HK1215040B - Method of isolating blue anthocyanin fractions - Google Patents

Abstract

The present invention is directed to a method of isolating fractions of anthocyanin molecules from anthocyanin-containing vegetable and fruit juices and extracts, or combinations thereof, at a select pH based on differences in polarity of the anthocyanin molecules in the anthocyanin-containing vegetable and fruit juices and extracts.

Description

Method for separating blue anthocyanin fractions

Background

Technical Field

The present invention relates to a method for obtaining natural blue anthocyanin-containing colorant compositions by selectively separating fractions of anthocyanin molecules from anthocyanin-containing vegetable and fruit juices and extracts.

Background

There is an increasing interest in the food industry to color food products with natural colorants instead of synthetic materials.

One challenge in replacing synthetic colorants with natural colorants is identifying natural colorants that provide color characteristics similar to those provided by synthetic colorants.

To date, no natural colorant has been found that provides the same color characteristics as the synthetic Blue colorant, U.S. Federal food, drug and cosmetic Law No.1 (FD & C Blue No. 1). The lack of a suitable natural bluish shade of colorant has also made it a challenge to obtain the desired natural green shade colorant from a mixture of natural blue and yellow colorants. Blue-green algae derived material Spirulina Blue (Spirulina Blue) is used as a natural Blue colorant, but it does not provide the same color characteristics as U.S. federal food, drug and cosmetic act Blue No. 1.

Anthocyanin (anthocyanin) is a water-soluble compound found in the cellular vacuoles of fruits, vegetables and petals, and sometimes the roots, leaves, stems and bracts of plants. Due at least in part to their wide availability, anthocyanin-containing vegetable and fruit juices and extracts have been used as natural edible colorants and to produce colorant compositions, particularly natural red, purple and blue-shade colorant compositions.

Anthocyanins (anthocyanidins) comprise anthocyanidins (aglycones) esterified with one or more sugar molecules (glycones) to form glycosides. The sugar molecule may be attached to the C-3, C-5, C-7, C-3 ', C-4 ' and/or C-5 ' position. Examples of sugar molecules found in the anthocyanin structure are: arabinose, galactose, glucose, rhamnose, rutinose, sambuciose (samubiose), sophorose and xylose.

Anthocyanin glycosides can also be acylated, i.e., they can have one or more molecules esterified with sugar molecules, usually at the 6-position of the monosaccharide, but also possibly at the 2-, 3-or 4-position. The most common acyl units include those from coumaric, ferulic, caffeic, sinapic, gallic, malonic, acetic, malic, succinic, vanillic and oxalic acids.

Yellow pigment with the structure of anthocyanidinThe cationic form of the salt (flavylium) shows that this form is the predominant form under acidic conditions. Anthocyanidins can be substituted with hydrogen, hydroxyl and/or methoxy at various positions:

wherein R is³Is a compound of formula (I) which is H or OH,

R⁵is H, OH or OCH₃，

R⁶Is a compound of formula (I) which is H or OH,

R⁷is OH or OCH₃，

R^3'Is H, OH or OCH₃，

R^4'Is OH or OCH₃And is and

R^5'is H, OH or OCH₃。

The following structures show the most common natural anthocyanidins:

thus, this class of compounds known as anthocyanin covers a large number of structurally diverse compounds based on differences in major structure, glycosylation and acylation patterns.

Known plant sources of anthocyanin include: (1) vegetables, for example, red cabbage, purple sweet potato, blue potato, red radish, black carrot, purple corn, red onion, purple broccoli, safflower broccoli, rhubarb, black soybean, red-leaf lettuce, black rice, and eggplant; and (2) fruit, e.g., strawberry, raspberry, cranberry, blueberry,Cowberry fruit, red grape, apple, blackcurrant, redcurrant, cherry, blueberry, elderberry, cowberry fruit, rock orchid, blackberry, chokeberry fruit, currant, and blackberryNectarine, peach, plum, blood orange and blue tomato. Each anthocyanin source contains varying amounts of a variety of different anthocyanin species, typically 15 to 30 structurally different anthocyanin molecules in a given plant source.

The color characteristics of anthocyanin-containing vegetable and fruit juices and extracts change as a result of changes in pH. Anthocyanin-containing juices and extracts generally exhibit a red hue at low pH and this hue shifts to purple as pH increases. With further increase in pH, only some juices and extracts exhibit a blue hue.

The color change of anthocyanin-containing juices and extracts caused by pH changes is associated with many secondary structures of anthocyanin which are associated with the predominant yellow in aqueous solutionThe salt cation structure exists in equilibrium. As the pH changes, the relative amounts of the different equilibrium structures will change. At a given pH, one or more structural forms may predominate, while other structural forms are present in lower amounts or are absent. E.g. yellow at very low pHThe salt cation form predominates. Yellow with increasing pHThe molecules in the form of salt cations can be deprotonated and converted to the methanopseudobase form, which can be further converted to the neutral and ionized quinoid base forms and to the chalcone form by loss of water molecules and protons, respectively. These transformations reduce yellowingThe molecular weight of the salt cation form and to varying degrees increases the amount of the other equilibrium forms. Thus, at higher pH, different equilibrium structures are present in different relative amounts compared to low pH. Anthocyanin in each structural form can absorb light differently, resulting in different perceived colors, including colorless. Thus, as the pH of the solution changes, changes in the relative amounts of the different structural forms can result in a change in the color of the solution.

The various anthocyanin molecules are characterized by a balanced molecular structure and a balanced constant set of reactions that transform one structure of its own to another. For example, a reaction that converts one anthocyanin equilibrium structure to another can have a particular acid dissociation constant, K, associated therewith_a. The reaction can also be based on the logarithmic constant pK_aIs described, i.e. defined as-log₁₀K_a。

Yellow colourThe salt cation and quinoid base structures have conjugated bonds linking all three rings of the anthocyanin molecule. Extensive delocalized pi-bonding to allow yellowThe salt cation and quinoid base absorb visible light, resulting in yellow at low pHThe perceived red hue of the salt cation, and the violet or blue hue that produces the ionized quinoid base at higher pH. In contrast, methanopseudobases and chalcone structures do not have delocalized pi bonds connecting all three rings, and are colorless or yellowish.

The substitution pattern of anthocyanin also affects color. For example, it is generally observed that when a hydrogen atom is substituted by a hydroxyl group, the hue shifts from pink to purple. Similarly, it was observed that the number of glycosyl (saccharide) units and the number and type of acyl units affected the color. However, these phenomena are not well understood or predictable.

In addition, intermolecular and intramolecular interactions also affect the color of anthocyanin. The same anthocyanin can produce different hues depending on the presence of other molecules. For example, it is believed that the acyl groups on anthocyanin sugars can fold and protect against yellow colorThe salt cation C-2 is not attacked nucleophilically. Thus, this intramolecular interaction prevents the formation of a colorless methanolic pseudobase structure. Similarly, it is believed that self-ligation (self-association) of anthocyanin molecules, as evidenced by the fact that anthocyanin concentration is increased by two-fold, can result in a 300-fold increase in chroma and can change hue and value. It is hypothesized that the self-ligation is similar to intramolecular stacking and prevents nucleophilic attack and formation of methanolic pseudobase structures.

While factors such as pH, anthocyanin chemical structure, substitution patterns, intermolecular and intramolecular interactions are known to all affect the color observed in anthocyanin-containing vegetable and fruit juices and extracts, the understanding of how these factors interact to change color is not sufficient, i.e., the specific causes and effects are unpredictable.

