JP3430922B2 - Soy protein and emulsifier containing the same as an active ingredient - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention has an advantage that is not obtained by the conventionally known soybean protein isolate and its fractions, glycinin and β-conglycinin, in terms of solubility and emulsification under neutral and acidic conditions. The present invention provides a soybean protein having a salt and an emulsifier containing the soybean protein as an active ingredient.

[0002]

2. Description of the Related Art Soybean protein contains glycinin which is 11S globulin and β-conglycinin which is 7S globulin as main components. The relationship between the structure and processing characteristics of glycinin has been analyzed in considerable detail by the present inventors, but little has been analyzed regarding β-conglycinin. It is known that this glycine and β-conglycinin can be fractionated by the method of THANH & SHIBASAKI. Glycinin consists of an acidic subunit and a basic subunit. An invention relating to this glycine is disclosed in, for example, Japanese Patent Application Laid-Open No. 63-36748, a method of preparing a sub-unit by separating soybean glycinin, and Japanese Patent Application Laid-Open No. 09-23821 discloses soybean 11
Method for separating S-globulin basic subunit
09-23837 discloses an emulsifier and oil-in-water emulsion composition containing a high concentration of 11S globulin basic subunit, and JP-A-09-25296 discloses a method for separating soybean 11S globulin basic subunit. Each is disclosed.

It has been confirmed by SDS-polyacrylamide electrophoresis that β-conglycinin is divided into three subunits of α, α'and β. But,
Purification of each subunit of β-conglycinin with high purity and no utilization of its properties have been found yet, and no invention of soybean protein having a large α subunit composition of β-conglycinin is known.

[0004]

In soybean seeds, there are molecular species in which these subunits (each subunit constituting glycinin and β-conglycinin) are almost randomly combined, so that a uniform molecule is obtained. It is extremely difficult to prepare seeds from soybeans. In the present invention, β-
The α subunit of conglycinin was genetically engineered in large amounts using Escherichia coli and the like, and the relationship between its content and properties was investigated, and the solubility and emulsifiability of soybean protein under neutral and acidic conditions were greatly improved. Aimed at emulsifiers.

[0005]

[Means for Solving the Problems] The present inventors decided to analyze the relationship between the structure of β-conglycinin and the processing characteristics, and prepare the target soybean protein. β-conglycinin is composed of three types of subunits, α, α ′, and β. Among these three types of subunits, the β subunit is composed only of a highly conserved core region,
It has been found that the α and α ′ subunits have an extension region rich in hydrophilic amino acids on the N-terminal side (see FIG. 1). In addition, we have found that sugar chains are added to each subunit.

Therefore, the present inventors decided to prepare uniform molecular species of each of these subunits using the Escherichia coli expression system. In the E. coli expression system, addition of sugar chains does not occur, so the effects of sugar chains could also be analyzed by comparing with the natural ones having sugar chains. Furthermore, for more detailed analysis, regarding the α and α'subunits, only the core region excluding the extension region could be expressed. It was found that the protein fraction containing a high content of such α-subunit is significantly improved in solubility and emulsification in solubility and emulsification under neutral and acidic conditions as compared with the whole β-conglycinin. To complete the present invention.

That is, according to the present invention, as the subunit composition of soybean protein β-conglycinin, the α subunit is 5
Soy protein containing 0% or more. Preferably, the soy protein β-conglycinin subunit composition is soy protein containing 70% or more, and more preferably 90% or more α subunit. Although the natural α subunit contains a sugar chain, the soybean protein of the present invention preferably also contains an α subunit having no sugar chain. Natural β-conglycinin randomly contains α, α ', and β subunits, but the soybean protein of the present invention may contain no β subunit. In addition, the present invention provides the α subunit as a subunit composition of soybean protein β-conglycinin.
It is an emulsifier containing 0% or more of soybean protein as an active ingredient. An emulsifier containing soybean protein containing 70% or more of α-subunit as an active ingredient is preferable. More preferably, an emulsifier containing soybean protein containing 90% or more of α subunit as an active ingredient is suitable. A soybean protein containing an α subunit having no sugar chain can be used as an emulsifier as an active ingredient. An emulsifier containing soybean protein containing no β subunit as an active ingredient is preferable.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The soybean protein of the present invention is a subunit composition α of β-conglycinin which is 7S soybean protein,
Of the α ′ and β subunits, 50 α subunits
It is important to include at least%. Preferably 70% or more,
More preferably, it is suitable to contain 90% or more. The smaller the β-subunit composition ratio and the higher the α-subunit composition ratio, the more soluble and emulsifiable soy protein can be obtained in neutral and acidic conditions. The α subunit is an α subunit having a sugar chain derived from natural β-conglycinin, and an α subunit not having a sugar chain prepared by genetic engineering (eg, Escherichia coli expression system) or an α subunit having a sugar chain (eg, Yeast expression system).
The molecular species constituting the soybean protein of the present invention prepared by genetic engineering are a trimer formed only from α subunits, a trimer formed from α, α, and α ′ subunits, α , May be a trimer formed from α, β subunits, but preferably α containing no β subunit,
A trimer formed exclusively of α'subunits is preferred.

