JP2015014589A - Method of analyzing protein in natural acid dye - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of performing precision analysis of protein in natural acid dye.SOLUTION: A method for analyzing protein in natural acid dye includes: (1) a pretreatment step A for pretreating a natural acid dye sample, which involves gel-filtration of the natural acid dye sample possibly containing protein using one or more cross-linked polysaccharides selected from dextran and derivatives thereof, and agarose and derivatives thereof as a stationary phase and aqueous solution having a salt concentration of 5 mM or more and pH in a range of 1.5-9 as a mobile phase to extract eluate possibly containing protein; and (2) a step for analyzing the natural acid dye sample that has gone through the pretreatment step A.

Description

  The present invention relates to a method for analyzing proteins in natural acid pigments.

Conventionally, various pigments are used as coloring agents for pharmaceuticals, cosmetics, and foods.
In recent years, the use of natural pigments has been declining due to the development of synthetic pigments. However, in recent years, natural pigments have come to be favored again along with the increase in nature orientation.
However, since natural pigments are obtained by extraction and purification from natural products such as animals and plants, remaining proteins and the like contained in natural products may be problematic.

  Cochineal pigment, which is a natural acidic pigment that has been widely used as a coloring agent for pharmaceuticals and foods in the past, parasitizes cactiaceae (Nopalea coccinellifera), etc. in the desert region of Mexico, Central America and South America. It is derived from the red pigment contained in the female body of Coccus cacti L .. For this reason, the cochineal pigment is prepared by extracting the dried product with water or alcohol using the above-mentioned enjim as a raw material. The main pigment component of cochineal pigments is carminic acid.

As described above, cochineal pigments prepared using insects as raw materials contain contaminating proteins, and it has been reported that these may cause allergies as allergens (Non-patent Document 1).
And 23 kD, 28 kD, 38 kD, and 50 kD proteins have been reported as allergens contained in the cochineal dye. (Non-patent documents 1 to 4)

  In view of this point, the applicant of the present application has developed a method for removing a specific protein (protein having a molecular weight of 6,000 or more) derived from enjim which causes allergies, and disclosed this (Patent Document 1, Patent Document 2). ).

By the way, the amount of protein in the cochineal pigment is stipulated in the Food Additives Official Version 8 (Ministry of Health, Labor and Welfare) as being 2.2% or less as a measured value by the semi-micro Kjeldahl method.
Conventionally, such cochineal pigments are considered not to cause allergic symptoms.

  However, in 2012, the Consumer Affairs Agency has confirmed that there are no cases of allergic symptoms caused by cochineal dyes in the National Living Center of Independent Administrative Institutions or consumer life centers of local governments. In response to the provision of domestic research information on beverages containing urine and acute allergic reactions to the Consumer Affairs Agency, a warning was issued regarding cochineal dyes.

Anthocyanin pigments that are natural acidic pigments that have been widely used as coloring agents for beverages and the like are, for example, mainly leaves of Brassica oleacea L. var. Capitata DC, or It is mainly derived from the pericarp of Vaccinium sp.
The safflower pigment, which is a natural acidic pigment that has been widely used as a coloring agent for beverages and the like, is a flavonoid pigment, and is mainly derived from the flower part of the safflower family (Carthamus tinctorius L.).
When anthocyanin pigments and safflower pigments are used as coloring agents for beverages or the like, precipitation may occur in the beverages due to the contaminating proteins they contain.
The amount of contaminating proteins in the anthocyanin pigment and in the safflower pigment is conventionally measured by, for example, the tannic acid turbidity method.
However, even in the case of using a pigment that is judged to have a low amount of contaminating protein by the tannic acid turbidity method, precipitation may occur in the beverage.

Japanese Patent No. 4129577 Japanese Patent No. 4184148

Lizaso, M. T. et al., Ann. Allergy Asthma Immunol., 84 (5), 549-552 (2000) Chung K. et al., Allergy, 56, 73-77 (2001) Japan Food Chemistry Research and Recovery Foundation 15th Research Report, 95-99 (2009) Japan Food Chemistry Research and Recovery Foundation 16th Research Report, 46-52 (2010)