For example, individual anthocyanin molecules have been isolated by HPLC, but the isolation always occurs at low pH, and the color characteristics of individual anthocyanin molecules are analyzed at low pH. Similarly, the effect of pH on the color characteristics of anthocyanin-containing vegetable and fruit juices and extracts has been studied, but these studies have analyzed the complex anthocyanin mixtures naturally occurring in such juices and extracts. However, how changes in pH affect the color characteristics of individual anthocyanin molecules or anthocyanin fractions from natural sources is not well understood or is unpredictable. The prior art discloses that the number and type of substituents (e.g. glycosyl and acyl) affect colour; however, it is not disclosed and known how these substituents affect the color with changes in pH. Finally, while the prior art hypothesizes that various intermolecular and intramolecular interactions affect color, it does not disclose how changes in pH affect these intermolecular and intramolecular interactions, and ultimately the color of the anthocyanin observed.

WO 2009/100165 a2 discloses a method for isolating anthocyanin from other phenolic molecules in the juice of anthocyanin-containing fruits and vegetables. WO 2009/100165 a2 does not disclose selectively separating fractions of anthocyanin molecules based on differences in charge and polarity of the molecules to produce fractions having a desired color different from that of the anthocyanin-containing juice.

The analytical scale separation of individual anthocyanin glycosides is described in journal of chromatography a (j. chromatography a.),1148(2007), 38-45. The separation is performed at low pH, i.e. pH less than 2, using HPLC to assist in the identification of individual anthocyanin glycosides. The method isolates anthocyanin molecules for detection rather than for production of fractions with mixtures of anthocyanin molecules.

WO 2004/012526 discloses a blue colorant solution of red cabbage anthocyanin at pH 7.9 for use in a syrup for coating a confectionery center. The red cabbage anthocyanin was not separated into fractions.

There is no example in the prior art of separating fractions of anthocyanin molecules from anthocyanin-containing vegetable and fruit juices and extracts at a selected pH based on differences in the charge and polarity of the anthocyanin molecules. Furthermore, no method has been disclosed for obtaining an anthocyanin fraction that provides a color characteristic that is different from the color characteristic provided by the source juice and extract. In particular, the prior art has not described a method for obtaining a natural anthocyanin-containing blue colorant composition that provides color characteristics similar to those provided by the synthetic blue colorant U.S. federal food, drug and cosmetic act blue No. 1.

It is desirable to have a variety of natural colorants that can be used to color foods. There is a long felt need for a natural blue colorant that provides color characteristics similar to those provided by the synthetic U.S. federal food, drug and cosmetic act blue No. 1. Therefore, methods of obtaining such natural blue colorants from anthocyanin-containing vegetable and fruit juices and extracts are desirable.

Disclosure of Invention

The present invention relates to a method for obtaining natural Blue anthocyanin-containing colorant compositions that provide color characteristics similar to those provided by the synthetic Blue colorant U.S. Federal food, drug and cosmetic Law No.1 (FD & C Blue No. 1). The natural blue anthocyanin-containing colorant is obtained by separating a fraction of a mixture containing anthocyanin molecules from anthocyanin-containing vegetable and fruit juices and extracts at a selected pH based on differences in anthocyanin molecule charge and polarity.

In one embodiment, the present invention relates to a method of separating a fraction of anthocyanin from a juice or extract of anthocyanin-containing vegetables or fruits or a combination thereof, the method comprising: a) loading anthocyanin-containing juice or extract of vegetables or fruits or a combination thereof onto an ion exchange column; b) selectively separating anthocyanin on the ion exchange column using a solvent of a selected pH based on differences in charge and polarity of anthocyanin molecules, wherein the pH is at least about 2; and c) selecting one fraction or combination of fractions comprising isolated anthocyanin such that the isolated anthocyanin in the one fraction or combination of fractions has a maximum absorbance of 615 to 635nm when in aqueous solution at pH 8.0. The selected anthocyanin-containing fraction or combination of fractions comprises isolated anthocyanin that provides color characteristics closer to those provided by U.S. Federal food, drug and cosmetic Act blue No.1, but that is different from, i.e., a subset of the fractions, of the anthocyanin-containing vegetable or fruit juice or extract or combination thereof.

Drawings

Figure 1 shows two perspective views of a three-dimensional representation of the color characteristics provided by american bangbang food, drug and cosmetic act blue No.1 in CIE 1976 CIELAB L a b color space as a function of concentration in an aqueous solution.

Fig. 2 shows two perspective views of a three-dimensional representation of the color characteristics provided by american bangbang food, drug and cosmetic act blue No.1 in CIE 1976 CIELCH L C h ° color space as a function of concentration in an aqueous solution.

Figure 3 shows two perspective views of the region of color in CIE 1976 CIELAB L a b color space that differs by Δ E of 3 or less from the color provided by federal food, drug and cosmetic act blue 1 in the united states, and a graphical representation of a segmented tube defined by color space data.

Figure 4 shows a comparison of the color provided by different fruit and vegetable extracts in aqueous solutions of different pH values.

Figure 5 shows two perspective views providing a three-dimensional representation of the color characteristics for the american bangbang food, drug and cosmetic act Blue No.1 in the color space as a function of concentration in an aqueous solution, and an area of color that differs by a Δ E of 3 or less from that provided by Blue No.1 (Blue No.1), and also shows two perspective views providing a three-dimensional representation of the color characteristics provided by spirulina Blue as a function of concentration in an aqueous solution (white line closer to the x-axis).

Figure 6 shows HPLC chromatograms of the red cabbage extract solution and two fractions separated from the red cabbage extract solution using a strong cation exchange column under 520nm detection.

Figure 7 shows HPLC chromatograms of the red cabbage extract solution and four fractions separated from the red cabbage extract solution using a strong cation exchange column under 520nm detection.

Figure 8 shows an HPLC chromatogram of a red cabbage extract solution at 520nm detection, identifying two sets of peaks for targeted separation. These two sets of peaks were separated into a "520-nm fraction" and a "530-nm fraction".

FIG. 9 provides a visual comparison of the color provided by the 520-nm and 530-nm fractions at different pH values. Fig. 9 also allows for a visual comparison of the color provided to the 520-nm and 530-nm fractions with the color of a confectionery product coated with syrup colored by U.S. federal food, drug and cosmetic act blue No. 1.

Figure 10 shows HPLC chromatograms of the red cabbage extract solution and two fractions separated from the red cabbage extract solution using semi-preparative HPLC under 520nm detection. FIG. 10 shows that the 520-nm fraction and the 530-nm fraction each contain three different anthocyanin compounds, and that the functional groups and sugars on these anthocyanin compounds are identified.

Detailed Description

Juices and extracts of vegetables and fruits containing anthocyanin are now used as natural edible colorants and for producing colorant compositions, especially natural red, purple and blue hues. The juices and extracts contain a mixture of all the anthocyanin molecules naturally present in vegetable and fruit sources, as well as many other kinds of compounds. Thus, currently available anthocyanin colorants are limited to those colors associated with anthocyanin mixtures naturally occurring in vegetable and fruit sources. The present invention relates to a method for isolating a mixture of anthocyanin molecules that is different from the complex mixture of anthocyanin molecules naturally present in juices and extracts of vegetables and fruits. The method involves separating fractions of anthocyanin molecules from complex mixtures in juices and extracts of vegetables and fruits at a selected pH based on differences in the charge and polarity of the anthocyanin molecules.

One aspect of the present invention relates to the isolation of anthocyanin molecular fractions from anthocyanin-containing vegetable and fruit juices and extracts to yield colorant compositions that provide specific, target color characteristics similar to those provided by the synthetic blue colorant U.S. Federal food, drug and cosmetic Act blue No. 1. As used herein, providing a color characteristic "similar" to U.S. federal food, drug and cosmetic act blue No.1 means that the color is closer in color characteristic than any other natural colorant (e.g., spirulina blue).

