WO2020193575A1

WO2020193575A1 - Colouring agent made from components of dates

Info

Publication number: WO2020193575A1
Application number: PCT/EP2020/058209
Authority: WO
Inventors: Fethi HAKKAR; Hedi HAMMOUDA
Original assignee: Mytamar Gmbh
Priority date: 2019-03-25
Filing date: 2020-03-24
Publication date: 2020-10-01
Also published as: CH715658B1

Abstract

The present invention relates to a colouring agent made from components of dates, fruits of the date palm Phoenix dactylifera, the components including polyphenol derivatives, and a method for producing such a colouring agent, the method being characterised in that it comprises the following steps of: a) selecting fruits depending on one or more stages of maturation, Hababouk, Kimri, Besser or Tamar, b) obtaining from the fruits one or more fractions of material containing components which include polyphenols, d) chemical or enzymatic processing of the polyphenols from one or a mixture of the fractions from step b), e) producing a colouring agent comprising components which include polyphenols and polyphenol derivatives.

Description

DESCRIPTION

TITLE: COLORING AGENT BASED ON DATE COMPONENTS

TECHNICAL AREA

The present invention relates to a coloring agent based on components of dates, fruits of the date palm Phoenix dactylifera.

Coloring agents are used in a large number of applications. Indeed, they are found in food, cosmetic, pharmaceutical and nutraceutical compositions, but also in textiles, inks or paints.

A coloring agent, in particular in food, must have certain characteristics such as high coloring power, high stability under specific conditions of use, for example pH or temperature conditions, a precise and stable color range.

Synthetic dyes have most of these characteristics, but suffer from a bad reputation with the general public and the reservations of the authorities in charge of the regulations push manufacturers to seek alternatives of natural origin. Indeed, studies on their impact on the environment or health, in particular on the hyperactivity of children caused by artificial dyes (McCann et al., 2007), lead to question their use.

For the sake of well-being and health, research and industrial innovation are now oriented towards the use of substances of natural origin, in particular in the field of nutrition, cosmetics or the pharmacy.

Several dyes of natural origin are now available.

Among them is the cochineal, a natural dye of animal origin. Also called carminic acid, this bright red colorant comes from the dried bodies of an insect from Central America, Coccus cacti L. It is used in sugary drinks, chewing gum, yoghurts, pastry, custards. , desserts, candies, confectionery. However, some studies have revealed the allergenic side of these compounds.

Madder roots are a source of dyes such as anthraquinones, classified as red dyes. However the extraction of these coloring compounds from madder roots is tedious and has been replaced by the production of synthetic dyes.

Betanin or betacyanin is a red pigment from the group of betacyanins, a subclass of betalains. It is the main colorant in beet juice, but it is also found in prickly pears. This dye has limited stability to heat, light, oxygen and sulfur dioxide. Betanin is also considered a dietary antioxidant. However, 10-14% of humans are unable to break it down.

Anthocyanins represent one of the alternatives for dyes in the color range from light red to purple. Anthocyanins can be found in most plant species. The color of anthocyanins varies from red to purple then to blue and is highly pH dependent. Generally, limitations exist linked to the conditions of use of these dyes, such as the pH and the temperature. Indeed, the maximum pH of use must be limited to 4.5 for the most stable anthocyanins, because beyond that their color degrades or turns blue, which makes their use impossible in products such as yogurt, for example. example. In addition, studies reveal the degradation of anthocyanins under the action of the rise in temperature.

In the range of yellow or orange colorings, we can cite curcumin, derived from the roots of Curcuma longa or even b-carotene extracted from carrots. These dyes of plant origin are used in particular in dairy products. However, these dyes are poorly soluble in aqueous liquid matrices, which limits their use for certain applications.

In the color range varying from light yellow to dark brown, mention will be made of the caramel coloring classified El 50 according to European regulations. It is a liquid or solid, water-soluble food coloring used to color a large number of foods such as flavored non-alcoholic drinks, confectionery, soups, seasonings and beer. It is a complex mixture of compounds obtained by the carefully controlled heat treatment of sugars. This type of caramel is made at higher temperatures (140 - 180 ° C) than aromatic caramel to obtain a stronger coloring character. These dyes are divided into four classes El 50a, El 50b, El 50c and E150d (Tomasik, 1989) according to their manufacturing process and in particular the chemicals used. Concerning the color characteristics of the caramel dyes, the absorbance measurement is carried out at 520 nm for the aromatic caramels on a 100 g / L solution and at 610 nm for the coloring caramels on a 1 g / L solution (Moretton , 2009). Caramel colors E150c and E150d are now suspected of being harmful to health. They would in fact be sources of carcinogenic compounds newly formed during their manufacturing process. Some jurisdictions have already imposed regulations on the use of these types of dyes.

Selecting a good candidate as a coloring agent therefore requires it to meet several criteria depending on the intended application.

Indeed, the non-toxicity of the coloring agent both to health and the environment is an important criterion. The latter should neither be toxic on its own, nor generate toxic neoformed compounds, either during its manufacture, during its use, or during its degradation in the environment. In addition, many traditional techniques for the production of commonly used dyes use solvents which are also toxic, polluting and difficult to neutralize.

The stability of the colorant in its destination product is also an important criterion, in particular in food products which are heat treated and have to be stored. Indeed, the conditions related to pH, acidity, oxidation, temperature used during heat treatment, packaging material, storage time and temperature, are likely to adversely affect to the stability of the coloring agent.

The production cost, linked to the source of raw material, to the solvents or other necessary products, to the materials, and the duration of the production chain are other criteria to be taken into account. The richness of the colors which can be provided by the coloring agent is another appreciable criterion. In fact, the majority of natural sources of dyes available today offer little variation in colors and hues.

Finally, the natural resource, the source of the coloring agent, must not be exhaustible or threatened.