From the foregoing background, the precise analysis (herein, the term “analysis”) includes detection and quantification of proteins in natural acid pigments (especially allergen proteins and contaminating proteins that cause precipitation). ) Is required.
The semi-micro Kjeldahl method described above is a method based on the measurement of the amount of nitrogen, not the amount of protein itself, and therefore the amount of protein as the measurement value is extremely large compared to the amount of actual allergen protein. Therefore, if the protein in the cochineal pigment is limited to a predetermined amount or less, the amount of the allergen protein becomes significantly smaller. This is consistent with the objective of keeping the amount of allergen protein contained in the cochineal dye low.
However, as can be simultaneously understood from this, the semi-micro Kjeldahl method is not suitable for precise analysis of proteins such as quantification of allergen proteins and detection of individual allergen proteins.
In general, in a precise protein analysis method, a Bradford method (Bradford method), which is a highly accurate quantitative method, and an SDS-Page method capable of separating proteins by molecular weight are used.
However, carminic acid, which is the main dye component of cochineal dye, interferes in principle with the Bradford method and SDS-Page method, so that these methods are conventionally used for the analysis of allergen proteins contained in cochineal dye. It was not possible to use it. Therefore, this problem is remarkable in a cochineal dye having a high color value.
On the other hand, according to the enzyme immunoassay (ELISA) method, it is possible to carry out precise analysis of single or plural allergens without using methods such as Bradford method and SDS-Page method. Since this method uses an antibody specific for each allergen, it is not suitable for simply or comprehensively analyzing a plurality of types of allergen proteins.

  Moreover, the tannic acid turbidity method is not a method that can accurately measure the amount of contaminating proteins in the pigment. This is considered to be one of the reasons that precipitation may occur in the beverage even when a dye determined to have a low amount of contaminating protein by the tannic acid turbidity method is used. .

  Accordingly, an object of the present invention is to provide an analytical method capable of precise analysis of proteins (particularly allergen proteins) contained in natural acidic pigments. The present invention also relates to proteins contained in natural acidic pigments (particularly, allergen proteins contained in cochineal pigments, anthocyanin pigments, and contaminating proteins that cause precipitation in safflower pigments). It is a further object to provide an analysis method capable of comprehensive or exhaustive and simple analysis.

As a result of intensive studies, the inventors of the present invention have included a natural acidic dye (eg, cochineal dye, anthocyanin dye, and safflower dye) in a cochineal dye by subjecting it to a predetermined gel filtration treatment (pretreatment stage A). To the extent that enables accurate analysis of the protein from the natural acid dye sample that interferes with the analysis of the protein. It was found that the proteins to be analyzed can be collected selectively, in other words, that they can be removed.
Here, as a result of the study by the present inventors, when the color value of the natural acidic pigment is high, the problem that the separation is insufficient only by the pretreatment stage A has been clarified. Thus, it was found that even in the case of a natural acidic dye sample having a particularly high color value, the separation can be performed by a predetermined ultrafiltration treatment (pretreatment stage B) prior to the pretreatment stage A.
Based on this finding, the present inventors have completed the present invention as a result of further research.

  The present invention includes the following aspects.

