WO2017156744A1

WO2017156744A1 - New gardenia blue pigment, preparation and use thereof

Info

Publication number: WO2017156744A1
Application number: PCT/CN2016/076596
Authority: WO
Inventors: Andre DUESTERLOH; Xiaoyan Wang; Xiaoping Huang; Huiwen DENG; Bingdong LIN; Qing He; Andrea BULBARELLO
Original assignee: Dsm Ip Assets B.V.
Priority date: 2016-03-17
Filing date: 2016-03-17
Publication date: 2017-09-21
Also published as: TW201735798A

Abstract

A gardenia blue pigment is derived from a reaction of genipin with an amino acid. It is brighter than commercial gardenia blue pigment and suitable for industrial application. A process for producing the gardenia blue pigment and the use thereof in food and beverage industries are also provided.

Description

[Title established by the ISA under Rule 37.2] NEW GARDENIA BLUE PIGMENT, PREPARATION AND USE THEREOF

Technical Field

The present application is related to a new gardenia blue pigment derived from a reaction of genipin with an amino acid, a process for the preparation and the use in food and beverage industries.

Background of the Invention

The gardenia blue pigment is a water-soluble natural pigment widely used in food, pharmaceutical and cosmetics industries.

Nowadays, the gardenia blue pigment is usually produced from the raw material geniposide contained in Gardenia Jasminoides Ellis of Rubiaceae by the process of treating geniposide with a β-glucosidase to obtain genipin which reacts with an amino acid to obtain the gardenia blue pigment. (see Shijing WU, et al., National Food Additive Communications, 1992 (3) : 90-93) However, the gardenia blue pigment obtained by this process is dark, it has a low color value, low quality and is easy to precipitate in beverage at low pH. So it is not suitable for some applications such as food and beverages. (see e.g. CN103509368A and CN102021210A)

Summary of the Invention

The present application provides a new gardenia blue pigment derived from a reaction of genipin with an amino acid, which is brighter than the known blue in the art.

The present application also provides a method for the preparation of the above gardenia blue pigment which comprises the following steps:

a) Treating geniposide with a glycosidase to obtain a hydrolysate； and

b) Extracting the hydrolysate obtained in step a) with a solvent and removing the solvent after the extraction to obtain a product comprising genipin； and

c) Reacting the product comprising genipin obtained in step b) with an aqueous solution of an amino acid and/or a salt thereof to produce the gardenia blue pigment, and preferably dissolving the product comprising genipin in a water-soluble solvent resulting in a solution and using the solution as such.

The present application further provides the use of the above gardenia blue pigment in food and beverage industries.

The present application further more provides beverages and food containing a gardenia blue pigment according to the present application.

Brief description of the drawings

Figure 1 shows the chemical formulae of the compounds of formulae (a) - (h) .

Figure 2 shows the HPLC-DAD-MS profile of the gardenia blue products according to the present application.

Figure 3 shows the molecular weight distribution profiles by GPC of the gardenia blue products according to the present application.

Figure 4A-4B show UV-VIS chromatograms of the gardenia blue products according to the present application.

Detailed Description of the Invention

In the first aspect, the present application provides a new gardenia blue pigment derived from a reaction of genipin with an amino acid, which is brighter than the gardenia blue commercially in the market.

In particular, the present application provides a new gardenia blue pigment which has a color shade h in the range of from 220 to 245, preferably from 230 to 240 at the CIELAB Color scale, and color value “a*” in the range of from -12 to -7, preferably from -11 to -9 at the CIELAB color scale.

preferably, the gardenia blue pigment according to the present application contains one or more of the compound (s) of formulae (I) - (IV) , a geometric isomer thereof, a tautomer thereof, a salt thereof, or a combination thereof:

Wherein: R is independently from each other H or C_1-7 alkyl, preferably methyl； and R’ is independently from each other selected from the group consisting of C_1-7 alkyl, preferably ethyl, propyl, butyl and pentyl, optionally substituted by a carboxyl, phenyl, imidazolyl or guanidino group.

Preferably, R’ is independently from each other selected from the group consisting of:

More preferably, the compound (s) of formulae (I) - (IV) is a compound of formulae (a) - (h) , a geometric isomer thereof, a tautomer thereof, a salt thereof, or a combination thereof:

The term geometric isomers as used herein mean isomers of identical structure except with different configurations at the double bond (s) (i.e., E or Z isomer, or cis/trans isomer) . For illustration, Scheme 1 shows an example of a pair of two geometric isomers, where the only difference between the two isomers is the configuration at the double bond.