EXPOSURE OF THE INVENTION

The present invention takes into account all of these problems and aims, in general, to provide a new coloring agent of natural origin which does not have the aforementioned drawbacks.

In particular, the aims of the present invention are to provide a coloring agent of natural origin obtained from a plant resource of easy and reproducible culture, which is non-toxic or cytotoxic, which exhibits stability under certain conditions, for example. facing heat treatments, and whose methods of obtaining are rapid, not very complex, inexpensive, even non-polluting, and finally which does not demonstrate toxicity on the environment or health, neither during its production nor during its use, nor during its degradation.

The object of the invention is also to propose an alternative to the dyes known today as harmful, such as caramel colorants El 50, that is to say to provide a coloring agent having values in the CIELAB space equivalent to those of caramel coloring E 150.

Finally, the aim of the invention is to provide a coloring agent making it possible to obtain a wide range of colors, tints and chromaticities, depending on the applications envisaged.

To this end, the invention relates to a coloring agent based on components derived from fruits of the Phoenix dactylifera date palm, said components including polyphenol derivatives.

Polyphenols are antioxidant compounds specific to the plant kingdom, and endowed with various biological activities demonstrated by numerous studies described in the scientific literature. Mention will in particular be made of their antioxidant, anti-inflammatory, antimicrobial, antiviral or even anticancer activities.

Chemical or enzymatic treatments imposed on the polyphenols will lead to the appearance of newly formed compounds, derived from polyphenols, and exhibiting coloring properties.

The two properties of polyphenols, the coloring property on the one hand, and the antioxidant property on the other hand, can be combined to provide products of great interest.

Dates are an interesting source of polyphenols. In addition, a significant part of the production of dates is transformed into derived products, such as jams, juices and syrups. Consequently, large quantities of tissue from dates and fruits not selected for sale are produced as waste which can be recovered on the basis of their high contents of compounds such as polyphenols.

The date palm fruit has three tissues: the stone, the pulp and the skin. It also evolves according to four stages of maturity: Hababouk, Blah (Kimri), Besser (Khalal) and Tamar. The selection of the fruit according to one of these stages, and according to the tissues, allows to obtain different results in terms of polyphenol content and therefore in terms of polyphenol derivatives produced during chemical or enzymatic treatment and responsible of the coloring provided by the coloring agent. Depending on the choice of tissues, the stages of maturity and the processing to generate the polyphenol derivatives, it will be possible to produce dyes with a varied chromatographic range.

Thus, advantageously according to the invention, the components obtained from fruits of the date palm Phoenix dactylifera are obtained from the whole fruit, the stone, the pulp, the skin or a mixture of these tissues, the fruit being selected from the Hababouk, Kimri, Besser, Tamar maturation stage, or a mixture of these stages.

According to a first embodiment of the invention, the components derived from palm fruits are obtained from the whole fresh fruit, without a tissue separation step, and after application of a chemical or enzymatic treatment of the polyphenols contained in this material. vegetable.

This embodiment is very advantageous because it makes it possible to generate directly from the fresh whole fruit polyphenol derivatives conferring the coloring activity.

In addition, as will be demonstrated by the examples which follow, the coloring agent obtained from fresh and whole date fruits can provide a coloring which is equivalent to that of caramel colorants El 50, depending on parameters that are simple to define such as pH. The equivalence is deduced from a simple comparison of the a and b values of the CIELAB system measured for the caramel dyes E150 and for the coloring agent according to the invention.

According to a second embodiment of the invention, the components obtained from palm fruits are obtained from the kernel, the pulp, the skin or one of their mixtures after separation of these tissues from one another and application. a chemical or enzymatic treatment of polyphenols.

This embodiment makes it possible, for example, to treat separately over time on the one hand the harvesting of the fruits and the separation of the tissues and, on the other hand, the preparation of a dye, the tissues being advantageously stored in the form of powders.

The coloring agent obtained from date tissue powders provides a color that changes between yellow with a brown tendency and red with a black tendency, depending on the pH and the concentration in the solution that contains it.

According to this embodiment, it is also possible to obtain a coloring agent having a coloring equivalent to that of caramel colorants El 50.

According to a third embodiment of the invention, the components obtained from palm fruits are obtained from polyphenols extracted, and preferably purified, from the kernel, from the pulp, from the skin of the fruit, or from a mixture of these. tissues, and after application of a chemical or enzymatic treatment of polyphenols.

The purified extract of polyphenols obtained can comprise at least 80% of polyphenolic compounds, by weight relative to the total weight of the purified dry extract, as well as at least 70%, preferably at least 95%, more preferably at least 97 %, even more preferably at least 99% of tannins, by weight relative to the total weight of polyphenols. An efficient extraction and purification method is for example described in Swiss patent application No. 01241/18.

This embodiment is advantageous for the production of dyes which must have high degrees of purity, for example for pharmaceutical, nutraceutical or cosmetic applications.

In this case, the coloring agent obtained from purified extracts of date polyphenols provides a color that varies between yellow and dark red, depending on the pH and the concentration in the solution that contains it.

Dates thus provide a source of polyphenols capable of giving a great variability of dyes, evolving from yellow to red or from yellow to brown. The dyes obtained thus frame a wide range of colors including that of the caramel dye (E150d).

Thus, according to an advantageous embodiment of the invention, the coloring agent has values of a and b in the CIELAB space of between 0 and 100.

More preferably, the coloring agent has values in the CIELAB space such that a is between 5 and 60, more preferably between 9 and 40 or between 10 and 40, and b is between 40 and 90, more preferably between 45 and 90.

According to another embodiment of the invention, the coloring agent comprises at least one compound chosen from caffeoylshikimic acid, chrysoriol, quercetin-hexo-sulfate, quercetin-3-O-rutinoside, quercetin 3- O-glucoside or one of its isomers, isorhamnetin-3-rutinoside, isorhamnetine hexoside, and isorhamnetine.