Item 1. A method for analyzing proteins in a natural acid dye sample,
(1) A natural acidic dye sample possibly containing a protein is used as a mobile phase using dextran and its derivatives as a stationary phase, and a cross-linked product of one or more polysaccharides selected from agarose and its derivatives. Before a natural acid dye sample comprising subjecting it to gel filtration using an aqueous solution having a salt concentration of 5 mM or more and a pH in the range of 1.5-9 and recovering an eluent that may contain protein An analysis method comprising: a treatment step A; and (2) a step of analyzing the natural acid dye sample that has undergone the pretreatment step A.
Item 2. Item 10. The analysis method according to Item 1, wherein the pH of the aqueous solution is in the range of 7-9.
Item 3.
Item 3. The analysis method according to Item 2, wherein the pH of the aqueous solution is in the range of 7.5 to 8.5.
Item 4. Item 4. The analysis method according to any one of Items 1 to 3, wherein the salt concentration of the aqueous solution is in the range of 5 to 55 mM.
Item 5. Item 5. The analysis method according to Item 4, wherein the salt concentration of the aqueous solution is in the range of 10 to 50 mM.
Item 6. Item 6. The analysis method according to any one of Items 1 to 5, wherein the aqueous solution is a phosphate buffer.
Item 7. The pretreatment step A further comprising the pretreatment step B, wherein the natural acidic dye sample is subjected to ultrafiltration having a molecular weight cut off of 3 kD or more to remove at least a part of the dye component. The analysis method according to any one of the above items.
Item 8. Item 8. The analysis method according to Item 7, wherein the pretreatment stage B is carried out when the natural acidic dye sample has a high color value.
Item 9. Item 9. The analysis method according to any one of Items 1 to 8, wherein the natural acidic dye sample is a cochineal dye sample, an anthocyanin dye sample, or a safflower dye sample.
Item 10. A method for preparing a natural acid dye sample for protein analysis, comprising:
A natural acidic dye sample that may contain a protein is used as a stationary phase using dextran and its derivatives and a cross-linked product of one or more polysaccharides selected from agarose and its derivatives, and having a mobile phase of 5 mM or more. A method of preparation comprising a gel filtration step that is subjected to gel filtration using an aqueous solution having a salt concentration and a pH in the range of 1.5 to 9 and recovering an eluent that may contain protein.
Item 11. Item 11. The method according to Item 10, wherein the pH of the aqueous solution is in the range of 7-9.
Item 12. Item 12. The preparation method according to Item 11, wherein the pH of the aqueous solution is in the range of 7.5 to 8.5.
Item 13. Item 13. The preparation method according to any one of Items 10 to 12, wherein the salt concentration of the aqueous solution is in the range of 5 to 55 mM.
Item 14. Item 14. The preparation method according to Item 13, wherein the salt concentration of the aqueous solution is in the range of 10 to 50 mM.
Item 15. Item 15. The preparation method according to any one of Items 10 to 14, wherein the aqueous solution is a phosphate buffer.
Item 16. Any one of paragraphs 10-15, further including an ultrafiltration step of removing the natural acid dye by subjecting the natural acid dye sample to ultrafiltration having a molecular weight cut off of 3 kD or more before the gel filtration step. The preparation method according to item.
Item 17. Item 17. The preparation method according to Item 16, wherein the ultrafiltration step is performed when the natural acidic dye sample has a high color value.
Item 18. Item 18. A method for preparing a protein in a natural acid dye sample, comprising analyzing the natural acid dye sample for protein analysis obtained by the preparation method according to any one of Items 10 to 17.
Item 19. Item 19. The preparation method according to any one of Items 10 to 18, wherein the natural acidic pigment sample is a cochineal pigment sample, an anthocyanin pigment sample, or a safflower pigment sample.
Item 20.
A method for pretreatment of a natural acid dye sample for the analysis of proteins in the natural acid dye,
A natural acidic dye sample that may contain a protein is used as a stationary phase using dextran and its derivatives and a cross-linked product of one or more polysaccharides selected from agarose and its derivatives, and having a mobile phase of 5 mM or more. A pretreatment method comprising a gel filtration step that is subjected to gel filtration using an aqueous solution having a salt concentration and a pH in the range of 1.5 to 9, and recovering an eluent that may contain protein.
Item 21. Item 21. The pretreatment method according to Item 20, wherein the pH of the aqueous solution is in the range of 7-9.
Item 22. Item 22. The pretreatment method according to Item 21, wherein the pH of the aqueous solution is in the range of 7.5 to 8.5.
Item 23. Item 23. The pretreatment method according to any one of Items 20 to 22, wherein the salt concentration of the aqueous solution is in the range of 5 to 55 mM.
Item 24. Item 24. The pretreatment method according to Item 23, wherein the salt concentration of the aqueous solution is in the range of 10 to 50 mM.
Item 25. Item 25. The pretreatment method according to any one of Items 20 to 24, wherein the aqueous solution is a phosphate buffer.
Item 26. Any one of paragraphs 20 to 25, further including an ultrafiltration step of removing the natural acid dye by subjecting the natural acid dye sample to ultrafiltration having a fractional molecular weight of 3 kD or more before the gel filtration step. The pretreatment method according to item.
Item 27. Item 25. The pretreatment method according to Item 24, wherein the ultrafiltration step is performed when the natural acidic dye sample has a high color value.
Item 28. Item 27. A method for pretreating a protein in a natural acid dye sample, comprising analyzing the natural acid dye sample for protein analysis obtained by the preparation method according to any one of Items 20 to 26.

  According to the present invention, a precise analysis method for proteins (particularly allergen proteins) in natural acid pigments is provided. Furthermore, according to the present invention, there is provided an analysis method capable of comprehensive or comprehensive and simple analysis of proteins (particularly allergen proteins) in natural acid pigments.

It is the photograph of the gel of SDS-PAGE of a cochineal dye sample. It is the photograph of the gel of SDS-PAGE of the anthocyanin dye sample (red cabbage dye sample). It is the photograph of the gel of SDS-PAGE of a safflower dye sample.