Applicants found that the use of a solvent to separate the anthocyanin at a selected pH and the difference in polarity of the anthocyanin molecules would result in a fraction comprising a mixture of anthocyanins that provides a color characteristic similar to that provided by the synthetic blue colorant U.S. federal food, drug and cosmetic act blue No. 1. Each anthocyanin source comprises a plurality of different anthocyanin molecules in varying amounts, and each molecule can exist in equilibrium with one or more secondary structures. There may be differences in charge and/or polarity between different anthocyanin molecules and their equilibrium molecular structure. By performing the separation at a selected pH based on the differences in charge and polarity of the anthocyanin molecules, applicants are able to separate anthocyanin fractions that have different spectral characteristics than complex mixtures of anthocyanins. The spectral characteristics of the fractions are different from those of the complex anthocyanin mixtures found in juices or extracts and do not exhibit the latter. Applicants have identified an anthocyanin fraction that provides a color characteristic that more closely approximates that provided by the synthetic U.S. federal food, drug and cosmetic act blue No.1 than any known natural blue colorant, including spirulina blue, can provide.

"anthocyanin-containing vegetable or fruit juice" can be obtained by pressing a fruit or vegetable out of a liquid. The "anthocyanin-containing vegetable or fruit extract" can be obtained by washing the impregnated fruit or vegetable with a solvent (e.g., water, alcohol). Juices and extracts contain anthocyanin and many other naturally occurring compounds including, for example, carbohydrates, acids, flavonoids, metal ions, phenolic acids, phenolates and vitamins. The term "juice or extract of vegetables or fruits" is equivalent to the following term "vegetable juice, fruit juice, vegetable extract or fruit extract" and includes treated juices and extracts, including, for example, reconstituted juices and extracts, deodorized juices and extracts, as well as juices and extracts that have undergone other methods to remove specific or broad classes of compounds.

"fractionation" is a process of selecting and separating a portion of anthocyanin from a complex mixture of anthocyanin in a juice or extract of anthocyanin-containing vegetables or fruits. The source of anthocyanin for use in the process of the invention is a juice or extract of a vegetable or fruit containing anthocyanin that provides a blue hue at high pH. In some embodiments, the anthocyanin source used in the methods of the invention is: red cabbage, purple sweet potato, blue potato, purple carrot or black carrot, or a combination thereof.

"fraction" is the fractionated product. The "anthocyanin fraction" comprises a different mixture of anthocyanin than the mixture of anthocyanin in the anthocyanin-containing juice or extract from which the fraction is isolated. Separating an anthocyanin fraction from the juice or extract at a selected pH based on differences in charge and polarity of the different anthocyanin molecules present.

In the context of both isolating anthocyanin and performing color characterization of anthocyanin, "selected pH" is a pH of 2 or higher, for example, a pH ranging from about 2 to about 9. In other embodiments, the pH can be 3 or greater, 4 or greater, 5 or greater, 6 or greater, or 7 or greater, for example, a pH in the range of from about 3 to about 9, from about 4 to about 9, from about 5 to about 9, from about 6 to about 9, or from about 7 to about 9.

"maximum ofAbsorbance "," Lambda max ", or" λ_max"is the wavelength in nanometers of the largest fraction of light absorbed by a substance. In general, the maximum absorbance can be used as a characteristic value of a comparative substance when measured with an ultraviolet/visible (UV/VIS) spectrophotometer or colorimeter.

Reference to "U.S. Federal food, drug and cosmetic act Blue No.1 (FD & C Blue No. 1)" includes different names that assign the same synthetic Blue colorant, Brilliant Blue FCF (Brilliant Blue FCF) and European Commission E133(European Commission E133). The maximum value of the Lanboda for U.S. Federal food, drug and cosmetic Act blue No.1 is 630 nm.

A "colorant" is any substance that imparts color by absorbing or scattering light of different wavelengths. A "natural colorant" is a colorant that is naturally occurring or naturally occurring, or of natural origin. A "blue colorant" is a colorant that reflects light having a wavelength in the region of 450nm to 495nm and has a maximum UV/VIS wavelength absorbance in the range of 615nm to 635 nm. "anthocyanin-containing natural colorant" is a natural colorant comprising anthocyanin derived from plants.

Anthocyanin-containing natural colorants are compositions that may contain only anthocyanin or may also contain other plant components. The composition may take a solid form, such as a powder, or a liquid solution, such as an aqueous liquid.

In one embodiment, the present invention relates to a method of separating a fraction of anthocyanin from a juice or extract of anthocyanin-containing vegetables or fruits or a combination thereof, the method comprising: a) loading anthocyanin-containing juice or extract of vegetables or fruits or a combination thereof onto an ion exchange column; b) selectively separating anthocyanin on the ion exchange column using a solvent of a selected pH based on the difference in charge and polarity of the anthocyanin molecule; and c) selecting a fraction or combination of fractions comprising isolated anthocyanin such that the isolated anthocyanin in the fraction or combination of fractions has a maximum absorbance of 615 to 635nm when in an aqueous solution at pH 8.0. In one embodiment, the anthocyanin-containing fraction is isolated from the anthocyanin-containing vegetable or fruit juice or extract with a solvent having a pH in the range of from about 2 to about 9, or the pH is in one of the following ranges, i.e., from about 3 to about 9, from about 4 to about 9, from about 5 to about 9, from about 6 to about 9, or from about 7 to about 9.

"hue" refers to the property of a color named for color, e.g., red, orange-red, blue, violet, etc.

"chroma" is a color property that indicates color purity, where higher chroma is associated with higher purity hues and is less diluted by white, gray, or black.

A "value" is a color property that represents the lightness or darkness of a color, where a higher value is associated with a higher lightness.

The terms "color" and "color feature" are used interchangeably and encompass color properties such as hue, chroma, and values and color model system parameters used to describe these properties, such as the international commission on illumination (commission international de L' Eclairage) CIE 1976 CIELAB color space L a b values and CIELCH color space L C h ° values. The CIELAB and CIELCH color models provide a visually more uniform color space than earlier color models. The colorants were analyzed with a spectrophotometer and CIELAB L a b values and CIELCH L C h ° values were calculated from the spectral data. The values of L a b and L C h provide a means of characterizing the color and assessing the magnitude of the difference between the two colors. Unless otherwise indicated, in all examples, the CIELAB L a b values and CIELCH L C h ° values shown herein were calculated from spectral data obtained from a Konica Minolta (Konica Minolta) spectrophotometer CM-3500D, which was operated in transmission mode with CIE standard illuminant D65 and an observation angle of 10 °.

The L a b value consists of a set of coordinate values defined in a three-dimensional cartesian coordinate system. L is a value coordinate, or a luminance coordinate. L provides the scale of brightness from black (0L units) to white (100L units) on the vertical axis. a and b are coordinates relating to hue and chroma. a provides a scale on the horizontal axis from green (-a units) to red (+ a units), with neutral color at the center point (0a units). b provides a scale from blue (-b units) to yellow (+ b units) on a second horizontal axis, with neutral color at the center (0b units), perpendicular to the first horizontal axis. The three axes intersect when L is 50 and a and b are both 0.