The dyes according to the invention thus contain a mixture of polyphenols and polyphenol derivatives which give it its dual activity as a dye and as an antioxidant.

In order to satisfy one of the other objects of the present invention, a production process is also proposed, which is intended to be not very complex, little or nontoxic, rapid and inexpensive.

The invention thus also relates to a process for obtaining a coloring agent based on fruit components of the date palm Phoenix dactylifera which is characterized in that it comprises the following steps of:

a) selection of fruits according to one or more of the Hababouk, Kimri, Besser or Tamar ripening stages,

b) obtaining, from the fruits, one or more fractions of matter containing components including polyphenols,

d) chemical or enzymatic treatment of the polyphenols of one or of a mixture of the fractions obtained in step b,

e) obtaining a coloring agent comprising components including polyphenols and polyphenol derivatives.

According to one characteristic of the invention, step (b) comprises obtaining one or more of the following fractions:

(i) a fraction of core tissue, (ii) a fraction of pulp tissue,

(iii) a fraction of skin tissue,

(iv) a whole fruit fraction.

The process according to the invention for obtaining a coloring agent can be implemented on fresh and whole fruits, therefore such as harvested for example, or on one of the three tissues of the fruit, that is to say ie, the core, the pulp or the skin, each of these fractions containing polyphenols capable of being treated chemically or enzymatically to generate the polyphenol derivatives.

It is also possible, from one or more of fractions (i), (ii) and (iii) to obtain, prior to the implementation of step (d) of chemical or enzymatic treatment, a purified polyphenol extract.

Thus, according to a specific embodiment of the invention, the method comprises a step (c) of obtaining a fraction containing an extract of polyphenols (v) from fractions (i), (ii), (iii ) and, or, (iv).

Such a process for extracting and purifying polyphenols essentially comprises the following steps:

obtaining and preparing the plant material,

solid-liquid extraction using a solvent, preferably polar, and recovery of an extract containing polyphenolic compounds,

purification of the polyphenolic compounds from the extract by a solid phase chromatography method, for example in reverse phase or by ion exchange,

recovery of a purified extract concentrated in polyphenolic compounds.

Step (d) of chemical or enzymatic treatment to generate the polyphenol derivatives can be carried out in different ways.

According to a first embodiment of the invention, step (d) consists in carrying out an oxidation, preferably in a basic medium.

As the examples which follow will demonstrate, this embodiment is very advantageous. It makes it possible, from whole date fruits or tissue powders, to obtain easily and in an uncomplicated way, a coloring agent whose L * a * b, C and h values of the CIELAB system are equivalent to those measured for El 50 caramel colors.

More generally, the oxidation carried out on the phenolic components allows to obtain dyes in a range of colors ranging from yellow to dark brown, passing through shades of mahogany red.

According to a second embodiment of the invention, step (d) consists in carrying out hydrolysis, preferably in an acidic medium.

The acid hydrolysis carried out on the phenolic components allows to obtain dyes in a range of red to brown colors.

According to a third embodiment of the invention, step (d) consists in carrying out an enzymatic oxidation in the presence of an activity of a polyphenoloxidase enzyme. According to this method, dyes in the colors light beige to brown are generated.

Thus, the method for preparing a coloring agent according to the invention is simple to implement and requires only a few steps.

According to a specific embodiment of the invention, the method comprises a step (f) of concentration of the polyphenols and polyphenol derivatives in the coloring agents obtained in step (e).

The concentration step can be a purification by a solid phase chromatography method of the polyphenols and polyphenol derivatives contained in the coloring agents obtained in step (e).

The purification step concentrates the polyphenols and polyphenol derivatives in the coloring agent. In the examples which follow, we will see that for two coloring agents obtained according to a production process which differs only by the addition or not of this step, the measured values L * a * b, C and h of the CIELAB system vary.

Advantageously, according to the invention, the method comprises adjusting the concentrations and adjusting the pH of the coloring agents to predetermined values.

These adjustments will depend on the color imparted by the dye.

The invention also relates to the use of fruits of the Phoenix dactylifera date palm as coloring agents or as sources of coloring agents.

The invention also relates to the use of components derived from fruits of the Phoenix dactylifera date palm, said components including polyphenol derivatives as coloring agents.

The invention finally relates to a composition comprising a coloring agent as defined above or obtained by a process as defined above.

The compositions targeted in the context of the invention are all the compositions requiring a coloring agent, such as food, cosmetic, pharmaceutical, nutraceutical, dyeing, ink or paint, liquid, solid or gel form compositions, without that this list be considered exhaustive.

DETAILED EXPOSURE OF EMBODIMENTS

The characteristics of the invention mentioned above, as well as others, will emerge more clearly on reading the following description of exemplary embodiments, said description being given in relation to the accompanying drawings, among which:

The [Fig. 1] is an LC-ESI-MS chromatogram of polyphenol derivatives obtained from date tissue powders, The [Fig. 2] is an LC-ESI-MS chromatogram of polyphenol derivatives obtained from whole date fruits.

Examples

Plant material used:

Different varieties of dates have been used. The examples which follow illustrate the results obtained from the variety deglet nour.

The fruits were calibrated manually.

In Examples 4, 7 and 8, the fruits are kept whole, without tissue separation.

In Examples 3 and 6, the skin, the pulp and the core are separated from each other and then reduced to powders.

In Examples 2 and 5, extraction and purification of polyphenols was performed from tissue powders.

Materials, chromatographic solvents and chemicals

• Laboratory equipment and glassware

Lyophilizer (LEYBOLD, model LYOVAC GT 2). HPLC (Waters, Milford, MA, USA). LC-ESI-MS LCQ Deçà ion trap mass spectrometer (ThermoFinnigan San José, CA, USA). Analytical balance (Adventurer, USA). Spectrophotometer: UV-visible (SHIMADZU 1605). Rotary Evaporator: LABOROTA 4000, Speed rpm.