The term tautomers as used herein means compounds that can be interconvertible through tautomerization. Tautomerization is known in the art and generally refers to a reaction as shown in Scheme 2. In most cases, group G in the reaction is hydrogen； and X, Y and Z are independently from each other a carbon atom.

As used herein, the term "salt" is understood to include salts having a cation as a counterion, such as an alkaline metal ion (e.g., Na, K, etc. ) , an alkaline earth metal ion (e.g., Mg²⁺, Ca²⁺, etc. ) , ammonium ion (e.g., NH₄, or an organic ammonium ion) , etc., and salts having an anion as a counterion, such as an inorganic anion (e.g., Cl, SO₄, Br, HSO₄, etc. ) or an organic anion (e.g., a carboxylic acid anion such as a formate, acetate, etc. ) .

In one embodiment of the gardenia blue pigment derived from a reaction of genipin with sodium glutamate or glutamic acid according to the present application, the gardenia blue pigment has further the following characterizations:

Molecular weight by GPC: 600-5000 Da, preferably 1000-4000 Da, more preferably 1200-3000 Da；

Content of bright blue fractions: 18-45wt％, preferably 20-40wt％, more preferably 25-35wt％, and/or；

Isoelectric point: 1.5-3.0, preferably 1.8-2.8, and more preferably 1.9-2.2.

In the present application, the bright blue fractions refers to the fractions which have the maximum absorption wavelength above 600 nm in the gardenia blue pigment obtained according to the below mentioned process.

In the second aspect, the present application provides a process for the preparation of the above gardenia blue pigment which comprises the following steps:

a) Treating geniposide with a glycosidase to obtain a hydrolysate； and

In the process, the geniposide used as raw material may be from various sources. It may be obtained by extracting the fruit Gardenia Jasminoides Ellis by any known process, for example, that as disclosed in Chinese patent publication CN102732050A. In addition, geniposide powders, which contain about 20wt％to 70wt％of geniposide and are commercially available, and the waste stream from the gardenia yellow production, which contains about 40wt％of geniposide and is also commercially available, may be used into the process directly or after simple refining by, for example, ultrafiltration. (see CN103509368A, CN103525883A etc. )

The glycosidase is an enzyme under EC 3.2.1 according to the Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes by the Reactions they Catalyse. Preferably, the glycosidase is cellulase (EC 3.2.1.4) or β-glucosidase (EC 3.2.1.21) or a mixture thereof. The examples of the glycosidase include but are not limited to cellobiase commercially available from Sunson Biotechnology Co. Ltd. (Guangzhou, China) , Cellulase 4000 commercially available from DSM (China) Ltd. (Shanghai, China) , and

commercially available from DSM (China) Ltd. (Shanghai, China) . The glycosidase may be added into the reaction of the step a) in an amount in the range of from 10 U to 1000 U, preferably in an amount in the range of from 50 U to 500 U, more preferably in an amount in the range of from 100 U to 400 U, the most preferably in an amount in the range of from 200 U to 300 U, per 1 g of geniposide.

The treatment of the step a) may be carried out at a pH in the range of from 3.0 to 6.5, preferably at a pH in the range of from 3.6 to 6.0, and more preferably at a pH in the range of from 4.0 to 4.6. Preferably, the treatment of the step a) is carried out in a buffer solution which can provide the above pH ranges. Such buffer solution is known in the art, and the examples include but are not limited to an aqueous HCOOH/NaOAc solution or an aqueous citric acid/Na₂HPO₄ solution. The buffer solution may be added in an amount in the range of from 8 mL to 80 mL, preferably in an amount in the range of from 10 mL to 50 mL, more preferably in an amount in the range of from 20 mL to 40 mL, per 1 g of geniposide.

The treatment of the step a) may be carried out at a temperature in the range of from about 20℃ to about 60℃, preferably at a temperature of about 50℃.

The reaction of the step a) may last about 10 hours to about 30 hours. The obtained hydrolysate contains genipin as main component and other components. The hydrolysate can be used for the extraction in step b) directly.

In the prior process, the hydrolysate obtained from the step a) is normally reacted with an amino acid directly to produce gardenia blue pigment. However, the gardenia blue pigment obtained from the prior process is dark and not good for some industrial applications such as food and beverages. The inventors of the present invention surprisingly discovered that an additional extraction of the step b) results in sky blue color, a bright blue pigment which is more applicable for food and beverage applications.

