomohiko Mori (New
According to Food Industry, 24 (6), 53, 1982), a low-concentration (about 0.5%) 11S globulin solution is converted into a soluble acidic subunit and an insoluble basic subunit after passing through a soluble aggregate by heating. Dissociation, and at a high concentration (5% or more), such dissociation does not occur, and the gel is formed from a soluble aggregate through a soluble giant aggregate. However, subsequent studies show that Hermansson, AM, (JAO
CS, 63 (5), 658, 1986) dissociates into acidic and basic subunits regardless of protein concentration, and states that only subsequent gelation depends on protein concentration. However, in any case, the acidic subunit and the basic subunit in the solution state can be fractionated only by means of low practicality such as ultracentrifugation.

As described above, it is a basic subunit which is difficult to fractionate only by heating.
magishi T. et al., (Agric. Biol. Chem., 44, 1575, 19
80) revealed that the acidic and basic subunits of 11S globulin are relatively clearly dissociated by heating in the presence of a reducing agent. The dissociation effect of acidic and basic subunits by the addition of this reducing agent is described in Damodaran, S. and Kinsella, E. (J. Agri
c. Food Chem., 30, 812, 1982) dissociate the acidic subunit and the basic subunit by heating at 90 ° C for 30 minutes, and then reduce the acidic subunit and the basic subunit based on the re-crosslinking between cystine with a reducing agent. The subunits are prevented from reassociating via disulfide bonds and the resulting complex polymerization, and only the basic subunits form hydrophobic aggregates, resulting in an easily separable cloudy precipitate. However, the reducing agent used here, 2-mercaptoethanol, cannot be used for food processing. Kikuchi et al. Have proposed sodium sulfite as a reducing agent that can be used in food processing.
Using (Na2SO3), the acidic subunit and the basic subunit are heated at 120 ° C. and 1 atm (gauge pressure) for 10 to 20 minutes by utilizing the aggregation effect of the basic subunit by using this reducing agent. Was found (Japanese Patent Application Laid-Open No. 63-36748).

However, the above method using sodium sulfite (Na2SO3), which is a reducing agent that can be used in food processing,
The added amount (0.5 mol) is the remaining allowable amount (3
0 ppm), and it is necessary to remove the remaining sulfurous acid through a complicated operation after separating the acidic subunit and the basic subunit. It is known that sodium sulfite may have acute or chronic toxicity depending on human intake.Thus, even if removal operation is added, using it in large quantities poses a problem in food hygiene. It can be said that there is. From the above background, when fractionating and preparing acidic subunits and basic subunits as constituents thereof from 11S globulin which is a main component of soybean protein,
From the viewpoint of food hygiene, a method for obtaining the compound by a simple method without using a reducing agent has been desired. The prior art described above uses indirect heating (hot water bath, autoclave, etc.) as a heating method, and further uses a reducing agent (2-mercaptoethanol or Na2SO3, etc.). Contact, blowing, etc.) and no reducing agent.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a food safe and safe product without using a reducing agent for 11S globulin.
It is an object of the present invention to provide a method for simply and efficiently fractionating and preparing an acidic subunit and a basic subunit and a product thereof.

[0007]

According to the present invention, 11S globulin which is a main component of soybean protein is prepared at 150 ° C. (at a saturated steam pressure gauge pressure of about 4 kgf / cm 2).
Abbreviated as "about 4 kgf / cm @ 2 in gauge pressure". In addition,
Absolute pressure corresponds to about 5 kgf / cm 2, but unless otherwise specified, it means gauge pressure. A) a method for fractionating an acidic subunit fraction and a basic subunit fraction, which is characterized by contacting with water vapor as described above. More specifically, no reducing agent is used at the time of contact with steam, centrifugation or filtration is performed after contact with steam, temperature is maintained at 120 ° C. or more for 1 minute or more after contact with steam, This is a method by using any one or more conditions such as performing the contact at a protein concentration of 10% or more. The present invention employs a heating method in which a high-concentration 11S globulin solution is brought into direct contact with water vapor to dissociate an acidic subunit and a basic subunit, and by centrifugation and filtration at a practically low rotational speed, The inventors have found that the two can be fractionated, and have achieved the present invention.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The present invention uses 11S globulin, which is a main component of soybean protein, by simply devising a heating method without adding a reducing agent, by aggregating a basic subunit to a fractionable level, and efficiently combining an acidic subunit with a base. This is a method for fractionating and preparing a sex subunit.

First, terms of the present invention will be described. 11S globulin is a globulin that is generally a generic name of soluble globular proteins, and has an ultracentrifugal sedimentation coefficient of molecular weight of 11S.
Say the equivalent of 2 in molecular weight distribution of globulin
S, 7S, 11S, and 15S exist, of which 7S and 1S
It is known that 1S is contained in large amounts in storage proteins of legumes such as soybeans. In addition, 11S of soybean
Globulin is also called glycinin. The acidic subunit is one of the components of the above 11S globulin, and is rich in acidic amino acids. The basic subunit is another component of the 11S globulin and is rich in basic amino acids. This basic subunit and the above-mentioned acidic subunit consist of one disulfide (S-
S) linked by a bond to form an intermediate subunit. 11S globulin is a combination of six of these intermediate subunits.