Electric plate. Ultrasonic bath (BRANSON 2200, USA). Water bath (ISOTEMP, Fisher scientific, U. S. A). Vortex agitator model Gilson GVlab. LmL injection vials (WISP 96 interchrom). Quartz cell: HELLMA, optical path 1 = 1 cm. Microplates Elisa 96 wells Greiner Bio-One. Sep pack Cl columns 8.

• Chromatographic solvents

The solvents used for the chromatographic analysis are HPLC grade, acetonitrile, methanol, formic acid and acetic acid (Fischer scientific, Loughborough, UK). Ultrapure water comes from a MilliQ system (Millipore SA, Molsheim, France).

• Chemical products

Ethanol, methanol and anhydrous methanol (Prolabo). Sulfuric acid (3M H2S04), sodium hydroxide (3M NaOH), glacial acetic acid.

• Standards (reference coloring products): 50% hydro carmine E120 supplied by the company Colorey (Lozanne), Black carrot type red (2.10592) and caramel E150d supplied by the company Dohler (Germany).

Buffers

Phosphate buffer (75 mM, pH: 7.4). CIE l * a * b System of Values:

The L * a * b * CIE 1976 (named CIELAB) color space is a color space commonly used for color characterization and well known to those skilled in the art.

The L * component is lightness, which ranges from 0 (black) to 100 (white).

The a * component represents the range from the red (positive value) green (negative) axis through the gray (0).

The component b * represents the range of the axis yellow (positive value) blue (negative) passing through gray (0).

La Chroma (C): points with the same chroma are located on a cylinder centered on the luminance axis that we call iso-chroma cylinder. The projections of these points on the chrominance plane define the iso-chroma circle.

The hue (H): the hues along the chromatic circle are displaced, with two orthogonal axes green-red (axis "a") and blue-yellow (axis "b"); the chroma, which replaces the saturation, is distributed very unevenly according to the hues: the blues with strong chroma are very dark, while the yellows with strong chroma are very light.

Example 1: Determination of the l * a * b c and h values of carmine red, black carrot and caramel dyes as a function of the pH (Controls)

Carmine red (lg / L): the l * a * b, c and h values of this standard dye diluted to lg / L show that it is redder towards the acidic pH values, and becomes black towards the pH values basic (Table 1). L values decrease from 37.56 to 15.57 with increasing pH from 4 to 12.

Table 1: l * a * b, c and h values of standard carmine red dye at lg / L. Absorbance measured at 520 nm after multiplying the dilution factors.

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Black carrot (1 g / L): this dye is red to pink at acidic pH between 4 and 6 but blue to blue black at basic pH (Table 2). Table 2: l * a * b values of standard black carrot dye at lg / L after lyophilization. Absorbance measured at 520 nm after multiplying the dilution factors.

Caramel E 150d (lg / L): The preparation of caramel as a standard colorant has been carried out according to TUE regulations and according to the manufacturing box. The solution is prepared at 1 g / L and the absorbance reading is taken at 610 nm. In order to study the stability of this standard during heat treatment and as a function of time, an incubation test at 80 ° C for 3 min was carried out.

Table 3: l * a * b values of caramel at lg / L and pH 3.5. Absorbance measured at 520 nm and 610 nm. t0: time at 0min of incubation; t3: incubation time at 3min

Example 2: obtaining dyes by chemical oxidation reaction of polyphenols previously extracted from date fruit tissue.

Example 2.1: process for obtaining coloring agents without a purification step for polyphenol derivatives

The method of obtaining dyes is as follows:

Steps (a): The fruits are harvested.

Step (b): The core, skin and pulp tissues are separated from each other to constitute independent fractions of plant material sources of components including polyphenols, then crushed and dried.

Step (c): a step of extraction and purification of the phenolic compounds is carried out here from each fraction of kernel, skin and pulp.

10 g of powder are brought into contact with 80 mL of hexane for 15 min., With stirring, then filtered, during a first extraction. The residue obtained is subjected to a second extraction by bringing it into contact with 80 ml of hexane for 15 min., With stirring and then filtered. The residue obtained is recovered and then dried under nitrogen for 1 h.

The dry residue from the extraction is brought into contact with 30 ml of polar solvent for 15 min., With stirring, then filtered, during a first extraction. The polar solvent is composed of ethanol / water / acetic acid in a ratio of 50 to 80 of ethanol: 1 to 10 of acetic acid: qs in water (V / V / V) (hydro-alcoholic solvent). For example, a ratio of 80: 19: 1 (Vol. Ethanol / Vol. Water / Vol. Acetic acid /) is suitable. Alternatively, a solvent composed of acetone / water / acetic acid in a ratio of 50 to 80 acetone: 1 to 10 acetic acid: qs water (V / V / V) (hydro-acetone solvent). For example, a ratio of 50: 49: 1 can be used.

The extract obtained, named El, is stored, and the residue obtained is subjected to a second extraction by bringing into contact with 30 mL of the same solvent for 15 min., With stirring and then filtered. The extract obtained, named E2, is stored and the residue obtained is recovered and subjected to a third extraction by contacting with 30 mL of the same solvent for 15 min., With stirring and then filtered. The extract obtained, named E3, is kept. The extracts E1, E2 and E3 are pooled. Filtration is carried out in order to recover the supernatant and then the phenolic compounds in the extract are concentrated by evaporation under vacuum.

The preparation of the extract by filtration just before concentration is an important step in preventing clogging of the separation system.

Various tests show that the purification protocol which follows cannot be applicable without carrying out these two stages of evaporation and filtration.

Purification: The extracts are centrifuged at 4500 rev / min for 10 min at 4 ° C in order to recover the supernatant which will be used for the Sep Pack 08 cartridge (marketed by the company Waters) for carrying out an extraction in phase solid (SPE).