Any person skilled in the art could understand that, the solvent used for extraction in the step b) is important and may be any one suitable for the purpose of the invention, and examples include but are not limited to diethyl ether, ethyl acetate, butanol, a mixture of butanol with petroleum and/or hexane, or mixtures thereof. In the embodiment that the solvent is a mixture of butanol with petroleum and/or hexane, the volume ratio of butanol with hexane and/or hexane is in the range of from 1: 5 to 5: 1, preferably in the range of from 1: 3 to 3: 1, and more preferably in the range of from 1: 2 to 2: 1.

The amount of the solvent used in the step b) may be from 1 mL to 5 mL, preferably from 1 mL to 3 mL, per 1 mL of the hydrolysate. The extraction of the step b) may be carried out at a temperature in the range of from 10℃ to 60℃, preferably at room temperature. According to the present invention, the extraction of the step b) may be repeated two to four times.

The product comprising genipin is obtained after the organic phases are collected and the solvent is removed in the step b) . The solvent may also be recycled. The extraction procedures and the procedures for collecting solvents and removing/recycling solvents during the extraction are known to the person skilled in the art. Therefore, they are not discussed in more detail here.

As known in the art, the gardenia blue pigment is the reaction product of genipin with an amino acid or a salt thereof. Accordingly, as the step c) of the process, the product comprising genipin obtained in the step b) is reacted with an amino acid or a salt thereof to produce the gardenia blue pigment. In the present invention, the amino acid suitable for the reaction may be selected from the group consisting of glutamate, phenylalanine, histidine, leucine, isoleucine, arginine and any mixture thereof. The salt may be any alkali metal salt such as sodium salt. Preferably the salt is sodium glutamate. The inventors of the present invention discovered that the amino acid and the salt used in the step c) are also important because they can provide sky blue color as disclosed in the present invention.

In the present invention, an aqueous solution of the amino acid or the salt thereof is used to react with the product comprising genipin to provide the gardenia blue pigment and the aqueous solution is added in an amount to provide a molar ratio of 1: 0.5-10, preferably 1: 1-8, more preferably 1: 2-6, and most preferably1: 3-4 between genipin and the amino acid in the reaction mixture of step c) .

In addition, the inventors of the present invention discovered that it would be helpful for the reaction in the step c) to dissolve the product comprising genipin in a water-soluble solvent to form a homogeneous reaction system. Accordingly, the product comprising genipin obtained from the step b) is preferably dissolved in a water-soluble solvent according to the present invention. The water-soluble solvent may be any one known in the art that can dissolve the product comprising genipin and examples include but are not limited to C_1-10 alkanols such as methanol and ethanol, and C_3-10 ketones such as acetone. Preferably, the water-soluble solvent is methanol, ethanol or acetone or any mixture thereof.

The reaction of the step c) may be carried out at about 40℃ to about 90℃, preferably about 60℃ to 80℃, such as 65℃, 70℃ and 75℃. The progress of the reaction can be monitored by any known method, such as HPLC and TLC.

Preferably, the step c) is carried out at a pH value in the range of from 7.0-11, more preferably 8-9.5. In some embodiments, a base selected from but not limited to NaOH, KOH, NaCO₃ and NaHCO₃ is added to adjust the reaction mixture of the step c) to an appropriate pH value.

After the reaction is complete, the gardenia blue pigment can be obtained as a solid by removing the organic solvent and water in the reaction mixture. Accordingly, the process of the present invention optionally further comprises the step of removing the solvent and water to provide a solid of the gardenia blue pigment by, for example, lyophilization or spray drying.

Optionally, the obtained gardenia blue pigment can be purified further by any procedures known in the art such as ultrafiltration to obtain an even purer gardenia blue pigment. The process of the present invention produces the gardenia blue pigment which is sky blue, brighter than the blue color such as ultramarine blue produced by the known processes and thus more popular for some industrial applications such as beverages.

In addition, most of the obtained gardenia blue pigment has a color value of >100, that means low dosage can be used in applications. Further, by an additional extraction step, the obtained gardenia blue pigments can surprisingly be easily separated and purified from the reaction mixture without complicated operations.

Accordingly, as the third aspect, the present application provides the use of the gardenia blue pigment according to the present invention in food and beverage industries.