The L C h ° value consists of a set of coordinates defined in a three-dimensional cylindrical coordinate system. L is a value coordinate, or a luminance coordinate. L provides a scale of brightness from black (0L units) to white (100L units) on the longitudinal axis. h ° is the hue coordinate. h ° is designated as an angle of 0 ° to 360 ° counter clockwise around the L-axis. Pure red has a hue angle of 0 °, pure yellow has a hue angle of 90 °, pure green has a hue angle of 180 °, and pure blue has a hue angle of 270 °. C-coordinate represents chromaticity and is assigned a radial distance from the L-axis. As the coordinates move away from the L axis (up to 100 or more C units), C provides a hue at the L axis (0C units) from achromatic, i.e., neutral white, gray, or black, to a higher purity. C and h can be calculated from a and b using equations 1 and 2:

C*＝(a*²+b*²)^0.5(1)

"Delta E (Delta E)", "Delta E_ab"or" Δ E "is a measure of the magnitude of the total color difference between two colors represented in the CIELAB L a b color space. It is reported that experienced color observers cannot distinguish any difference between the two colors when the Δ Ε is about 2.3 or less. Using equation 3, the value of L a b is calculated as L₁a*₁b*₁And L₂a*₂b*₂Δ E of two different colors:

the CIELAB L a b and CIELCH L C h values for U.S. federal food, drug and cosmetic act blue No.1 are shown in table 1 at seven different concentrations in aqueous solution. These values were calculated from spectral data obtained using the transmission settings from the cunica minolta spectrophotometer CM-3500 d.

TABLE 1

Concentration of	L*	a*	b*	C*	h°
						1000ppm	10.49	15.82	-44.99	47.69	289.37
500ppm	24.07	9.80	-58.18	59.00	279.56
						100ppm	52.43	-29.57	-57.38	64.55	242.74
50ppm	63.64	-43.71	-48.31	65.14	227.86
						10ppm	84.25	-37.23	-23.42	43.99	212.17
5ppm	90.65	-24.40	-14.28	28.27	210.33
						1ppm	97.69	-6.43	-3.57	7.36	209.02

These L a b and L C h values for U.S. federal food, drug and cosmetic law blue No.1 represent the ideal target values for these natural blue colorant alternatives for U.S. federal food, drug and cosmetic law blue No. 1. Natural blue colorants having a L a b value that falls within a Δ E of 2.3 or less compared to these target values would be expected to provide color characteristics sufficiently similar to those provided by U.S. federal food, drug and cosmetic act blue No.1 so that the human eye cannot distinguish the differences in color provided by natural colorants from synthetic colorants. It is clear that the natural blue colorant's value of la b is closer to the synthetic target value (i.e., resulting in a smaller Δ Ε), then the natural blue colorant will better replace U.S. federal food, drug and cosmetic act blue No.1 in comestible applications.

Fig. 1 shows two perspective views, which are three-dimensional representations of the values of la b of the aqueous solution at the seven concentrations reported in table 1, which values are connected by line segments. Figure 2 shows two perspective views, which are three-dimensional representations of the L x C h ° values of the aqueous solution of federal food, drug and cosmetic act blue No.1 at the seven concentrations reported in table 1, which values are connected by line segments.

Mathematical models can be generated to represent the color characteristics provided by federal food, drug and cosmetic act blue No.1 in any concentration in the L a b and L C h color spaces. For example, the color feature may be represented by a segmented line model connecting the L a b data points or L C h ° data points of table 1. The line (L) in space connecting two points (P1 and P2) representing two different concentrations of federal food, drug and cosmetic act blue No.1 in the united states can be calculated using equation 4 below:

L＝{P₁+t*(P₂-P₁)} (4)

wherein P is₁Is (L₁,a*₁,b*₁)；P₂Is (L₂,a*₂,b*₂) (ii) a And t is any real number.

Thus, the segment line model for U.S. federal food, drug and cosmetic act blue No.1 in L a b color space can be interpolated using equation 4 below based on L a b values for seven different concentration points:

for concentrations between 500ppm and 1000ppm, 0< t < 1:

L*＝10.49+13.58*t

a*＝15.82+-6.02*t

b*＝-44.99+-13.19*t

for concentrations between 100ppm and 500ppm, 0< t < 1:

L*＝24.07+28.36*t

a*＝9.80+-39.37*t

b*＝-58.18+0.80*t

for concentrations between 50ppm and 100ppm, 0< t < 1:

L*＝52.43+11.21*t

a*＝-29.57+-14.14*t

b*＝-57.38+9.07*t

for concentrations between 10ppm and 50ppm, 0< t < 1:

L*＝63.64+20.61*t

a*＝-43.71+6.48*t

b*＝-48.31+24.89*t

for concentrations between 5ppm and 10ppm, 0< t < 1:

L*＝84.25+6.40*t

a*＝-37.23+12.83*t

b*＝-23.42+9.14*t

for concentrations between 1ppm and 5ppm, 0< t < 1:

L*＝90.65+7.04*t

a*＝-24.40+17.97*t

b*＝-14.28+10.71*t

the U.S. federal food, drug and cosmetic act blue No.1 segmented line model in L a b space is depicted in fig. 1.

In addition, colors whose values fall within the specified Δ E ranges of the U.S. federal food, drug and cosmetic act blue No.1 model can be mathematically modeled in la b color space. A particular value of Δ E, e.g., 3, relative to U.S. federal food, drug and cosmetic act blue No.1 was selected and plotted in L α b color space, resulting in a tubular structure surrounding U.S. federal food, drug and cosmetic act blue No.1 model, as shown in fig. 3. It is noted that any color having a Δ E value of about 2.3 or less from any point on the model is indistinguishable from the color provided by U.S. Federal food, drug and cosmetic Act blue No. 1.

To determine points (X) of the color space₀) Whether it falls within a specified Δ E value from the U.S. Federal food, drug and cosmetic Act blue No.1 model, the point and model must be calculated (from line segment X)₁To X₂Representation) of the minimum distance d between_min。

D can be calculated using equation 5_min：

Where x represents the cross product of two vectors and the vertical line represents the size of the vector expression.

If d is_minIs less than or equal to the selected delta E value, then the point in the L a b color space falls within the specified delta E value of the U.S. federal food, drug and cosmetic act blue No.1 model.

For example, it may be determined whether spirulina blue provides a color having a Δ E of 12 or less compared to the color provided by U.S. federal food, drug and cosmetic act blue No. 1. Table 2 shows the color characteristics provided by the known natural blue colorant spirulina blue at two different concentrations in aqueous solution.

TABLE 2

Concentration of	L*	a*	b*	C*	h°
						(404.8mg/L)	69.97	-29.69	-43.56	52.72	253.72
(206mg/L)	80.3	-23.97	-29.39	37.92	230.8

Solution of 404.8mg/L spirulina blue in L a b color spaceX of (2)₀Comprises the following steps:

X₀＝(69.97，-29.69，-43.56)

x of 206mg/L Spirulina blue solution in L a b color space₀Comprises the following steps:

X₀＝(80.3，-23.97，-29.39)

X₁and X₂Are two points from the U.S. federal food, drug and cosmetic act blue model No.1 at concentrations of 10ppm and 50ppm, respectively, in aqueous solution.

X₁＝(63.64,-43.71,-48.31)

X₂＝(84.25,-37.23,-23.24)

Calculating d using equation 5_minFor a solution of 404.8mg/L Spirulina blue, d_minIs 12.4, and for a 206mg/L spirulina blue solution, d_minIt was 14.4. Thus, the spirulina blue solution does not provide a color having a Δ E of 12 or less when measured against a segment line defined by the value of la b in the federal food, drug and cosmetic act blue No.1 at 10ppm and 50ppm in aqueous solution, as compared to the federal food, drug and cosmetic act blue No.1 in aqueous solution.

As shown in table 3, the spectral characteristics of several different spirulina blue solutions were determined.