In the present specification, the “natural acidic pigment” means that an appropriate amount of water is added to an animal or plant sample containing pigment components and a crushed, pulverized or dried product thereof, and the pH of the resulting water extract is less than 7 (preferably Is a dye that is 6 or less, more preferably 4 or less. The lower limit of the pH is not limited, but is usually 1. Here, the appropriate amount of water can be, for example, 10 times the amount (weight) of water of the “animal and plant sample and its crushed product, pulverized product, or dried product”.
Examples of the “natural acidic pigment” include “cochineal pigment”, “anthocyanin pigment”, and “safflower pigment”.
In the present specification, the “cochineal pigment” is a pigment obtained from Coccus cacti L ..
The main pigment component of cochineal pigments is carminic acid.
In the present specification, examples of the “anthocyanin pigment” include a pigment obtained from Brassica oleracea L. var. Capitata DC, and a blueberry (Vaccinium sp) of the Azalea family. Can be mentioned.
The main pigment component of anthocyanin pigments (eg, red cabbage pigment, blueberry pigment) is cyanidin acyl glucoside.
In the present specification, the “safflower pigment” is a flavonoid pigment derived from the safflower family (Carthamus tinctorius L.).
Examples of the “safflower pigment” include safflower yellow pigment and safflower red pigment.
The main pigment component of safflower yellow is the flavonoid safurin (eg, safurin-A).
The main pigment component of safflower red pigment is cartamine, a flavonoid.
In the present specification, “natural acid dye sample”, “cochineal dye sample”, “anthocyanin dye sample”, and “safflower dye sample” are other than the dye sample used in the analysis method of the present invention. In some cases, it means a sample that has undergone each stage in the analysis method of the present invention. These meanings are understood by the context before and after.

Method for analyzing protein in natural acid dye sample The method for analyzing protein in the natural acid dye sample of the present invention comprises:
(1) A natural acidic dye sample possibly containing a protein is used as a mobile phase using dextran and its derivatives as a stationary phase, and a cross-linked product of one or more polysaccharides selected from agarose and its derivatives. Before a natural acid dye sample comprising subjecting it to gel filtration using an aqueous solution having a salt concentration of 5 mM or more and a pH in the range of 1.5-9 and recovering an eluent that may contain protein Processing stage A, and (2) a stage of analyzing the natural acid pigment sample that has undergone the pretreatment stage A (analysis stage)
including.

Pretreatment stage A
In pretreatment stage A, a natural acidic dye sample that may contain protein is used as a stationary phase with dextran and its derivatives, and a cross-linked product of one or more polysaccharides selected from agarose and its derivatives, and Gel filtration using an aqueous solution having a salt concentration of 5 mM or more and a pH in the range of 1.5-9 as the mobile phase is recovered and the eluent that may contain protein is recovered.

The analysis target of the analysis method of the present invention is a protein in a natural acid dye sample.
The protein to be analyzed is preferably a protein that is not covalently bound to the natural acidic dye.
When the analysis target of the analysis method of the present invention is a protein in a cochineal pigment, the protein is usually a protein derived from Coccus cacti L.
When the analysis target of the analysis method of the present invention is a protein in an anthocyanin dye sample, the protein is, for example, a protein mainly derived from leaves of red cabbage (Brassica oleracea L. var. Capitata DC) and / or blueberry. It is a protein mainly derived from the pericarp part of the genus (Vaccinium sp.).
When the analysis object of the analysis method of the present invention is a protein in a safflower pigment sample, the protein is usually a protein mainly derived from a flower part of safflower (Carthamus tinctorius L.).
The sample used for the analysis method of the present invention is usually a natural acid dye sample containing protein (eg, a cochineal dye sample, an anthocyanin dye sample, and a safflower dye sample), but the analysis method of the present invention is a natural acid dye sample. It can also be used for the purpose of confirming that the acidic dye sample does not contain protein (or that the protein content is below the detection limit).
Therefore, the analysis method of the present invention can be applied to any natural acid dye sample that may contain proteins.

In gel filtration in the pretreatment stage A, a cross-linked product of one or more polysaccharides selected from dextran and derivatives thereof, and agarose and derivatives thereof is used as a stationary phase. That is, the gel in the gel filtration is a gel of the crosslinked product.
Preferred examples of the crosslinked product include 1-chloro-2,3-epoxy-propane crosslinked dextran gel.