In the fourth aspect, the present application provides beverages and food containing a gardenia blue pigment according to the present application.

The present invention is illustrated further by the following Examples. These Examples are not intended to limit the invention in any way.

Examples

In the following examples, the maximum absorption wavelength and the color value were measured according to the national standard GB 28311-2012 of China.

Example 1

6.7 g of geniposide powder (35.5wt％) purchased from Jiatian Biotechnology Co., Ltd. (Xi’ an, China) was added into 47 ml of an aqueous citric acid/Na₂HPO₄ buffer solution (pH 4.0) . 685.7 mg (1000 U/g) of cellobiase purchased from Sunson Biotechnology Co. Ltd. (Guangzhou, China) was further added for reaction for 19 hours at 50℃. After having cooled down the reaction mixture to room temperature, the reaction mixture was extracted with 90 ml of ethyl acetate twice. The ethyl acetate phase was concentrated under vacuum to obtain rude genipin.

The obtained rude genipin was dissolved in 16 ml of absolute ethyl alcohol to obtain 19.4 g of a solution (4.79wt％genipin by HPLC) . 970 mg of sodium glutamate in 10 ml of deionized water was added into the solution for reaction for 25 hours at 70℃. TLC indicated that the genipin had been converted completely.

The reaction liquid was lyophilized to obtain 2.1 g of gardenia blue pigment as solid powder, with a maximum absorption wavelength of 601 nm and a color value of 155.

Example 2

140 g of liquid geniposide (40wt％) purchased from Zhongda HengYuan Biotechnology Co., Ltd. (He’ nan, China) was mixed with 980 ml of aqueous citric acid/Na2HPO₄ buffer solution (pH 4.0) . The mixture was heated to 50℃ and then 42 g (400 U/g) of cellobiase purchased from Sunson Biotechnoloty Co. Ltd. (Ningxia, China) was added. After stirring for about 12 hours at 50℃, the mixture was filtrated to remove the insoluble solid while the filtrate was extracted with about 1 L ethyl acetate twice. Ethyl acetate was removed by evaporation to provide crude genipin which was 92.7％purity by HPLC

6 g of the obtained crude genipin was dissolved in 90 ml of absolute ethyl alcohol. 18.5 g of sodium glutamate in 90 ml of deionized water was mixed with the crude genipin ethanol solution. The mixture was stirred at 75℃ for about 6 hours.

The reaction solution was evaporated to remove ethyl alcohol and then lyophilized to provide about 19 g of gardenia blue product as solid powder with a maximum absorption wavelength of 601 nm and a color value of about 80.

Example 3

Color (lightness, Chroma, and hue) of the gardenia blue pigment according to the present application was determined with a HunterLab Ultra Scan Pro spectrocolorimeter (Hunter Associates Laboratory, Reston, VA, USA) and expressed on basis of the CIELAB color scale. The mode used was RSIN which stands for Reflectance -Specular Included. The small area view (SAV) with a diameter of 4.826 mm (0.190 inch) was chosen. Color measurements are carried out after CIE guidelines (Commission International d’ Eclairage) . Values can be expressed as planar coordinates as L*, a*, b* with L* being the measuring values for lightness, with a* being the value on the red-green axes and b* being the value on the yellow-blue axes.

The Chroma (C*) sometimes called saturation describes the vividness or dullness of a color which can be calculated as followed:

C*＝√ (a*²+b*²)

The angle called hue (h) describes how we perceive an object’s color and can be calculated as followed:

h＝tan (b/a) ^(-1)

Instruments settings:

· Color scale is the CIE L*a*b*/L*C*h

· Light source definition: D65 daylight equivalent

· Geometry: Diffuse/8°

· Wavelength: scan 350-1050 nm

· Sample measurement area diameter: 4.826 mm

· Calibration mode: Reflection/Specular-included

The gardenia blues obtained as Example 1 and 2 were dissolved in deionized water to provide a test solution with the maximum absorbance of 0.337 for color test. The test results are summarized in the Table 1 as below compared to two gardenia blue pigments commercially available (GB1 from Qiangjiang Green Sea Treasure Biotechnology Co. Ltd. and GB2 from Zhuhai Golden Land Natural Colors Co., Ltd. ) .