SPE cartridges containing 5 g of gel 08 (Sep Pack 08 cartridge sold by the company Waters) are conditioned in ethanol (20 mL) and then equilibrated with acidified water (40 mL) before the aqueous extracts of each sample (40 ml), previously centrifuged at 4500 rpm for 10 min at 4 ° C. A rinse is performed by 40 mL of acidified water (with 2% acetic acid), then the retained fraction is eluted with 40 mL of ethanol / water / acetic acid mixture (50 to 80 ethanol: 1 to 50 acetic acid: qs water), preferably 50 : 49: 1 (V / V / V). The column is then washed with 30 ml of ethanol to remove the very hydrophobic polyphenols which can still be fixed.

Alternatively, the purification can be carried out using an FPX 66 ion exchange column (Rohm & Haas France SAS).

Concentration of the extracts is achieved by evaporation of the solvent and a purified fraction of polyphenols is obtained. The fraction is then dried.

Step (d): a chemical oxidation treatment, in a strong base medium using 3M sodium hydroxide, is carried out so as to generate polyphenol derivatives for each fraction of purified extracts of polyphenols obtained from skin tissues (Pe), pulp (Pu) and kernel (N).

In a 10ml glass tube, 5mg of phenolic extract and 5ml of NaOH (3M) are placed. The solutions are stirred briefly and then placed in a water bath at 90 ° C. for 2 h.

Step (e): coloring agents comprising components including polyphenol derivatives are thus obtained.

The concentrations are adjusted to lg / L, with water, and the pH is stabilized at values of 6, 8, 9 and 12.

The effects of variations in pH and incubation time at 80 ° C have been realized. The determination of the l * a * b values was carried out after reading the absorbance in scanning between 380 and 720 nm. The results are shown in Tables 4 to 6.

Table 4: l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the nuclei in stage 2 of maturation (N2) (1 g / L). Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0 min of incubation; t3: incubation time at 3min.

Table (5): l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the skins in stage 2 (Pe2) (1 g / L). Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table (6): l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the pulps in stage 2 (Pe) (1 g / L). Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

The results show that the products obtained are stable after incubation at 80 ° C. The effect of varying pH between 3 and 12 is very significant, a strong variability was observed on the l * a * b values.

The coloring agents obtained exhibit a tint located in the color red.

Example 2.2: process for obtaining coloring agents with a solid phase purification step of polyphenol derivatives

The method for obtaining the dyes is carried out as in Example 2.1 except that it comprises a step of (f) purification of the dyes after step (e) by a solid phase chromatography method.

This purification step removes the salts formed and other uncolored compounds.

The solutions thus obtained after oxidation are stabilized at neutral pH. After stabilization of the pH, centrifugation at 4500 rev / min for 10 min at 4 ° C. is carried out in order to recover the supernatant.

SPE cartridges containing 5 g of gel 08 (Sep Pack 08 cartridge marketed by the Waters company) are conditioned in ethanol (20 mL) then equilibrated with acidified water (40 mL) before the aqueous extracts are deposited ( 40 ml) centrifuged from each sample. Rinsing is carried out with 40 mL of acidified water (at 2% acetic acid), then the retained fraction is eluted with 40 mL of ethanol / water / acetic acid mixture (50: 49: 1; V / V / V ). The column is then washed with 30 mL of ethanol to remove the very hydrophobic polyphenols which may still be fixed.

Evaporation of the extracts obtained after purification is carried out at a temperature of 48 ° C. The evaporation step is stopped when the volume reaches approximately half. Suspensions aqueous obtained are frozen at -80 ° C then lyophilized for 3 days to obtain dry powders.

The concentrations are adjusted to lg / L or 3 g / L with water and the pH is stabilized at values of 4, 6, 8, 9 and 12.

Coloring agents comprising components including purified and concentrated polyphenol derivatives are thus obtained.

The effects of variations in pH and incubation time at 80 ° C have been realized. The determination of the l * a * b values was carried out after reading the absorbance in scanning between 380 and 720 nm. The results are shown in Tables 7 to 12.

Table (7): l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the nuclei in stage 2 (N2) and purification by Cl 8. Solution at 1g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table (8): l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the skins in stage 2 (Pe2) and purification by 08. Solution at lg / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table (9): l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the pulps in stage 2 (Pu2) and purification by Cl 8. Solution at lg / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table (10): l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the nuclei and purification by Cl 8. Solution at 3 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table (11): l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the skins and purification by Cl 8. Solution at 3 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table (12): l * a * b values of the products obtained after auto-oxidation of the phenolic extracts of the pulps and purification by Cl 8. Solution at 3 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

The values of the l * a * b system and the absorbance at 520 nm vary depending on the concentration of the solutions and the pH of the solution to be prepared. Colors vary between yellow and dark red. A decrease in L values was observed from 1 to 3 g / L.

The color closest to that of the caramel colorant is obtained for the 3g / L solutions at acidic pHs between 4 and 6. At more basic pHs, the coloring solutions obtained are very close to the colorants located between red-brown and dark red.

Example 3: obtaining dyes by chemical oxidation reaction of polyphenols contained in date fruit tissue powders.

Example 3.1: process for obtaining coloring agents without a purification step for polyphenol derivatives

The method for obtaining the dyes takes place in steps (a) and (b) as for example 2.1 but without implementing step (c) of extracting the polyphenols prior to their chemical treatment of step (d).

In step (e): the concentrations are adjusted to 4 g / L and 6 g / L with water and the pH is stabilized at values of 3, 4, 6, 8, 9 and 12.

Coloring agents comprising components including derivatives of polyphenols are thus obtained.

The effects of variations in pH and incubation time at 80 ° C have been realized. The determination of the l * a * b values was carried out after reading the scanning absorbance between 380 and 720 nm. The results are shown in Tables 13 to 18.