In the gel filtration in the pretreatment stage A, an aqueous solution having a salt concentration of 5 mM or more and a pH in the range of 1.5 to 9 is used as the mobile phase.
The pH of the aqueous solution as the mobile phase is preferably in the range of 7 to 9, more preferably in the range of 7.5 to 8.5.
When the pH is within such a range, the dye component and the protein in the natural acidic dye sample are sufficiently separated in the gel filtration in the pretreatment stage A.
The pH may be adjusted by a conventional method such as addition of an acidic substance, a basic substance, and a buffer (eg, phosphate buffer). These substances can be used alone or in combination of two or more.
The salt concentration of the aqueous solution as the mobile phase is preferably in the range of 5 to 55 mM, more preferably in the range of 10 to 50 mM.
When the salt concentration is within such a range, the dye component and the protein in the natural acidic dye sample are sufficiently separated in the gel filtration in the pretreatment stage A.
The salt concentration may be adjusted by a conventional method such as addition of salt. Examples of such salts include NaCl, and buffers (eg, phosphate buffers). These substances can be used alone or in combination of two or more.

  The aqueous solution is preferably a phosphate buffer. Thereby, salt concentration and pH can be adjusted in a suitable range simultaneously. The phosphate buffer contains a phosphate buffer. The phosphate buffer may further contain a salt (eg, sodium chloride) for adjusting the salt concentration.

The gel filtration in the pretreatment stage A may be carried out in the same manner as a general gel filtration method.
Specifically, a natural acidic dye sample is loaded on a gel that is a stationary phase, and the eluate is collected through the mobile phase through the gel.
The stationary phase is preferably equilibrated with an aqueous solution of the same composition as the aqueous solution used for the mobile phase prior to loading with the natural acid dye.

  Usually, the natural acidic pigment is in the form of powder, lump, liquid or paste. The natural acid dye sample can be loaded on the gel, for example in these forms, in the form of its aqueous solution, or in the form of its suspension, but is preferably pretreated in the form of its aqueous solution or in the form of its suspension. It is attached to stage A. That is, the natural acidic dye sample is dissolved or suspended in an aqueous solution and subjected to pretreatment stage A. Suitable examples of the aqueous solution include the aqueous solution as the mobile phase described above.

In the gel filtration, the protein contained in the natural acid dye tends to elute before the dye component of the natural acid dye.
Based on this tendency, by selecting and collecting the fraction containing the protein (or the fraction that may contain the protein), the dye component that interferes with the analysis of the protein is extracted from the natural acid dye sample. It can be removed to the extent that precise analysis of proteins is possible.
That is, the natural acid dye sample that has undergone the pretreatment stage A has a reduced dye component content relative to the protein content as compared to the original natural acid dye sample.

Pretreatment stage B
When the color value of a natural acid dye sample is high, the pretreatment stage A alone allows a dye component that interferes with the analysis of the protein in the natural acid dye from the natural acid dye sample to the extent that enables precise analysis of the protein. May not be removed.
Here, “when the color value of the natural acidic dye sample is high” can be, for example, a case where the color value is 20 or more, or a case where the color value is 40 or more.
“When the color value of the natural acidic dye sample is high” can be set according to the type of the dye.
For example, when the natural acid dye sample is a cochineal dye, “when the color value of the natural acid dye sample is high” can be a case where the color value is 40 or more.
Further, for example, when the natural acidic dye sample is an anthocyanin dye, the color value can be 20 or more.
Further, for example, when the natural acidic dye sample is a safflower dye, the color value can be 20 or more.
In this case, the natural acidic dye sample can be subjected to ultrafiltration having a fractional molecular weight of 3 kD or more to remove at least a part (in other words, part or all) of the dye component in the natural acidic dye sample. That is, the natural acid dye sample that has undergone the pretreatment stage B has a reduced dye component content relative to the protein content as compared to the original natural acid dye sample. As a result, in the subsequent pretreatment stage A, the dye component that interferes with the analysis of the protein in the natural acidic dye can be removed to the extent that the protein can be accurately analyzed. In addition, when all the pigment components of the natural acidic pigment are removed in the pretreatment stage B, it is not necessary to perform the pretreatment stage A, but even in such a case, the pretreatment stage A is within the scope of the present invention. It is.
The ultrafiltration can be performed using an ultrafiltration membrane.
In the ultrafiltration, the passage of the liquid containing the natural acidic pigment through the ultrafiltration membrane can be promoted by centrifugation or the like. The centrifugation conditions can be appropriately set according to common technical knowledge, but are, for example, about 4000 × g for about 40 minutes.
The protein contained in the natural acid dye does not pass through the ultrafiltration membrane and remains in the natural acid dye sample on the ultrafiltration membrane.
Preferably, the natural acid dye sample is washed by adding a phosphate buffer to the natural acid dye sample and repeating ultrafiltration, in other words, the dye component can be further removed. The washing is preferably repeated, for example, until the color value is not high (that is, until, for example, the color value is less than 40 or the color value is less than 20). Repeated 5 times.
The ultrafiltration includes, for example, a commercially available centrifugal filtration unit (eg, Amicon-15 (trade name, Millipore)) that includes an ultrafiltration membrane and can be used for ultrafiltration. Can be implemented according to