When the amount of α-subunit is low, the solubility and emulsification under neutral or acidic conditions are less different from that of natural β-conglycinin. As the proportion of the α subunit increases, the soybean protein can be improved in solubility and emulsifiability under neutral and acidic conditions. Usually, soybean protein is mainly composed of 7S soybean protein and 11S soybean protein, but the soybean protein of the present invention contains α, α ′, and β which are subunits constituting β-conglycinin which is 7S soybean protein. The feature is that the α subunit, which is one of the types, is 50% or more. The conventionally known 7S protein has an α subunit ratio of about 37%. Ordinary isolated soybean protein consisting of 11S protein, 7S protein, etc. has a much smaller α subunit.

The soybean protein of the present invention is extremely difficult to obtain by a conventional fractionation method. The soybean protein of the present invention is suitably obtained using a genetic engineering technique. Alternatively, a soybean protein obtained by using the genetic engineering technique and natural β-conglycinin can be combined and prepared. or,
The present invention is an emulsifier containing, as an active ingredient, soybean protein containing 50% or more of α subunit as a subunit composition of soybean protein β-conglycinin. An emulsifier containing soybean protein containing 70% or more of α-subunit as an active ingredient is preferable. More preferably, an emulsifier containing soybean protein containing 90% or more of α subunit as an active ingredient is suitable. As in the above case, the larger the proportion of the α subunit, the more emulsifying the emulsifier can be. An emulsifier containing a soybean protein containing an α subunit having no sugar chain as an active ingredient can be used, and an emulsifier containing a soybean protein containing no β subunit as an active ingredient is preferable because of excellent emulsifying property. Further, the soybean protein and the emulsifier of the present invention are genetically engineered by combining α subunit and α ′ subunit to form a trimer, and the trimer alone or in combination with natural β-conglycinin, or natural separation. It can be prepared in combination with soy protein. Examples of the trimer include α, α, α and α, α, α, α ′, α, α ′ and α ′, which are combinations of α subunit and α ′ subunit. Also, α
Even if the combination of subunit and β subunit is large, the combination of α, α, and β subunits, which has a large proportion of α subunit, has a larger proportion of α subunit than natural β-conglycinin, so it is used as the target soybean protein or emulsifier. You can The embodiments of the present invention will be specifically described below with reference to examples.

[0011]

EXAMPLES Examples of the present invention will be described below with reference to examples. Example 1 Using E. coli expression vector pET21d, α, α ′, β,
Expression plasmids for the α and α ′ cores were constructed and expressed in E. coli BL21 (DE3). In Figure 2,
The results of subjecting the E. coli whole cell protein to SDS-PAGE are shown.

C is the case where pET24d is transformed, as is clear by comparison with this,
α, α ', β, α core, α'core is 10-3 of whole cell protein
It could be expressed at a level of 0%. All were expressed in a soluble state. Next, each expressed protein could be purified almost uniformly by ammonium sulfate fractionation, Q-Sepharose and mono-Q column chromatography. On the other hand, it was found that β-conglycinin prepared from natural soybean contained 1.3 ': 2' of α ', α and β. The structure-forming ability of the expressed protein was analyzed using these purified preparations.