Table 1

	Example 1	Example 2	GB1	GB2
L*	84.50	85.02	88.95	89.22
a*	-7.68	-9.56	-3.44	-3.35
b*	-9.16	-13.55	-10.13	-9.87
C*	11.96	16.59	10.70	10.43
h	230.01	234.80	251.27	251.25

Example 4

The formula of the compounds contained in the gardenia blue according to the present application were identified as below:

15.5 mg of the gardenia blue product obtained as in Example 1 or 2 were dissolved with a mixture of acetonitrile/water (50/50, V/V) and made up to volume in a 10 ml volumetric flask to receive a test solution. 3 μl of the test solution were injected for HPLC-DAD-MS analysis.

The HPLC system consisted of a 1260 quaternary pump, 1290 autosampler, 1290 thermostatted column compartment and 1290 diode array detector (Agilent Technologies, Waldbronn, Germany) . The mass spectrometer used was a Bruker Maxis 4G UHR-QTOF-MS equipped with an Apollo II electrospray ion source and the steering software was Compass QTOF (quadrupole-Time-of-Flight mass spectrometer) control version 3.2. (all Bruker Daltonik GmbH, Bremen, Germany) .

The test solution sample was separated on a YMC Pro C4 3.0 x 150 mm column at a constant flow rate of 0.5 ml/min by applying a ternary mobile phase gradient. Mobile phases were (A) water, (B) water containing 0.1％ methanesulfonic acid and (C) acetonitrile. The time table for the gradient programming was as follows (Table 2) :

Table 2

Time [min]	A [％]	B [％]	C [％]
0.00	10.0	85.0	5.0
5.00	10.0	85.0	5.0
20.00	10.0	0.0	90.0
25.00	2.0	0.0	98.0
35.00	2.0	0.0	98.0
35.20	10.0	85.0	5.0
40.00	10.0	85.0	5.0

The obtained UV-VIS spectra were recorded at 5Hz from 200-600 nm. The ion source parameters of the mass spectrometer were as follows: end plate offset 500 V, capillary voltage 3500 V, nebulizer pressure 3.0 bar, drying gas 9.0 l/min, drying temperature 250℃. The mass spectrometer was scanning in positive MS mode, the scan range was 60-2000 m/z and the spectra rate was 1 Hz. Calibration of the mass axis was done using the reference masses of sodium acetate clusters which were produced in the source after infusion of 20 μl of a 10 mM sodium acetete solution at the beginning of each analysis. In addition a lock mass calibration at m/z 622.0290 was performed using hexakis (2, 2-difluoroethoxy) phosphazene. For this purpose 20 μl of a 1000 μg/ml solution of the phosphazene was added to the lock mass container before analysis. Data analysis software version 4.3 (Bruker Daltonik GmbH, Bremen, Germany) was used for deconvolution and peak detection (see Figure 2) and the Bruker smart formula algorithm was used for the computation of ion formulas from the MS data.

In Figure 2, the extracted peaks at 65 and 80 obtained the ion formula C₃₂H₃₁N₂O₁₂ with m/z 635.4872, the extracted peaks at 55, 69, 71 obtained the ion formula C₃₃H₃₃N₂O₁₂ with m/z 649.2029, and the extracted peaks at 61, 76 obtained the ion formula C₃₄H₃₅N₂O₁₂ with m/z 663.2183.

Example 5

The molecular weight of the gardenia blue according to the present application were determined by GPC as below:

The GPC system (Polymer Standards Service GmbH, Germany) was calibrated using Pullulan/Dextran standards from 180 to 47100 Da. The gardenia blue product obtained as Example 1 or 2 was dissolved in GPC eluent (see Table 3) and filtrated through a 1 μm PTFE filter. The GPC was conducted with the conditions indicated in table 3:

Table 3

The molecular weight distribution was calculated using PSS-WinGPC UniChrom Version 8.2 and the results were indicated in Figure 3.

Example 6

The content of the brighter blue fraction of the gardenia blue pigment according to the present application were determined as below:

HPLC analysis was conducted in Agilent HPLC 1260 with the DAD detector system (Agilent, USA) and 6130 Quadrupole MS system (Agilent, USA) .

5 mg of gardenia blue obtained as Example 1 or 2 was dissolved in 1 mL of water as a test solution. The test solution sample was separated on a Supelco C18, 75 mm x 4.6 mm x 5 um column at a constant flow rate of 1.0 ml/min phase gradient described in Table 4. Mobile phases were (A) 0.01％MSA in Water, (B) 0.01％MSA in ACN. The time table for the gradient programming was as follows (Table 4) :

Table 4

Time [min]	A [％]	B [％]
0.00	95	5
30.00	50	50
35.00	0	100
44.00	0	100
45.00	100	0

The obtained UV-VIS chromatograms were recorded at 600 nm as Figure 4A and Figure 4B.