For example, the pretreatment stage B defines an analysis scheme to be performed when the natural acid dye sample has a high color value (for example, when the color value is 40 or more, when the color value is 20 or more). However, the present invention is not limited to the color value of the natural acidic dye sample.
In the present specification, the color value is a numerical value obtained by measuring the absorbance at the maximum absorption wavelength in the visible part of the colorant solution and converting it to the absorbance of a 10 w / v% solution. , Measured according to the method described in the 8th edition Food Addendum.

Protein Analysis Stage If desired, natural acid dye samples that may contain the protein may be optionally concentrated after an appropriate stage prior to the protein analysis stage, preferably after pretreatment stage A. Good. The concentration may be performed by a conventional method such as ultrafiltration. The ultrafiltration can be performed, for example, using a commercially available centrifugal filtration unit (for example, Amicon-4 (trade name, Millipore)) that can be used for ultrafiltration according to the description of the manual.

  Protein analysis may be carried out by selecting an appropriate method according to the purpose. For example, natural acid dye samples after the pre-treatment A (or after the pre-treatments B and A) are obtained by Bradford method (Bradford method), which is a highly accurate quantitative method, and SDS-Page that can separate proteins by molecular weight. Since the dye components that interfere with the method have been reduced, high accuracy analysis of proteins in natural acidic dyes is possible using one or more of the optional methods used for protein analysis, including these methods It is. In the analysis method of the present invention, preferably, the Bradford method (Bradford method), the SDS-Page method, or both are used.

A method for preparing a natural acid dye sample for protein analysis, and a pretreatment method for a natural acid dye sample for analysis of proteins in the natural acid dye . In another aspect, the present invention provides a natural acid dye sample for protein analysis. It can be a preparation method or a pretreatment method of a natural acid dye sample for analysis of proteins in the natural acid dye. These methods can be performed in the same manner as the pretreatment method described in the analysis method.

  Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

1) Preparation of natural acidic pigment extract and measurement of color value To 10 g of dried powder of Coccus cacti L. parasite, 100 mL of water was added, and the mixture was stirred and extracted at room temperature for 60 minutes, and filter paper (ADVANTEC The extract filtered with 5A (110 mm, Toyo Roshi Kaisha, Ltd.) was used as the cochineal dye extract.
The main pigment component of this extract is anthraquinone carminic acid.
Anthocyanin pigment (red cabbage pigment): 100 g of water was added to 10 g of leaves of Brassica oleracea L. var. Capitata DC, and the mixture was extracted by stirring at room temperature for 60 minutes and filtered through filter paper The solution was used as an anthocyanin pigment extract. The main pigment component of this extract is cyanidin acylglycoside.
Safflower pigment (Safflower yellow): 100 g of water is added to 10 g of dried flower of safflower (Carthamus tinctorius L.), and the mixture is stirred and extracted at room temperature for 60 minutes. An extract was obtained.
The main pigment component of this extract is saflomin, a flavonoid.
Each dye extract was appropriately diluted with 0.1N hydrochloric acid and the absorbance at the maximum absorption wavelength (410 nm) in the visible region was measured. A value obtained by converting the absorbance into the absorbance of a 10 w / v% solution was defined as a color value.

  Preliminary studies using cochineal dye extracts of various color values (0.31, 0.63, 1.25, 2.5, 5, 10, 20, or 40) revealed that When the value exceeds 2.5, it was confirmed that protein quantification (Bradford method) was interfered with by the dye component (see Table 1). Furthermore, when the color value of anthocyanin dye and safflower dye exceeded 2.5, it was confirmed that the protein quantification (Bradford method) was interfered by the dye component.