Table 13: l * a * b values of the products obtained after auto-oxidation reaction of date powders from the stones (N) at 4 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table 14: l * a * b values of the products obtained after auto-oxidation of date powders from the stones (N) at 6 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table 15: l * a * b values of the products obtained after auto-oxidation of date powders on the skins (Pe) at 4 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table 16: l * a * b values of the products obtained after auto-oxidation of date powders on the skins (Pe) at 6 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table 17: l * a * b values of the products obtained after auto-oxidation of date pulp powders (Pu) at 4 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table 18: l * a * b values of the products obtained after auto-oxidation of date pulp powders (Pu) at 6 g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

The results show that the products obtained are stable after incubation at 80 ° C. Indeed, the l * a * b * c, h values do not vary significantly between a zero incubation time and an incubation time of 3 minutes, for the same pH.

The effect of varying the pH between 6 and 12 on the product obtained is very significant, a strong variability was observed on the l * a * b values. By comparison between the solutions at different pH, the measurements show that in the case of the dyes obtained from powder of cores the values l * a * b * C and h are lower in the case of more acidic solutions. On the other hand, the optical densities at 520 and 610 nm are higher in the case of more basic solutions.

In this example, the coloring agents obtained exhibit colors in a range from yellow to dark brown.

Example 3.2: process for obtaining coloring agents including a stage of purification of polyphenol derivatives

The method of obtaining the coloring agents is carried out as in Example 3.1 but including a step (f) of purification of the polyphenol derivatives of the dyes obtained in step (e).

Step (f): This step proceeds as in Example 2.2. Coloring agent solutions are prepared at 0.45g / L with water for measurements of l * a * b values. This concentration is optimized by comparison with the caramel coloring agent E 150 d at lg / L and at pH 3.5 of Example 1.

The effects of variations in pH and incubation time at 80 ° C have been realized. The determination of the l * a * b values was carried out after reading the absorbance in scanning between 380 and 720 nm. The results are shown in Tables 19 to 21.

Table 19: l * a * b values of the products obtained after auto-oxidation of date powders on the skins (Pe) at 6 g / L and purification by Cl 8. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table 20: l * a * b values of the products obtained after auto-oxidation of the date powders from the nuclei (N) at 6 g / L and purification by Cl 8. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. tO: time at 0min; t3: time at 3min.

Table 21: l * a * b values of the products obtained after auto-oxidation of the date powders of the pulps at 6 g / L and purification by Cl 8. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication dilution factors. tO: time at 0min; t3: time at 3min.

20

The results show that the l * a * b values of the coloring agents obtained from the skin and pulp tissues purified and adjusted to pH 3 are very close to those of caramel E 150d (ex.l). Incubation for 3 min at 80 ° C has no significant effect on pH stability or l * a * b and C and h values.

Increasing the pH from 3 to 12 changes the color from a yellow brown to a black red. By comparison between the tissues studied, the nuclei have low OD compared to the skins or the pulps. Therefore, the consumable part composed of the skins and pulps is the most suitable for preparing powdered colorings with characteristics very close to the characteristics of caramel color. Example 3.3: ORAC index value of the dyes obtained from date fruit tissue powders in Example 3.2.

The ORAC index makes it possible to evaluate the antioxidant capacity of a component. It is calculated by means of a test which bears the same name, and which is well known to those skilled in the art.

The ORAC number is expressed in pmole Trolox / g of sample, that is to say in micromole of Trolox equivalent per gram of sample. The larger the value, the more antioxidant the sample.

The results show that the ORAC values found for dyes obtained from date powders are very high and reach 10195 pmole Trolox / g.

The dyes obtained thus exhibit a dual activity: a dye activity and an antioxidant activity. This dual activity is proving to be extremely interesting in the formulation of numerous products, such as food, pharmaceutical and cosmetic products.

Example 4: Obtaining coloring agents by oxidation of whole fresh fruits of dates.

Example 4.1: Obtaining Dyes Without a Step for Purifying Polyphenol Derivatives

The method of obtaining dyes is as follows:

Steps (a) and (b): The fruits are selected and harvested and constitute source fractions of components including polyphenols.

Step (d): a chemical oxidation treatment of the fruit polyphenols, in a strong base medium using 3M sodium hydroxide, is carried out so as to generate polyphenol derivatives. To do this, solutions containing 500 g of fresh material per liter of sodium hydroxide are prepared and then incubated for 1 hour at 80 ° C.

Step (e): the solutions are diluted to 1/80 and coloring agents comprising components including polyphenol derivatives are thus obtained.

The pH is stabilized at values of 3, 3.5, 5, 5, 6, 7, 8 and 12.

The effects of variations in pH and incubation time at 80 ° C have been realized. The results are shown in Table 22.

Table 22: l * a * b values of the solution obtained after auto-oxidation of date fruits at 500g / L and dilution at 1/80. Absorbance measured at 520 nm and 610 nm. tO: time at 0min; t3: time at 3min.

Depending on the pH, the color varies considerably between a caramel-like color at acidic pH and brownish red at basic pH.

When the pH is set between 3 and 4, the absorbance measured as well as the l * a * b, C and h values are very close to those measured for the caramel color E150d (example 1).

At a pH value of 3.5, the colorant obtained from fresh fruit according to the invention merges with the colorant E 150d. The absorbance spectra of the E150d dye and of the dye according to Example 4.1 at pH 3.5 are comparable, as shown in Fig. 3.

The dye thus obtained can therefore be used as a substitute for the dye E150d.

Example 4.2: process for obtaining coloring agents including a step of purification of polyphenol derivatives

The method for obtaining the dyes is carried out as for Example 4.1 but includes a step (f) of purification and concentration of the polyphenol derivatives of the coloring agents obtained in step (e). Tl

Step (f): To do this, following the incubation of step (d), the solutions are filtered and passed through a Sep pack 08 purification cartridge, as in Example 2.2. Concentration is then carried out in order to evaporate the alcohol. The evaporated solutions are freeze-dried for three days.

Solutions of coloring agents are then prepared at 0.35 g / L for the measurements of the l * a * b values. This concentration is optimized by comparison with the caramel coloring agent E 150 d at lg / L and at pH 3.5 of Example 1.