First, a CD spectrum was measured in order to compare the secondary structures. α, α ′, and β showed mutually characteristic spectra. From these spectra, the spectrum calculated from the composition ratio of each subunit of natural β-conglycinin was very similar to that of natural β-conglycinin. Therefore, α, α ', β
It was considered that each of the expressed proteins of 1) formed the original secondary structure. On the other hand, the α core and the α ′ core gave spectra similar to those of β, although there were some differences.
Further, the difference spectrum between the α and α cores and the difference spectrum between the α ′ and α ′ cores, that is, the spectra derived from the α and α ′ extension regions were very similar. That is, it was suggested that the core region and extension region of each subunit form secondary structures similar to each other.

Next, in order to analyze the molecular assembly ability of each expressed protein, sucrose density gradient centrifugation was performed at an ionic strength of 0.5. It was revealed that α, α ′, β and α form a trimer like the natural β-conglycinin.
Further analysis was performed by gel filtration. This gel filtration was also performed under the condition of an ionic strength of 0.5 as in the density gradient centrifugation. The α'core determined to form a hexamer by density gradient centrifugation eluted at the position of 83.6 minutes. Then, α, α ', β determined to form a trimer,
Among the α cores, the β and α cores were later than the α ′ core and eluted at the positions of 92 to 96 minutes. In addition, since the α'core also eluted at the position of 94.9 minutes at an ionic strength of 1.0, the α'core was similar to that of natural β-conglycinin in 3
It has been found that a mer can be formed. On the other hand, although α and α'form a trimer, they were eluted earlier than the core of the hexamer α '. Therefore, it was considered that the extension regions of α and α'protrude on the surface of the molecule and, as a result, contribute greatly to the dimension.

The results of the above CD, density gradient centrifugation and gel filtration indicate that the expressed protein in E. coli can form a higher-order structure similar to that of natural β-conglycinin. Therefore, these expressed proteins can be used to analyze the correlation between the structural and functional properties of β-conglycinin.

Therefore, first, ANS (1-anilinonaphthale
ne-8-sulfonate) was used to examine the surface hydrophobicity. As a result, it was found that the surface hydrophobicity became stronger in the order of β, α, α ′. The spectrum calculated from the abundance ratio of each of these subunits was close to that of the natural one. This supports that each subunit forms the original structure. Also, α '
The core of α was given a value close to that of α, and the core of α was given a value close to α. Therefore, it was considered that the surface hydrophobicity of each subunit was mainly based on the core region. Next, the denaturation temperature was examined by DSC measurement. α is 78.6 ° C, α'is 82.7 ° C, β is 90.8
It was found that the denaturation temperature was higher in the order of α, α ′ and β. The core of α was 77.3 ° C, the core of α ′ was 83.3 ° C, the core of α was α, and the core of α ′ was almost the same as α ′. In other words, it can be said that the thermal stability of each subunit is determined by the core region. On the other hand, natural β-conglycinin gave two peaks of 79.0 ° C and 83.1 ° C, and these values corresponded to the denaturation temperatures of α and α '.
Therefore, it was considered that the sugar chain does not affect the thermostability, and that the thermostability of the heterotrimer is dominated by the subunit having a low denaturation temperature.

Summarizing the above results, the extension regions of α and α ′ containing a large amount of acidic amino acids have a structure protruding from the surface of the molecule. Therefore, α and α ′ trimers are Was considered to have a larger dimension. In addition, structural features such as surface hydrophobicity and thermal stability were determined by the core region of each subunit, which was revealed to be different for each subunit.

Example 2 α of soybean β-conglycinin using an E. coli expression system,
It was clarified that the three types of subunits, α'and β, differ in structural features such as surface hydrophobicity and thermal stability, and that they are mainly based on the difference in the core region. In addition, the α and α ′ subunits had hydrophilic extension regions that were not present in the β subunit. Due to such differences in structural characteristics, it was expected that the functional characteristics reflecting the structure would also be different in each subunit. Therefore, for the purpose of clarifying the structure-function characteristic correlation of β-conglycinin at the subunit level, recombinant α, α ', β subunit and α
The functional properties of the core region of α and the core region of α ′ were investigated.