[0010] The water vapor of 150 ° C or higher is defined as a gas, ie, a water vapor having a saturated vapor pressure of about 4 k.
gf / cm2 (gauge pressure. Absolute pressure is about 5 kgf / cm2)
2) Say what you have. Reducing agents are reagents that cause a reduction reaction in chemistry, and include various metals, metal hydrides, and low-valent metal ions (Fe ++, Sn ++, Ti + 3).
), Low oxidation state anions (S--, SO3-, S
2 O3-), hydrogen, carbon, carbon monoxide, silicon, sulfur, hydrogen iodide and the like are typical reducing agents. In the production of food, limited use is approved with conditions such as substance names, quality standards, and use standards. For example, sodium sulfite (standard of use: 30 ppm or less as SO2 for general foods other than limited foods), sulfur disulfide (standard of use: SO2 for general foods other than limited foods)
30 ppm or less).

The term "fractionation" refers to the separation of a solid and a liquid.
Examples include centrifugation and filtration. What is centrifugation?
It refers to a method of separating and fractionating solid particles in a liquid by a difference in density using centrifugal force. In the present invention, any method capable of performing this centrifugation can be used without limitation. Filtration refers to an operation of separating a liquid and a solid contained therein by passing through a filter which is a porous object. As the filter, a filter cloth, sand, a porous body such as spongy metal or the like is industrially used. The protein concentration is determined by quantifying nitrogen by a known Kjeldahl method or a method analogous thereto using a constant amount of the test substance, and determining the nitrogen coefficient (6.
25).

The fraction containing substantially no reducing agent is a fraction fractionated and prepared without using a reducing agent not derived from the raw material soybean at the time of contact with steam of 11S globulin of soybean. It means that the residue is not detected or about 30 ppm or less, preferably 10 ppm or less. The analytical measurement method is, for example, an alkali titration method (test method A) or a colorimetric method (test method B.
B is based on "Food Additive Analysis Method in Food" edited by Food Chemistry Division, Ministry of Health and Welfare.

According to the study by the present inventors, the following has been found. 1) To fractionate the acidic subunit and the basic subunit at a temperature of 120 ° C. or lower, sodium sulfite must be at least 35%.
Mmol must be added. 2) In this case, about 4,000 to 5,000 ppm of sulfurous acid remained in the separated acidic subunit and basic subunit. 3) An operation of removing the residual sulfurous acid, that is, in order to reduce the residual sulfurous acid to below the allowable amount in food, in the case of an acidic subunit, a 5% aqueous solution is heated at 80 ° C. or more for 10 minutes,
It is necessary to repeat the operation of adjusting the H to 4.0 to 5.0 to precipitate the acidic subunit and recovering by centrifugation three times or more. 4) In the case of the basic subunit, it is necessary to repeat the operation of heating the 5% suspension at 80 ° C. or higher for 10 minutes and collecting the basic subunit by centrifugation at least three times. The above was found.

The present inventors have assiduously studied and found the following. 5) The acidic subunit and the basic subunit are separated by heating at 100 ° C. or more for 10 minutes or more without adding a reducing agent. 6) However, when heated at 100 ° C. for about 10 minutes, the basic subunit does not aggregate and precipitate, and it is not easy to fractionate the heated solution into an acidic subunit and a basic subunit. 7) An aggregate precipitate of the basic subunit was obtained by direct contact (eg, by blowing the steam) with steam at 150 ° C. (about 4 kgf / cm 2 at a gauge pressure) for 1 minute. . We have found the above. Therefore, when the acidic subunit and the basic subunit are fractionated, the basic subunit is aggregated to the extent that it can be fractionated by simply devising a heating method without adding a reducing agent, and the acidic subunit is efficiently separated. To find a method for fractionating / preparing units and basic subunits, to obtain acidic subunits and basic subunits substantially free of residual reducing agent concentration, or 30 ppm or less, and often 10 ppm or less. Was completed.
The above 150 ° C. (gauge pressure of about 4 kgf / cm 2) or higher may be any temperature at which the product temperature of the 11S globulin solution can reach 150 ° C. or higher.
(About 60 Kgf / cm @ 2), preferably 150
C. to 214.degree. C. (about 20 kgf / cm @ 2). The direct contact time may be any time during which the temperature of the 11S globulin solution can reach 150 ° C. or higher. In practice, it will be about 2 to 3 seconds or more in an instant, but about 1 hour is preferable in terms of production efficiency. May be from 2 seconds or more to about 10 minutes. Conventional indirect heating (hot water bath, autoclave, etc.)
The difference in the effect of the direct heating (water vapor contact, blowing, etc.) of the present invention, compared with that of the present invention, was agglomeration precipitation and fractionation of the basic subunit, which was an unexpected finding. The mechanism of this effect difference has not been elucidated. It is presumed that precipitation occurs.