  Pretreatment stage A shown below was performed on cochineal dye extracts with various color values exceeding 2.5 (color values: 10, 20, 30, 40, 50, 60, 70, 80).

2) Pretreatment stage A
As a gel filtration column, a commercially available gel filtration chromatography column (PD-10 column / GE health care) in which 8.3 mL of 1-chloro-2,3-epoxy-propane crosslinked dextran gel was packed in a column 1.45 × 5.0 cm. Company name).
The column was pre-equilibrated with 50 mM phosphate buffer (pH 8.0) and loaded with 1 mL of cochineal dye extract.
Subsequently, 50 mM phosphate buffer (pH 8.0) was injected into the column, and the eluate was fractionated into 13 fractions of 1 mL each.
Each protein-eluted fraction was concentrated to 1 mL using a regenerated cellulose membrane ultrafiltration unit (Amicon Ultra / Merck, trade name) having a fractionation performance at a molecular weight of 3 kD.
A part of the concentrated solution was subjected to protein quantification by the Bradford method, and the presence or absence of protein in each fraction was evaluated. As a result, the protein elution fraction was No. There were 4 to 8 fractions. On the other hand, the elution of the dye component interfering with the protein quantification is no. Fractions after 9 The elution of the dye component was detected by absorbance (620 nm) (2 significant digits) (50 mM phosphate buffer (pH 8.0) was used as a blank for absorbance measurement).
In Table 1, regarding the color value, the symbols in the table indicate the following.
−: Absorbance of 0.00
±: Absorbance of 0.01 or more and less than 0.10 +: Absorbance of 0.10 or more and less than 0.40 +++: Absorbance of 0.40 or more When the absorbance of the fraction is 0.01 or more and less than 0.10 The fraction contains cochineal, but with such small amounts, the interference of the cochineal dye with protein analysis is negligible.
That is, as understood from Table 1, when the upper limit of the color value of the cochineal dye extract is 10 to 40, the pretreatment separates the protein in the cochineal dye extract from the dye component that interferes with the analysis. It was confirmed that the dye component that interferes with the analysis of the protein can be removed to the extent that the protein can be precisely analyzed.
In other words, this means that the upper limit of the color value of the cochineal dye extract capable of separating the protein from the cochineal dye component by the treatment was 40.

  As a result of conducting the same test for the anthocyanin dye and safflower dye, the upper limit of the color value of the dye extract capable of separating the protein was 20 in all cases.

For the cochineal dye extract with a color value greater than 40, the following pretreatment stage B was performed.
3) Pretreatment stage B
Using an ultrafiltration unit (trade name: Amicon-15, Millipore) having a molecular weight cut off of 3 kD, ultrafiltration (4 000 × g, 40 minutes 4-5 times) and subjected to pretreatment stage A.

  In addition, as a result of conducting an addition recovery test using a commercially available standard protein mixture (Precision Plus Protein Standards / BioRad: trademark) in pretreatment stage A and pretreatment stages B and A, The recovery rate was 84% or more, which was sufficiently high. (90% for pretreatment stage A alone, 84% for pretreatment stages B and A)

  On the other hand, for the anthocyanin dye and safflower dye, the pretreatment stage B was carried out for the dye extract with the color value exceeding 20, respectively, in the same manner as the cochineal dye extract.

3) Protein quantification Samples that have undergone the pretreatment stage A (samples that have not undergone the pretreatment stages B and A) or samples that have undergone the pretreatment stages B and A are added with a 50 mM phosphate buffer (pH 8.0). The protein was quantified according to the Bradford method (Anal. Biochem., 72, 248 (1976).). For preparing a calibration curve, bovine serum albumin aqueous solution was used as a standard protein.
As a result, the amount of protein in the cochineal dye extract was 110 μg / mL.
As a result of repeating the quantification, when the sample that has undergone the pretreatment steps B and A is analyzed by the Bradford method, the cochineal dye extract (the sample that has not undergone the pretreatment step B or A) is used as a conventional protein quantification method ( The reproducibility was higher than that in the case of analysis according to the method described in the Food Additives Official Version 8 (Ministry of Health, Labor and Welfare).

  In addition, as for the anthocyanin pigment and safflower pigment, the amount of protein in the anthocyanin pigment extract was 250 g / mL as a result of the same protein quantification, and the amount of protein in the safflower pigment extract was , 100 μg / mL.