First, the solubility was investigated. Natural β-
Since conglycinin is a salt-soluble globulin, it is known that it tends to precipitate at low ionic strength, but has high solubility at high ionic strength. Therefore, the solubility was investigated under the conditions of high ionic strength and low ionic strength. First, at high ionic strength, all expressed proteins were soluble at all pH levels, similar to natural β-conglycinin. Next, under low ionic strength, natural β-conglycinin is at pH
Only in the range 4.0 to 6 is insoluble and shows a very sharp pattern. α and α ′ were also insoluble only in the pH range of 4.0 to 6, but the pH range in which they were completely insoluble was wider than that of natural β-conglycinin. It was considered that this difference was affected by the natural β-conglycinin sugar chain. On the other hand, the core regions of β and α, and the core region of α'are pH 4
It was insoluble above 5 and above. From this result, pH 6.0
It was suggested that the extension region influences the solubility above. In addition, for the β and α core regions and α ′ core region, which are insoluble at pH 4 to 5 or higher,
The solubility with respect to ionic strength was examined under the conditions of 7.6.

Further, the emulsifiability was compared by measuring the particle size distribution of the emulsion prepared by homogenizing and sonicating soybean oil under the same conditions as the heat association property. The horizontal axis represents particle size and the vertical axis represents frequency. These values are the average size of the particles, and fine particles were formed in the order of α, α ′, α core region, α ′ core region, and β. In particular, α was close to that of BSA, which has high emulsifiability. Since the emulsifying property requires a flexible structure that is easily modified on the oil droplet interface, the structural stability and the emulsifying property may be correlated. Therefore, the average particle size obtained from the particle size distribution was plotted against the denaturation temperature of each subunit.

When the core regions were compared, fine particles were formed in the order of α core region, α ′ core region, and β, which corresponded to the order of low denaturation temperature. Furthermore, between α and α'having extensions, the order of modification temperature also corresponded, and the modification temperature was correlated with the particle size of the emulsion. Further, α and α ′ formed finer particles than the respective core regions, which indicated that the extension region greatly contributed to the emulsification property. The above results indicate that the functional properties of each subunit are different from each other, which is due to the contribution of sugar chains and extension regions, and to the great difference in the structural stability of the core region. It was

Example 3 (Preparation of protein containing a large amount of α subunit) In order to prepare a protein fraction containing a large amount of α subunit, the following genetic engineering technique was used. First, a cDNA library was prepared from seeds of the soybean variety Wasethalis during the ripening stage. Next, the cDNA for the α subunit was cloned from the obtained cDNA library. And ma
The region encoding the ture portion was amplified by PCR and inserted into the downstream of the T7 promoter of the expression vector pET21d to construct an expression plasmid for the α subunit. This plasmid was used for E. coli BL21
(DE3) was expressed to prepare a protein solution containing a high amount of α fraction. A protein fraction was precipitated from the obtained protein solution with 70% ammonium sulfate (ammonium sulfate), dialyzed to remove ammonium sulfate, and freeze-dried to obtain a target sample. This sample is S
As a result of examination by DS-polyacrylamide gel electrophoresis, no protein bands other than the α subunit were detected.

(Basic Properties) The basic properties of this sample were investigated.
For comparison, β-conglycinin prepared by the method of THANH & SHIBASAKI was used. The results are shown in (Table 1). The solubility and emulsifying property of a mixture of β-conglycinin containing about 38% of α-subunit and soybean protein consisting of α-subunit at the ratio shown in Table 1 were examined. The emulsifying property was measured by the method of Kinseler.
Solubility was measured as follows. Setting ionic strength, p
The sample was added to the phosphate buffer adjusted to H so that the final concentration was 1%, and the mixture was stirred uniformly. Leave for 30 minutes at 37 ° C in a water bath. 3000G * 10 minutes centrifugation Measure the amount of protein with BIOLAD's protein measurement kit.