The effects of variations in pH and incubation time at 80 ° C were realized. The results are shown in Table 23.

Table 23: l * a * b values of the solution obtained after auto-oxidation of date fruits at 500g / L and purification by 08. Concentration at 0.35g / L. Absorbance measured at 520 nm and 610 nm, El% determined at 610 nm and after multiplication of the dilution factors. t0: time at Omin; t3: time at 3min

When the pH is between 3 and 6, the absorbance measured as well as the l * a * b, C and h values are very close to those measured for the caramel color E150d (example 1). The dye thus obtained can therefore be used as a substitute for the dye E150d.

Example 4.3: ORAC index value of the colorings obtained from fresh date fruits in Example 4.2.

The results show that the ORAC values found for dyes in Example 4.2 are very high and reach 6615 pmole Trolox / g.

Example 4.4: another example of obtaining a brown coloring agent

In this example, two brown dyes are obtained, one from date fruit including the stones, the other from date fruit without the stones.

The method of obtaining dyes is as follows:

Steps (a) and (b): 1 kilogram of whole date fruit has been finely ground.

Step (d): a chemical oxidation treatment of the fruit polyphenols, in a strong base medium using 3M sodium hydroxide, is carried out so as to generate polyphenol derivatives.

To do this, after grinding, 2 liters of sodium hydroxide (3M NaOH) were added. After incubation for 45 min at 80 ° C, the pH correction is made to 3.5 on the one hand, and to 7 on the other hand. Centrifugation is carried out for 3 min at 4000 rpm and after filtration through membranes at 5 μm, the extracts are passed to a step of cooling to 10 ° C. in order to precipitate the part which is still insoluble. An enzymatic treatment step with pectinase enzymes from Aspergillus niger and Xylanase Trichodermareesei was carried out in order to remove all traces of precipitation. The dye obtained (step e) is concentrated by concentration in a rotary evaporator (water temperature 99 ° C). The concentration ends at 85% volume step f).

The same work was done with fruit without pits. In this case, 1 kilogram of dates provides 885 grams of material fraction without pits.

The coloring agents obtained are in liquid form of the order of 1.2 L.

Reading the absorbance:

Preparation of diluted solutions and passage to UV.

Wavelength scan between 380 and 720 nm.

Reading at a wavelength of 610nm to compare with the caramel E150d at lg / L (example 1) The results are shown in Table 24.

Table 24

The measurements of the l * a * b values are shown in Table 25

Table 25

In Tables 24 and 25, the expression "with pits" denotes the colors obtained from fruits including the pits. The term "pits-free" refers to colors obtained from fruits not including pits.

At a pH value of 3.5 or 7, the dyes merge with the dye E 150d (example 1). The absorbance spectra of the dye E150d and the dyes according to Example 4.4 at pH 3.5 and 7 are comparable.

The dye thus obtained can therefore be used as a substitute for the dye E150d.

Example 5: Obtaining dyes by acid hydrolysis of polyphenols previously extracted from the tissues of date fruits.

Example 5.1: acid hydrolysis with sulfuric acid

The method for obtaining the dyes is carried out as in Example 2.1 concerning steps (a) to (c).

Step (d): The chemical method of treating the polyphenols to generate polyphenol derivatives providing the dye activity is, on the other hand, carried out by hydrolysis in an acidic medium. For each fraction containing a phenolic extract purified from the skins (Pe), pulps (Pu) and stones (N) of dates, an acid hydrolysis is carried out using sulfuric acid (3M). In a 10ml glass tube, 5mg of fraction and 5ml of H2S04 (3M) are placed. The tubes are closed with a screw cap. The solutions are stirred briefly and then placed in a water bath at 90 ° C. for 2 h. Once the heating step is complete, the pH of each treated fraction is stabilized at values of 2, 3, 4, 5, 6 and 7 and the concentrations are adjusted to lg / L. After filtration, the absorbance of the solutions is read in cuvettes. The spectrophotometer scan is set between 350nm and 800nm. The l * a * b, c and h values are determined using a macro. The effects of variations in pH and incubation time at 80 ° C. have been realized. The results are shown in Tables 24-28.

Table 26: l * a * b values of the products obtained after acid hydrolysis of the phenolic extracts of the date stones at lg / L. tO: time at 0min; t3: time at 3min.

Table 27: l * a * b values of the products obtained after acid hydrolysis of the phenolic extracts of the date skins at lg / L. tO: time at 0min; t3: time at 3min.

Table 28: l * a * b values of the products obtained after acid hydrolysis of the phenolic extracts of the date pulps at lg / L. tO: time at 0min; t3: time at 3min.

The results show that the dyes obtained are stable after incubation at 80 ° C. for 3 min. The study shows that the variation in pH between 3 and 6 on the product obtained gives rise to low variability in the l * a * b values.

This method produced brown dyes.

Example 5.2: acid hydrolysis using a mixture of ethanol and hydrochloric acid

The method for obtaining the dyes is carried out as in Example 5.1, on fractions containing extracts of polyphenols at 1g / L., Except that the hydrolysis reaction is carried out using an ethanol mixture. and hydrochloric acid (ratio 95: 5).

The dye solutions are centrifuged following the heating step at 4500 rpm, and diluted to ^1/25 in water.

The pH of each solution is stabilized at values of 2, 3, 4, 5 and 9. The absorbance reading of the solutions is taken in cuvettes. The spectrophotometer scan is set between 350nm and 800nm. The l * a * b, c and h values are determined using a macro.

The effects of variations in pH and incubation time at 80 ° C have been realized. The results are shown in Tables 29 to 31.

Table 29: l * a * b values of the products obtained after acid hydrolysis of the phenolic extracts of dates (Nuclei) at lg / L with 1/25 dilution. tO: time at 0min; t3: time at 3min.