TABLE 3

Spirulina solution data

Data name	ppm	L*(D65)	a*(D65)	b*(D65)	C*(D65)	h°(D65)
							0.04% of spirulina	400	67.69	-30.25	-45.87	54.94	236.6
0.03% of spirulina	300	72.77	-29.43	-39.52	49.27	233.32
							0.02% of spirulina	200	78.87	-25.56	-30.99	40.17	230.49
0.015% Spirulina	150	82.98	-21.82	-25.29	33.4	229.22
							0.01% of spirulina	100	87.77	-16.29	-18.32	24.52	228.35
0.0075% of spirulina	75	90.46	-12.94	-14.27	19.27	227.79
							0.005% Spirulina	50	93.23	-9.26	-10.13	13.72	227.59

Data for spirulina blue has been plotted against the U.S. federal food, drug and cosmetic act blue No.1 data in the color chart shown in fig. 5.

The differences between the color characteristics provided by spirulina blue and U.S. federal food, drug and cosmetic act blue No.1 are shown in fig. 5. Figure 5 shows a segmented line model of the color characteristics provided by U.S. federal food, drug and cosmetic act blue No.1 at a concentration of 1ppm to 1000ppm in aqueous solution in L a b color space surrounded by tubes representing regions of Δ E that differ by 3 or less from the color provided by blue No. 1. For comparison, fig. 5 also shows a piecewise linear model of the color characteristics provided by spirulina blue at a concentration of 50ppm to 400ppm in aqueous solution in L a b color space. The spirulina blue model did not cross any point in L a b color space with the blue No.1 model or the associated tube.

The present invention includes selecting a fraction or combination of fractions having a natural blue anthocyanin-containing colorant derived from a vegetable, a fruit, or a combination thereof. The fraction or combination of fractions comprises a selectively isolated anthocyanin mixture, wherein at least one concentration of the colorant provides a color characteristic having a delta E value of 12 or less when in aqueous solution at a pH of 8.0 as compared to a color characteristic defined by a segmentation line defined by the L a b values of U.S. federal food, drug and cosmetic act blue No.1 at 5ppm and 10ppm in aqueous solution. In other embodiments, the Δ Ε value may be less than 11, 10, 9, 8, 7, 6, 5, 4, or 3. At least one concentration of colorant may also be, for example, 1ppm and 5ppm, 10ppm and 50ppm, 50ppm and 100ppm, 100ppm and 500ppm, 500ppm and 1000ppm, or any combination selected therefrom, if desired as measured against a plurality of segmented lines defined by the U.S. Federal food, drug and cosmetic Act blue No.1 at different concentrations in an aqueous solution. For example, when not required, at least one concentration of colorant can be defined as having a Δ E value of 12 or less for the first segmentation line at 5ppm to 10ppm, a Δ E value of 8 or less for the segmentation line at 1ppm to 5ppm, and a Δ E value of 12 or less for the segmentation line at 10ppm to 50 ppm. However, if Δ E values are used to describe the colorants of the present invention, only one segmented line is required to define the colorant.

Although spirulina blue is considered to be the natural colorant that provides the closest color matching U.S. federal food, drug and cosmetic act blue No.1, the anthocyanin-containing natural blue colorant derived from vegetables, fruits or combinations thereof, i.e., the selectively isolated anthocyanin mixture in the fractions or combinations of fractions obtained according to the process of the present invention, is a better color match. In particular, when at least one concentration of said colorant in a selected fraction or combination of fractions is in an aqueous solution at pH 8.0, based on a series of aqueous solutions of U.S. federal food, drug and cosmetic act blue No.1 with different concentrations defined in L a b color space, the aqueous colorant solution provides color characteristics that match the blue No.1 segment line of the U.S. federal food, drug and cosmetic act, wherein match means that at least one concentration of said colorant in an aqueous solution at pH 8.0 has a Δ E value measured against the U.S. Federal food, drug and cosmetic Act blue No.1 segment line, that is, it is at least one unit less than the delta E value of a blue fraction line of spirulina based on a series of aqueous solutions having different concentrations of blue spirulina, as defined in the same la b color space, as measured against the U.S. federal food, drug and cosmetic act blue No.1 fraction line. In other embodiments, the delta E value of at least one concentration of a colorant in an aqueous solution at pH 8.0 measured against the U.S. federal food, drug and cosmetic act blue No.1 segment line is at least 2, 3, 4, 5, or 6 units less than the delta E value of a spirulina blue segment line measured against the U.S. federal food, drug and cosmetic act blue No.1 segment line. In other embodiments, the delta E value of at least one concentration of a colorant in an aqueous solution at pH 8.0 measured against the U.S. federal food, drug and cosmetic act blue No.1 segment line is at least 7, 8, 9, 10, or 11 units less than the delta E value of a spirulina blue segment line measured against the U.S. federal food, drug and cosmetic act blue No.1 segment line.

Various anthocyanin-containing fruit and vegetable extracts were analyzed to identify the source of anthocyanin that would provide the closest color characteristics to those provided by the synthetic blue colorant U.S. Federal food, drug and cosmetic Act blue No. 1. Figure 4 shows six different commercially available extracts of red cabbage, purple sweet potato, black carrot, purple corn and grape in five aqueous solutions of different pH values. Visually, it can be seen that anthocyanin from red radish, purple corn and grape does not provide a blue hue in aqueous solutions at any pH ranging from pH6 to pH 8. Anthocyanin from red cabbage, sweet potato and black carrot provides a blue hue in aqueous solutions at higher pH ranges.

Any anthocyanin-containing fruit or vegetable juice or extract that provides a blue hue at high pH can be used as a source of anthocyanin to produce the anthocyanin fraction of the invention. In some embodiments, the anthocyanin fraction is isolated from an extract of red cabbage, purple sweet potato, blue potato, purple carrot or black carrot, or a combination thereof.

In one embodiment, the method involves selectively separating an anthocyanin-containing fraction from red cabbage extract to produce an anthocyanin-containing natural colorant that provides a color characteristic similar to that provided by synthetic U.S. federal food, drug and cosmetic act blue No. 1.

An ion exchange column or semi-preparative HPLC column may be used to separate anthocyanin-containing fractions of selected anthocyanin-containing fruit and vegetable juices and extracts. Suitable ion exchange media include cation and anion exchange media. Suitable semi-preparative HPLC columns include C-18 columns. In one embodiment, the ion exchange column is activated with a solvent suitable for the ion exchange medium prior to loading the juice or extract of the vegetable or fruit.

Separating the anthocyanin-containing fraction from the anthocyanin-containing vegetable or fruit juice or extract with a solvent having a pH of at least about 2, preferably at least about 4. In some embodiments, the anthocyanin fraction is isolated with a solvent having a pH of about 2 to about 9. In another embodiment, the anthocyanin fraction is isolated with a solvent having a pH of about 3 to about 9. In another embodiment, the anthocyanin fraction is isolated with a solvent having a pH of about 4 to about 9. In another embodiment, the anthocyanin fraction is isolated with a solvent having a pH of about 5 to about 9. In other embodiments, the anthocyanin fraction is separated with a solvent having a pH of about 6 to about 9. In other embodiments, the anthocyanin fraction is separated with a solvent having a pH of about 7 to about 9.

Solvents suitable for eluting the selected anthocyanin-containing fraction include methanol, acetonitrile, water and mixtures thereof, selected depending on the polarity of the column medium and the solubility of the anthocyanin-containing juice or extract. In some embodiments, the solvent is aqueous methanol.

Suitable agents that may be added to the solvent to adjust the pH include potassium phosphate, sodium hydroxide, and the like.