For the solubility, the ratio of the amount of protein in the solution before (the conditions of) centrifugation and the solution after the removal of the conditions (the conditions) was determined, and the pH of β-conglycinin was 7.0 and the ionic strength was 0.5.
The value in 100 is shown as a relative value. Table 1 (mixing ratio of β-conglycinin and α-subunit protein and α-subunit content (% by weight)) ------------------------- ------------------------------------------- No. β Conglycinin α subunit Proportion of protein α subunit -------------------------------------------- ------------------------ 1 100 0 37.7 2 0 100 100 100 3 100 0 37.7 4 50 50 50 68.9 5 30 30 70 81 3 6 10 90 93.8 7 0 100 100 -------------------------------------- ------------------------------

Table 2 (Relationship between composition ratio of β-conglycinin-α subunit and solubility / emulsification property) -------------------------- -------------------------- No. of Table 1, PH Ionic strength Solubility Emulsification ------------ ------------------------------------------ 1 7.0 0.5 100 100 2 7.0 0.5 100 250 3 3 5.0 0.3 30 30 25 4 5.0 0.3 50 50 75 5 5.0 0.3 0.3 70 120 6 5.0 5.0 0.3 90 150 7 5.0 0.3 90 150 --------------------------------------------- ------------ Thus, it was found that the α subunit has a high emulsifying property and has a high solubility in the acidic region as compared with β conglycinin.

Example 4 (Application) Using this sample, a beverage containing fats and oils was prepared with the composition as shown in (Table 2). The resulting solution is 0.2 for each pH.
It was dropped in M phosphate buffer and the state was observed. As a result β
It was found that the stability in the low PH region was better than that using conglycinin. Table 3 ---------------------- Composition / mixing rate ----------------------- Protein 5% Palm oil 7% Sugar 3% Water 85% ------------------------ (Preparation method) Mix the raw materials with a propeller stirrer at 60 ℃. After homomixer (manufactured by Tokushu Kika) 7,000 rpm * 10 minutes emulsification

Table 4 (Solubility and coagulability of emulsified protein drink) ---------------------------------- ------------------ PH 6.0 5.0 4.5 4.0 ------------------- ---------------------------------- α subunit ○ ○ △ × β Conglycinin block ○ × × ×- -------------------------------------------------- --However, ○: dissolved, △: partially aggregated, ×: aggregated. As described above, the protein solution containing a high content of α subunit is superior in solubility and emulsification in the low PH region as compared with the conventional isolated soybean protein, and can be expected to be effective as a functional agent for foods.

[0028]

As described above, according to the present invention, β-
It is possible to obtain a soybean protein having a high α subunit content in the subunits constituting conglycinin, and having greatly improved solubility and emulsification under neutral and acidic conditions. The soybean protein is also excellent in gelling property. Further, an emulsifier having an excellent emulsifying property under neutral and acidic conditions has become possible.

[Brief description of drawings]

"

1] is a drawing schematically showing each subunit of α, α ′, and β, and is a drawing showing a trimer listed from each subunit in the lower column. "

[Fig. 2] is an E. coli whole strain in which an expression plasmid for α, α ', β, α core and α'core was constructed using E. coli expression vector pET21d and expressed in E. coli BL21 (DE3). It is a figure which shows the result of having applied the body protein to SDS-PAGE.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ identification code FI B01F 17/00 A23L 2/00 J C07K 14/415 L (58) Fields investigated (Int.Cl. ⁷ , DB name) A23J 1 / 00-3/16 A23L 1/035 C07K 14/415 BIOSIS (DIALOG) CA (STN) JISC file (JOIS)

Claims (10)

(57) [Claims] 1. A soybean protein containing 50% or more of α subunit which is a subunit of soybean protein β-conglycinin. 2. A soybean protein containing 70% or more of α subunit which is a subunit of soybean protein β-conglycinin. 3. A soybean protein containing 90% or more of α subunit which is a subunit of soybean protein β-conglycinin. 4. The soybean protein according to claims 1 to 3, which contains an α subunit having no sugar chain. 5. The soybean protein according to claim 1, which does not contain β subunit. 6. An emulsifier comprising soy protein as an active ingredient, which comprises 50% or more of α subunit which is a subunit of soy protein β-conglycinin. 7. An emulsifier containing, as an active ingredient, soybean protein containing 70% or more of α subunit which is a subunit of soybean protein β-conglycinin. 8. An emulsifier containing soy protein as an active ingredient, which contains 90% or more of α subunit which is a subunit of soy protein β-conglycinin. 9. The emulsifier according to claim 6, which comprises soybean protein containing an α subunit having no sugar chain as an active ingredient. 10. The emulsifier according to claim 6, which contains soybean protein containing no β subunit as an active ingredient.