The gardenia blue peak was integrated dividedly with 0.5 min per portion. Average the UV-Vis absorption spectrum of each portion and identify the portions with λmax≥600 nm. Content of λmax≥600 nm fraction is calculated as:

As shown in Figure 4A and Figure 4B, the contents of the fractions with the maximum

absorption wavelength above 600 nm in the gardenia blue products obtained as the Example 1 and 2 were 23％and 41％ (area％) respectively.

Example 7

10 mg of the gardenia blue obtained as Example 1 or 2 was dissolved into 100 mL of demineralized water at room temperature under agitation. Afterwards, this solution is further diluted until the final solution hardly shows any color shade. The isoelectric point of the solution was determined by a Zetasizer Nano ZS (Malvern, England) in accordance with the guidance provided by the manufacture. Every sample was tested twice and the data were averaged as below (Table 5) :

Table 5

No.	First test	Second test	Average
1	2.02	2.02	2.02
2	1.95	1.99	1.97
3	2.03	2.11	2.07
4	2.14	2.07	2.11
5	2.15	2.13	2.14

Claims

A gardenia blue pigment, which has a color shade h in the range of from 220 to 245, preferably from 230 to 240 at the CIELAB Color scale, and color value “a*” in the range of from -12 to -7, preferably from -11 to -9 at the CIELAB color scale.
The gardenia blue pigment according to claim 1, which derives from a reaction of genipin with an amino acid.
The gardenia blue pigment according to claim 1 or 2, which contains one or more of the compound (s) of formulae (I) - (IV) , a geometric isomer thereof, a tautomer thereof, a salt thereof, or a combination thereof:

Wherein: R is independently from each other H or C_1-7 alkyl, preferably methyl； and R’ is independently from each other selected from the group consisting of C_1-7 alkyl, preferably ethyl, propyl, butyl and pentyl, optionally substituted by a carboxyl, phenyl, imidazolyl or guanidino group.
The gardenia blue pigment according to any one or more of claims 1 to 3, wherein the compound (s) of formulae (I) - (IV) is a compound of formulae (a) - (h) , a geometric isomer thereof, a tautomer thereof, a salt thereof, or a combination thereof:
The gardenia blue pigment according to any one or more of claims 2 to 3, wherein the amino acid is sodium glutamate.
The gardenia blue pigment according to claim 5, which has further the following characterizations:

Molecular weight by GPC: 600-5000 Da, preferably 1000-4000 Da, more preferably 1200-3000 Da；

Content of bright blue fractions: 18-45wt％, preferably 20-40wt％, more preferably 25-35wt％, and/or；

Isoelectric point: 1.5-3.0, preferably 1.8-2.8, and more preferably 1.9-2.2.
A process for the preparation of the gardenia blue pigment according to any one or more of claims 1-6 which comprises the following steps:

a) Treating geniposide with a glycosidase to obtain a hydrolysate； and

b) Extracting the hydrolysate obtained in step a) with a solvent and removing the solvent after the extraction to obtain a product comprising genipin； and

c) Reacting the product comprising genipin obtained in step b) with an aqueous solution of an amino acid and/or a salt thereof to produce the gardenia blue pigment, and preferably dissolving the product comprising genipin in a water-soluble solvent resulting in a solution and using the solution as such.
The process according to claims 7, wherein the glycosidase is cellulase (EC 3.2.1.4) or β-glucosidase (EC 3.2.1.21) or mixture thereof.
The process according to claim 7 or 8, wherein the solvent used in the step b) is diethyl ether, ethyl acetate, butanol, a mixture of butanol with petroleum and/or hexane, or mixtures thereof.
The process according to any one or more of claims 7 to 9, wherein the amino acid used in step c) is selected from the group consisting of glutamate, phenylalanine, histidine, leucine, isoleucine, arginine and mixture thereof.
Use of the gardenia blue pigment according to any one or more of claims 1-6 in food and beverage industries.
Beverages containing a gardenia blue pigment according to any one or more of claims 1-6.
Food containing a gardenia blue pigment according to any one or more of claims 1-6.