In another embodiment, the present invention relates to a method of isolating a second fraction of anthocyanin from a juice or extract of anthocyanin-containing vegetables or fruits or combinations thereof, the method comprising: a) selectively separating anthocyanin on the ion exchange column using a second solvent of a selected pH based on differences in charge and polarity of anthocyanin molecules, wherein the pH of the second solvent is different from, preferably higher than, the pH of the solvent used to elute the first fraction; and b) selecting a second fraction or combination of fractions comprising isolated anthocyanin such that the isolated anthocyanin in the second fraction or combination of fractions provides those color characteristics provided by U.S. Federal food, drug and cosmetic Act blue No.1 when in aqueous solution at pH 8.0 as measured by having a maximum absorbance between 615nm and 635 nm. In this embodiment, the first fraction separated from the ion exchange column may not provide a mixture of isolated anthocyanin with a selected pH of first solvent, for example a solvent having a pH of at least about 2, i.e., which provides those color characteristics provided by U.S. federal food, drug and cosmetic act blue No.1, when the mixture of isolated anthocyanin is in an aqueous solution at pH 8.0, as measured by having a maximum absorbance between 615nm and 635 nm. In one embodiment, the selected second anthocyanin-containing fraction is separated from the anthocyanin-containing vegetable or fruit juice or extract using a solvent having a pH of from about 2 to about 9, or the pH is in one of the more preferred ranges of from about 3 to about 9, from about 4 to about 9, from about 5 to about 9, from about 6 to about 9, or most preferably from about 7 to about 9.

Additional anthocyanin-containing fractions can be isolated by further fractionating selected anthocyanin-containing fractions using an ion exchange column or a semi-preparative HPLC column. Suitable ion exchange media include cation and anion exchange media. Suitable semi-preparative HPLC columns include C-18 columns.

For example, in another embodiment, the classification method may further include the steps of: c) loading the selected fraction or combination of fractions comprising isolated anthocyanin onto an ion exchange column; d) selectively separating the anthocyanin loaded on the ion exchange column in step c) using a solvent of a selected pH based on the difference in charge and polarity of the anthocyanin molecules; and e) selecting the fraction or combination of fractions isolated in step d) comprising isolated anthocyanin such that the isolated anthocyanin selected in step e) provides a maximum absorbance of 620nm to 635nm when in aqueous solution at pH 8.0. If desired, third, fourth and further additional anthocyanin-containing fractions can be produced in a similar manner. In another embodiment, the isolated anthocyanin selected in step E) of an aqueous solution at pH 8.0 provides a color characteristic having a Δ E value of 12 or less compared to the color characteristic defined by the segmentation line defined by the L a b values of U.S. federal food, drug and cosmetic act blue No.1 at 5ppm and 10ppm in the aqueous solution.

In another embodiment, the step of selectively separating the anthocyanin on the ion exchange column based on the difference in charge and polarity of the anthocyanin molecule comprises the steps of: (i) first obtaining a first fraction using a solvent at a selected pH, and (ii) using a second solvent at a second selected pH, wherein the pH of the second solvent is different from the pH of the first solvent, to obtain a subsequent fraction as one fraction or to obtain a combination of subsequent fractions as a combination of fractions, wherein when the isolated anthocyanin in the one fraction or combination of fractions is at least one concentration in an aqueous solution at pH 8.0, it provides a color profile having a Δ E value of 12 or less compared to a color profile defined by a segmentation line defined by L a b values of 5ppm and 10ppm of U.S. federal food, drug and cosmetic act blue No.1 in the aqueous solution.

In another embodiment, the step of selectively separating the anthocyanin on the ion exchange column based on the difference in charge and polarity of the anthocyanin molecule comprises the steps of: (i) first obtaining a first fraction using a solvent of a selected pH, said solvent being a first elution solvent, and (ii) obtaining a combination of said one or more fractions using one or more subsequent elution solvents of a selected pH, wherein each elution solvent is different, and said difference may be independently selected from the group consisting of pH, solvent composition, and combinations thereof. Preferably, the selected pH of the first elution solvent is lower than the selected pH of the one or more subsequent fractions. Preferably, the selected pH will be in the range of about 2 to about 9, or in one of the following more preferred ranges, i.e., about 3 to about 9, about 4 to about 9, about 5 to about 9, about 6 to about 9, or most preferably about 7 to about 9. In another embodiment, the isolated anthocyanin in the one fraction or the combination of fractions provides a color profile having a Δ E value of 12 or less when at least one concentration in an aqueous solution at pH 8.0 as compared to a color profile defined by a segmentation line defined by the L a b values of U.S. federal food, drug and cosmetic act blue No.1 at 5ppm and 10ppm in an aqueous solution.

The isolated anthocyanin fraction can be used as a colorant or can be subjected to further processing, e.g., purification, concentration, deodorization or color stabilization.

The selective separation process can be carried out on a scale that produces commercially useful amounts of the natural blue colorant.

The natural blue anthocyanin-containing colorants prepared by the methods of the present invention can be applied to or incorporated into all types of edible products, including foods, beverages, and medicaments for human and animal consumption. Examples of edible products include: pet foods and snacks, dry goods (e.g., rice, cereals, and cereals), soups and sauces, confectionery products (e.g., all types of chocolate, sugar and sugarless candies, chewing gum, sugar bars, and sugar coated confections), dessert products (e.g., puddings, frostings, sugar powders, and toppings), baked goods (e.g., cakes, cookies, crackers, and biscuits), dairy products (e.g., yogurt, whipped cream, and cheese), beverages (e.g., dairy beverages, water, juices, tea, and soda), snack products (e.g., crackers, snack bars (snackbars), crackers, and chips), and pharmaceutical forms (e.g., tablets, suspensions, chewable agents, and syrups). The natural blue anthocyanin-containing colorant can also be incorporated into food grade colorant compositions, coatings, and caramel juices (ink). In one embodiment, the blue anthocyanin-containing colorant is included in a coating or caramel juice that coats the surface of the confectionery article. In another embodiment, the blue anthocyanin-containing colorant is included in a coating or caramel juice that coats the surface of a confectionery product, wherein the confectionery product is a confectionery center having a soft panned or hard panned sugar-based coating. In another embodiment, the blue anthocyanin-containing colorant is included in a coating or caramel juice that coats the surface of the confectionery product, wherein the confectionery product is a confectionery center having a soft panned or hard panned sugarless coating.

In one embodiment, the red cabbage extract is fractionated using a strong cation exchange column. The first fraction was eluted with 75% v/v of 0.1M potassium phosphate buffer pH 8 and 25% v/v methanol. The second fraction was eluted with 30% v/v of 0.1M potassium phosphate buffer pH 8 and 70% v/v methanol.

In another embodiment, the red cabbage extract is fractionated using a strong cation exchange column. The first fraction was eluted with 75% v/v of 0.1M potassium phosphate buffer pH6 and 25% v/v methanol. The second fraction was eluted with 75% v/v of 0.1M potassium phosphate buffer pH 7 and 25% v/v methanol. The third fraction was eluted with 75% v/v of 0.1M potassium phosphate buffer pH 8 and 25% v/v methanol. The fourth fraction was eluted with 30% v/v of 0.1M potassium phosphate buffer pH 8 and 70% v/v methanol.

In another embodiment, the red cabbage extract is separated using a C-18 semi-preparative HPLC column.

A method for selectively separating anthocyanin fractions from complex mixtures of anthocyanin in juices and extracts of vegetables and fruits based on differences in the charge and polarity of the anthocyanin molecules produces colorants that provide color characteristics that are different from those provided by the complex mixtures.