[0015] The soybean 11S globulin used for the present invention is prepared by converting defatted soybean to a pH-fractionation method of Tan Shibasaki (Thanh,
VH, and Shibasaki, K., J. Agric. Food Chem., 2
4, 117,1976), but without using the buffer containing a reducing agent described in this document.
The pH of defatted soy milk extracted with water from defatted soybeans,
It is possible to obtain 11S globulin simply by adjusting to pH 6.4, which is the isoelectric point of 11S globulin, and cooling.

The present invention relates to 10% by weight prepared as described above.
As described above, preferably, the 11S globulin aqueous solution concentrated to about 13 to 16% is brought into direct contact with water vapor,
The acidic subunit and the basic subunit are separated from each other. At this time, conditions such as protein concentration, temperature, and heating time are set. According to the experimental results of the inventors, a protein concentration of 10% or more, preferably 13.5 to 15.2%, and a pH of 11S globulin aqueous solution adjusted to a neutral region, for example, 7.0 to 8.0, were added at 150 ° C. 4 kgf
/ cm2) or more, and then the liquid temperature is set to 120
It has been found that holding at a high temperature of at least 120 ° C., for example, preferably at a temperature of about 120 to 150 ° C. for at least 1 minute is the optimal condition for efficient fractionation of acidic subunits and basic subunits. It is preferable to set heating conditions based on these conditions.

In the present invention, the reaction solution obtained by heating 11S globulin under the above conditions is separated into a supernatant and a precipitate by centrifugation or filtration, and the supernatant fraction is directly spray-dried or frozen. After drying, the precipitate fraction is also suspended in water and then spray-dried or freeze-dried to obtain the supernatant fraction as an acidic subunit and the precipitate fraction as a basic subunit.

The fractions thus obtained do not contain the respective subunits and do not use a reducing agent, so that a complicated operation of the reducing agent after heating is unnecessary.

[0019]

EXAMPLES The effectiveness of the present invention will be described below together with examples, but the technical idea of the present invention is not limited by these examples.

[0020]

Example 1 Preparation of 11S globulin Water was added to defatted soybean at a weight ratio of 1:10, and the pH was adjusted to 7.0 as needed.
Stir for 1 hour while adjusting the mixture to centrifuge.
(4,000 rpm, 20 ° C for 10 minutes), adjust the resulting supernatant to pH 6.4, leave at 4 ° C overnight, and centrifuge
(4,000 rpm, 4 ° C. for 10 minutes), and the obtained precipitate was defined as 11S globulin. 11 obtained in this way
S-globulin was subjected to SDS-polyacrylamide gel electrophoresis, and thereafter, the degree of staining of the stained protein band was measured. From the result, it was confirmed that the purity was 80% or more, and the purity was sufficient to withstand the following examination.

[0021]

Example 2 Separation and fractionation of each subunit from 11S globulin The 14.2% concentration 11S globulin solution obtained above was supplied to a steam sterilizer having a steam pressure of 6 kgf / cm2, and steam was blown for 2 seconds. The high temperature was maintained at 140 ° C. for 1 minute. After the solution has cooled to around 50 ° C,
The mixture was centrifuged (8,000 rpm, about 20 ° C. for 10 minutes) to fractionate into a supernatant and a precipitate. The supernatant was freeze-dried as it was. The precipitate was suspended by adding water in an amount substantially equal to the weight of the precipitate, and then freeze-dried. Each subunit obtained in this way is
The pattern was examined by subjecting to S-polyacrylamide gel electrophoresis. As a result, most of the supernatant fraction was composed of acidic subunits, and no contamination with basic subunits was observed. The precipitated fraction was occupied by basic subunits, and no acidic subunit was detected.

[0022]

According to the present invention, from 11S globulin,
It is possible to provide an acidic subunit and a basic subunit safely, easily and efficiently in terms of food hygiene such as steam blowing heating without using a reducing agent.

[Brief description of the drawings]

FIG. 1 shows an acidic subunit separated according to the present invention (
The electrophoresis pattern of SDS-polyacrylamide gel electrophoresis of 3) and basic subunit (4) is shown, including the sample of each purification step.

Claims (6)

[Claims] (1) 11S globulin which is a main component of soybean protein is prepared at 150 ° C. (at a saturated steam pressure gauge pressure of about 4 ° C.).
A method for fractionating an acidic subunit fraction and a basic subunit fraction, which is carried out by contacting water vapor of not less than KGf / cm2). 2. The method according to claim 1, wherein the fraction is substantially free of a reducing agent without adding a reducing agent when contacting 11S globulin with water vapor. 3. The method according to claim 1, wherein after contacting with water vapor, a precipitate as a basic subunit fraction and a supernatant as an acidic subunit fraction are fractionated. 4. After contact with water vapor, the temperature of the
The method according to any one of claims 1 to 3, wherein the method is held for at least one minute. 5. The method according to claim 1, wherein the contact with the water vapor is carried out at a protein concentration of 11S globulin of 10% by weight or more and at a neutral pH. 6. A product obtained by the method according to claim 1.