4) SDS-polyacrylamide gel electrophoresis was carried out as follows for the samples that had undergone protein detection pretreatment stages B and A and the cochineal dye extract (samples that had not undergone pretreatment stage B or A).
To 100 μl of the sample, 95 μl of Laemmli sample buffer (BioRad, trade name) and 5 μl of 2-mercaptoethanol were added and boiled for 5 minutes to obtain a sample loaded on the SDS-polyacrylamide gel. The electrophoresis conditions were in accordance with the Laemmli method (Nature, 227, 680 (1970)) (the conditions are described later). The gel after electrophoresis was stained by the CBB staining method which is a conventional method.
The results of the CBB staining method of the electrophoresis gel are shown in FIG. 1 (FIG. 1 left: sample without pretreatment stage B and pretreatment stage A. FIG. 1 right: sample with pretreatment stage B and pretreatment stage A) . In the case of the sample subjected to the pretreatment stage B and the pretreatment stage A, the bands of proteins of 17, 23, 28, 38 and 50 kD reported as allergens were clearly observed. In the case of the sample that was not subjected to the pretreatment stage A, the band became smeared, and each protein could not be detected.
<Electrophoresis conditions>
Gel: 10-20% polyacrylamide gel (TEFCO, trade name)
Electrophoretic conditions: 200 V constant pressure, about 40 minutes Electrophoresis buffer: 10 × Tris / Glycine / SDS premix buffer (BioRad, trade name) was diluted 10 times with ultrapure water.
・ Molecular weight marker: Precision Plus Protein Standards (BioRad, trade name)
Sample load: 3 μg protein / 10 μl / well

Similarly, with respect to the anthocyanin dye and safflower dye, SDS-polyacrylamide gel electrophoresis is similarly performed for the sample that has undergone the pretreatment stages B and A and the dye extract (the sample that has not undergone the pretreatment stage B and A). Carried out.
FIG. 2 shows the results of CBB staining of an electrophoresis gel for an anthocyanin dye (FIG. 2 left: a sample in which neither pretreatment stage B nor pretreatment stage A was performed. FIG. 2 right: pretreatment stage B and pretreatment stage A. Sample performed). In the case of the sample subjected to the pretreatment stage B and the pretreatment stage A, the protein band was clearly observed, but in the case of the sample where neither the pretreatment stage B nor the pretreatment stage A was performed, the band became smeared, Each protein could not be detected.
The results of CBB staining of the electrophoresis gel for safflower dye are shown in FIG. 3 (FIG. 3 left: sample without pretreatment stage B or pretreatment stage A. FIG. 3 right: pretreatment stage B and pretreatment stage A. Sample performed). In the case of the sample subjected to the pretreatment stage B and the pretreatment stage A, the protein band was clearly observed, but in the case of the sample where neither the pretreatment stage B nor the pretreatment stage A was performed, the band became smeared, Detection as a specific protein could not be performed.

  The present invention can be used for analysis of proteins in natural acid pigments.

Claims (9)

A method for analyzing proteins in natural acid pigments,
(1) A natural acidic dye sample possibly containing a protein is used as a mobile phase using dextran and its derivatives as a stationary phase, and a cross-linked product of one or more polysaccharides selected from agarose and its derivatives. Before a natural acid dye sample comprising subjecting it to gel filtration using an aqueous solution having a salt concentration of 5 mM or more and a pH in the range of 1.5-9 and recovering an eluent that may contain protein An analysis method comprising: a treatment step A; and (2) a step of analyzing the natural acid dye sample that has undergone the pretreatment step A. The analysis method according to claim 1, wherein the pH of the aqueous solution is in the range of 7-9. The analysis method according to claim 2, wherein the pH of the aqueous solution is in the range of 7.5 to 8.5. The analysis method according to any one of claims 1 to 3, wherein the salt concentration of the aqueous solution is in the range of 5 to 55 mM. The analysis method according to claim 4, wherein the salt concentration of the aqueous solution is in the range of 10 to 50 mM. The analysis method according to claim 1, wherein the aqueous solution is a phosphate buffer. The pretreatment step B further includes a pretreatment step B in which the natural acidic dye sample is subjected to ultrafiltration having a molecular weight cut off of 3 kD or more to remove at least a part of the dye component before the pretreatment step A. The analysis method according to any one of the above. The analysis method according to claim 7, wherein the pretreatment stage B is performed when the natural acidic dye sample has a high color value. The analysis method according to claim 1, wherein the natural acidic dye sample is a cochineal dye sample, an anthocyanin dye sample, or a safflower dye sample.

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