The method of selectively separating anthocyanin fractions from complex mixtures of anthocyanins based on differences in polarity of the anthocyanin molecules enables a long felt approach to obtaining natural colorants that provide color characteristics similar to those provided by the synthetic colorant U.S. Federal food, drug and cosmetic Act blue No. 1.

Specific embodiments of the present invention will now be illustrated with reference to the following examples. It is to be understood that these embodiments are disclosed merely for purposes of illustration and that variations within the spirit of the invention are contemplated.

Example 1

Fractionating a red cabbage extract using a strong cation exchange column

Activation of Philomen Using pure methanol(Torrans, Calif.) SCX (Strong cation exchange) solid phase extraction column (cartridge). The column (cartridge) was washed with 0.01% v/v acidified water. An aqueous solution of red cabbage extract was loaded onto the column and washed with 0.01% v/v acidified water. Potassium phosphate buffer (0.1M) at pH 8 was passed through the column. Elution was performed with 25% v/v methanol solution at pH 8 and fraction 1 was collected. Elution was performed with 70% v/v methanol solution at pH 8 and fraction 2 was collected.

Fraction 1 and fraction 2 were acidified with 2ml to 5ml 88% v/v formic acid. Methanol was removed using a rotary evaporator.

To remove any salts, fraction 1 was loaded onto a C-18 column and eluted with 0.01% v/v acidified water. The eluate was collected in 0.01% v/v acidified water and the residual methanol was evaporated. Fraction 2 was also passed through a C-18 column using the same procedure outlined for fraction 1.

As shown in table 4 below, Red Cabbage Extract (RCE) and fractions 1 and 2 were analyzed for maximum ultraviolet/visible (UV/VIS) wavelength absorbance and color characteristics at different pH values.

TABLE 4

Fraction 2 at pH 7.6 and pH 8.0 provided a λ max value closest to the λ max value of synthetic U.S. federal food, drug and cosmetic act blue No.1 (λ max 630nm), i.e., the λ max values were 616.40 and 619.40, respectively.

The color characteristics provided by fraction 2 at pH 7.6 and at pH 8.0 can also be compared to synthetic U.S. Federal food, drug and cosmetic Law No.1 (FD)&C Blue No.1) to calculate the Δ E value. The Δ E value is equal to the minimum distance between the fraction 2 color point in the L a b color space and the U.S. federal food, drug and cosmetic act blue model No. 1. Thus, d is calculated from the following data using equation 5_minValue or Δ E value:

x of fraction 2 at pH 7.6 in L a b color space₀Comprises the following steps:

X₀＝(91.62,-4.17,-5.68)

x of fraction 2 at pH 8.0 in L a b color space₀Comprises the following steps:

X₀＝(91.56，-5.80，-5.81)

X₁and X₂Is from U.S. Federal food, drug and cosmetic act blue No.1 (FD)&C Blue No.1) two points of the model:

X₁＝(90.65,-24.40,-14.28)

X₂＝(97.69,-6.43,-3.57)

calculated d for fraction 2 at pH 7.6_minValue or Δ E value of 6.7, d calculated for fraction 2 at pH 8.0_minThe value or Δ E value is 6.0.

FIG. 6 provides high performance liquid chromatograms (HPLC chromatograms) of Red Cabbage Extract (RCE) and fraction 1 and fraction 2 under 520nm detection. Fig. 6 shows that fraction 2 from the red cabbage extract has a higher concentration of the post-elution peak (later-eluting peak).

Example 2

Fractionation of red cabbage extracts using strong cation exchange columns and solvents of different high pH values

Using a Feilou door(Torrans, Calif.) SCX (Strong cation exchange) solid phase extraction column (cartridge). The red cabbage extract diluted in 0.01% v/v acidified water (10ml to 15ml) was loaded onto a column and washed with 0.01% v/v acidified water. Potassium phosphate buffer (0.1M) at pH6 was passed through the column. Fraction 1 was eluted with 25% v/v methanol solution at pH6 and collected. Potassium phosphate buffer (0.1M) at pH 7 was passed through the column. Fraction 2 was eluted with 25% v/v methanol solution at pH 7 and collected. Potassium phosphate buffer (0.1M) at pH 8 was passed through the column. Fraction 3 was eluted with 25% v/v methanol solution at pH 8 and collected. Fraction 4 was eluted with 70% v/v methanol solution at pH 8 and collected.

Fractions 1 to 4 were acidified with 20% v/v formic acid. Methanol was removed using a rotary evaporator.

To wash the salts, fraction 1 was loaded onto a C-18 column and eluted with 0.01% v/v acidified water. The eluate was collected in 0.01% v/v acidified water and the residual methanol was evaporated. Fractions 2 to 4 were also passed through a C-18 column using the same procedure outlined for fraction 1.

The Red Cabbage Extract (RCE) and fractions 1 to 4 were analyzed for maximum ultraviolet visible (UV/VI) S wavelength absorbance and color characteristics at different pH values, as shown in table 5 below.

TABLE 5

Fraction 4 at pH 8.0 provides a comparison with synthetic U.S. Federal food, drug and cosmetic Law No.1 (FD)&Lambda of CBlue No.1)_maxValue (lambda)_max630nm) nearest λ_maxValue, i.e. λ_maxThe value is 622.2.

The Δ Ε value may also be calculated by comparing the color characteristic provided by fraction 4 at pH 8.0 to the color characteristic provided by the synthetic U.S. federal food, drug and cosmetic act blue No. 1. The Δ E value is equal to the minimum distance between the fraction 4 color point in the L a b color space and the U.S. federal food, drug and cosmetic act blue model No. 1. Thus, d is calculated from the following data using equation 5_minValue or Δ E value:

x of fraction 4 at pH 8.0 in L a b color space₀Comprises the following steps:

X₀＝(90.08，-7.87，-7.20)

X₁and X₂Are two points from the U.S. federal food, drug and cosmetic act blue No.1 model:

X₁＝(90.65,-24.40,-14.28)

X₂＝(97.69，-6.43，-3.57)

calculated d for fraction 4 at pH 8.0_minThe value or Δ E value was 6.7.

FIG. 7 provides HPLC chromatograms of red cabbage extract solution (RCE) and fractions 1 through 4, detected at 520 nm. Fig. 7 shows that fraction 4 from the red cabbage extract has a higher concentration of the post-elution peak (later-eludingpeak).

Example 3

Separation of peak groups of cabbage extract using semi-preparative High Performance Liquid Chromatography (HPLC)

As shown in the chromatogram of fig. 8, semi-preparative HPLC can be used to separate and collect fractions associated with two specific peak groups from red cabbage extracts. The red cabbage extract was loaded onto a C-18 semi-preparative HPLC column and two fractions, 520-nm fractions (. lamda.) (lambda.) were eluted using an acidic acetonitrile and water gradient_max524nm) and 530-nm fractions (. lamda.)_max532 nm). The residual acetonitrile was evaporated from each fraction using a rotary evaporator.

Characterization of the color characteristics was performed on the samples after adjusting the concentration of the fractions and mixing the separated fractions aliquots with a buffer to produce 5 aliquots of pH6, pH 6.6, pH 7, pH 7.6 and pH 8. The maximum UV/VIS wavelength absorbance and color characteristics of the 520-nm fraction aliquot and the 530-nm fraction aliquot were analyzed and the results are provided in table 6.

TABLE 6

¹ND denotes that the absorption spectrum of the sample does not show the maximum peak in the visible range.

The 530-nm fraction has a maximum absorbance at pH 7.6 and pH 8.0 of about 621nm and provides for the synthesis of U.S. Federal food, drug and cosmetic Law blueLambda of number 1_max(λ_max630nm) nearest λ_max。

The Δ E values may also be calculated by comparing the color characteristics provided by the 530-nm fractions at pH 7.6 and pH 8.0 with the color characteristics provided by the synthetic U.S. Federal food, drug and cosmetic Act blue No. 1. The Δ E value is equal to the minimum distance between the 530-nm fraction color point in the la b color space and the U.S. federal food, drug and cosmetic act blue model No. 1. Thus, d is calculated from the following data using equation 5_minValue or Δ E value:

530-nm fractions X at pH 7.6 in L a b color space₀Comprises the following steps:

X₀＝(87.67,-5.44,-9.90)

530-nm fractions of X at pH 8.0 in L a b color space₀Comprises the following steps:

X₀＝(86.39，-11.79,-11.98)

X₁and X₂Are two points from the U.S. federal food, drug and cosmetic act blue No.1 model:

X₁＝(84.25，-37.23，-23.42)

X₂＝(90.65，-24.40，-14.28)

calculated d for the 530-nm fraction at pH 7.6_minValue or Δ E value of 12.1, calculated d for the 530-nm fraction at pH 8.0_minThe value or Δ E value was 9.9.

FIG. 9 provides a visual comparison of 520-nm and 530-nm fractions for different pH values. The concentration of the 520-nm fraction was 107.7mg/L (Cyn-3-glu), and the concentration of the 530-nm fraction was 55.6mg/L (Cyn-3-glu). At neutral and higher pH, it can be seen that the 530-nm fraction provides two to four times the color (Chroma, measured by C) of the 520-nm fraction at half the colorant concentration.

FIG. 10 provides HPLC chromatograms of red cabbage extract and 520-nm and 530-nm fractions detected at 520 nm. FIG. 10 shows that each fraction contains three different anthocyanin compounds.

Comparative examples

Several different concentrations of red cabbage anthocyanin solutions as disclosed in the examples of WO 2004/012526 were prepared at pH 8.0. The fractions were not separated and the separated anthocyanin-containing colorant was collected. The maximum absorbance of the resulting solution was 610 nm. This color is not considered an acceptable match for the color of U.S. Federal food, drug and cosmetic Law No.1 (FD & C blue No. 1).

Claims (17)

1. A method of separating a fraction of anthocyanin from a juice or extract of anthocyanin-containing vegetables or fruits or combinations thereof, the method comprising:

a) loading anthocyanin-containing juice or extract of vegetables or fruits or a combination thereof onto an ion exchange column;

b) selectively separating anthocyanin on the ion exchange column using a solvent of selected pH based on difference in charge and polarity of the anthocyanin molecule; and

c) selecting a fraction or a combination of fractions comprising isolated anthocyanin such that the isolated anthocyanin in the fraction or the combination of fractions has a maximum absorbance of 615nm to 635nm when in an aqueous solution at pH 8.0.

2. The method according to claim 1, wherein the isolated anthocyanin in the one fraction or combination of fractions at least one concentration in an aqueous solution at pH 8.0 provides a color characteristic having a Δ Ε value of 12 or less compared to the color characteristic defined by the segmentation line defined by the U.S. federal food, drug and cosmetic act blue No.1 values of 5ppm and 10ppm in the aqueous solution.

3. The method of claim 1, wherein the anthocyanin-containing vegetable or fruit juice or extract is derived from a source selected from the group consisting of red cabbage, purple sweet potato, blue potato, black carrot, purple carrot and combinations thereof.

4. The method according to claim 3, wherein the source of the anthocyanin-containing vegetable or fruit juice or extract is red cabbage.

5. The method of claim 1, wherein the selected anthocyanin-containing fraction is separated from the anthocyanin-containing vegetable or fruit juice or extract or combination thereof with a selected solvent having a pH of from 4 to 9.

6. The method of claim 5, wherein a first anthocyanin-containing fraction is eluted with a 25% v/v methanol solution at pH 8 and a subsequent anthocyanin-containing fraction is eluted with a 70% v/v methanol solution at pH 8, the subsequent anthocyanin-containing fraction being one fraction or a plurality of subsequent fractions that are a combination of fractions.

7. The method of claim 6, further comprising the step of purifying the fraction or the combination of fractions.

8. The method according to claim 7, wherein the isolated anthocyanin in at least one concentration of the one fraction or the combination of fractions in aqueous solution at pH 8.0 provides a color characteristic having a Δ E value of 12 or less compared to the color characteristic defined by the segmentation line defined by the U.S. Federal food, drug and cosmetic Act blue No.1 values of 5ppm and 10ppm in aqueous solution.

9. The method of claim 1, wherein the ion exchange column is a cation exchange column.

10. The method of claim 1, wherein the step of selectively separating the anthocyanin on the ion exchange column based on the difference in charge and polarity of anthocyanin molecules comprises the steps of: (i) first obtaining a first fraction using a solvent of a selected pH, and (ii) using a second solvent of a second selected pH, wherein the pH of the second solvent is different from the pH of the solvent of the selected pH, to obtain a subsequent fraction as the one fraction or a combination of multiple subsequent fractions as a combination of the multiple fractions, wherein the isolated anthocyanin provides a color profile having a Δ Ε value of 12 or less when the isolated anthocyanin in the one fraction or combination of multiple fractions is at least one concentration in an aqueous solution of pH 8.0 compared to a color profile defined by segmentation lines defined by L a b values of 5ppm and 10ppm U.S. federal food, drug and cosmetic act blue No.1 in the aqueous solution.

11. The method of claim 1, further comprising the steps of:

d) loading the selected fraction or combination of fractions comprising isolated anthocyanin onto an ion exchange column;

e) selectively separating the anthocyanin loaded onto the ion exchange column in step d) using a solvent of a selected pH based on the difference in charge and polarity of the anthocyanin molecules; and

f) the fraction or combination of fractions isolated in step e) comprising isolated anthocyanin is selected so that the isolated anthocyanin selected in step f) provides a maximum absorbance of 620nm to 635nm when in aqueous solution at pH 8.0.

12. The method according to claim 11, wherein the isolated anthocyanin selected in step f) in an aqueous solution at pH 8.0 provides a color profile having a Δ E value of 12 or less compared to the color profile defined by the segmentation line defined by the L a b values of 5ppm and 10ppm of U.S. federal food, drug and cosmetic act blue No.1 in the aqueous solution.

13. The method of claim 1, wherein the step of selectively separating the anthocyanin on the ion exchange column based on the difference in charge and polarity of anthocyanin molecules comprises the steps of: (i) first obtaining a first fraction using a solvent of a selected pH, said solvent being a first elution solvent, and (ii) obtaining a combination of said one or more fractions using one or more subsequent elution solvents of a selected pH, wherein each elution solvent is different, and said difference may be independently selected from the group consisting of pH, solvent composition, and combinations thereof.

14. The method according to claim 13, wherein the isolated anthocyanin provides a color profile having a Δ E value of 12 or less when the isolated anthocyanin is at least one concentration in an aqueous solution at pH 8.0 as compared to a color profile defined by a segmentation line defined by the L a b values of U.S. federal food, drug and cosmetic act blue No.1 at 5ppm and 10ppm in an aqueous solution.

15. The method of claim 14, wherein the first elution solvent is a mixture of organic solvent and water at a first concentration of organic solvent, and the subsequent elution solvent used is a second mixture of organic solvent and water having a second concentration of organic solvent, wherein the first concentration is different from the second concentration.

16. The method of claim 13, wherein the first elution solvent has a selected pH that is different from the selected pH of the subsequent elution solvent.

17. The method of claim 16, wherein the selected pH of the first elution solvent is lower than the selected pH of the subsequent elution solvent.