Table 30: l * a * b values of the products obtained after acid hydrolysis of the phenolic extracts of dates (skins) at lg / L with 1/25 dilution. tO: time at 0min; t3: time at 3min.

Table 31: l * a * b values of the products obtained after acid hydrolysis of the phenolic extracts of dates (pulps) at lg / L with dilution 1/25. tO: time at 0min; t3: time at 3min.

The hydrolysis carried out makes it possible to produce red dyes in an acidic medium. The tannins in an acidic and heated alcoholic medium are transformed into cyanidins, compounds of the anthocyanidol family, which are red in color in an acidic medium.

The Carmine Red and Black Carrot standards of Example 1 and the natural dyes obtained from the phenolic extracts obtained from the stones, skins and pulps of dates have very similar behavior in an acid environment. They evolve, between acidic and basic pH, from red colors to brown-black colors through purple.

The dyes obtained from the phenolic extracts are characterized by very high El% compared to the values found in the other dyes according to the invention and the reference Black Carrot dye.

Example 6 Obtaining dyes by acid hydrolysis of polyphenols contained in date fruit tissue powders.

Example 6.1: acid hydrolysis with sulfuric acid

The method for obtaining the dyes takes place in steps (a) and (b) as for Example 3.1 except that step (d) is performed as by chemical treatment of acid hydrolysis using sulfuric acid.

In step (e): the concentrations are adjusted to 6 g / L with water and the pH is stabilized at values of 3, 4, 5 and 6.

The effects of variations in pH and incubation time at 80 ° C have been realized. The determination of the l * a * b values was carried out after reading absorbance in scanning between 380 and 720 nm The results are shown in Tables 32 to 34.

Table 32: l * a * b values of the products obtained after acid hydrolysis of date powders from the stones at 6 g / L. tO: time at 0min; t3: time at 3min.

Table 33: l * a * b values of the products obtained after acid hydrolysis of date powders from the skins at 6 g / L. tO: time at 0min; t3: time at 3min.

Table 34: l * a * b values of the products obtained after acid hydrolysis of the date powders of the pulps at 6 g / L. tO: time at 0min; t3: time at 3min.

Example 6.2: acid hydrolysis using a mixture of ethanol and hydrochloric acid.

The method for obtaining the dyes is carried out as in Example 6.1, except that step (d) is carried out as by chemical treatment of acid hydrolysis of a mixture of ethanol and hydrochloric acid and on powder fractions. of skins and pulps mixed in a single fraction (PC Powder).

The dye solutions are concentrated to 1 g / 50 mL. The pH of each solution is stabilized at values of 2, 3, 4, 5 and 9. The absorbance reading of the solutions is taken in cuvettes. The spectrophotometer scan is set between 350nm and 800nm. The l * a * b, c and h values are determined using a macro.

The effects of variations in pH and incubation time at 80 ° C have been realized. The results are shown in Table 35.

Table 35: l * a * b values of the products obtained after acid hydrolysis of date powders (PC: consumable part, ie tissue, skin and pulp mixed) at lg / 50 mL with 1/50 dilution. tO: time at 0min; t3: time at 3min.

It is observed that the colors vary between red at acidic pH and green-black at basic pH, passing through brown at pH 5-6.

Example 6.3: acid hydrolysis using a mixture of ethanol and hydrochloric acid previously boiled.

The chemical method of treating polyphenols is carried out by hydrolysis in an acidic medium using an acidified and hot (boiled) alcoholic solvent.

Step (d) proceeds as follows: a quantity of the lyophilized tissue powder is dispersed in a volume of ethanol (96%) acidified with hydrochloric acid (EtOH (96%) + 1% HCl) (V / P) previously brought to the boil, at a concentration of 120 g / L. After stirring, the mixtures are immediately cooled and placed at 4 ° C overnight. After maceration, the mixture is filtered.

Step (e): once the filtration step is complete, centrifugation is carried out for 10 min at 4500 rev / min. Dilution operations at 1/50 are carried out on the dyes obtained, then the pH of each solution is stabilized at values of 2, 3, 4, 5, 7 and 9. The absorbance reading of the solutions is taken in cuvettes. The spectrophotometer scan is set between 350nm and 800nm. The l * a * b, c and h values are determined using a macro. The effects of variations in pH and incubation time at 80 ° C. have been realized. The results for a dye obtained from a core tissue fraction are shown in Table 36.

Table 36: l * a * b values of the products obtained after acid hydrolysis of date stone powders at 120 g / L with 1/50 dilution. tO: time at 0min; t3: time at 3min.

The results show that the products obtained are stable after incubation at 80 ° C. for 3 min. The effect of varying the pH between 2 and 9 on the product obtained is significant and makes it possible to obtain dyes of various colors. The colors turn between red in an acidic medium and brown-black in a basic medium.

Example 7: Obtaining dyes by acid hydrolysis of polyphenols from whole fresh fruit of dates.

Steps (a) and (b) proceed as for Example 4.1.

Step (d): on each fraction of fresh fruit (400 g / L of fresh material) an acid hydrolysis is carried out using a mixture of ethanol and hydrochloric acid (95: 5). The solutions are stirred briefly and then placed in a water bath at 80 ° C. for 30 min. Once the heating step is complete, centrifugation is carried out for 10 min at 4500 rpm. Dilution operations at 1/25 are carried out on the dyes obtained and finally the pH of each solution is stabilized at values of 2, 3, 4, 5 and 9. The absorbance reading of the solutions is taken in cuvettes. The spectrophotometer scan is set between 350nm and 800nm. The l * a * b, c and h values are determined using a macro.

The results are shown in Table 37.

Table 37: l * a * b values of the products obtained after acid hydrolysis of fresh date fruits at 400g / L with 1/25 dilution. tO: time at 0min; t3: time at 3min.

The effect of varying the pH between 2 and 9 on the product obtained is significant and makes it possible to obtain dyes of various colors. The colors turn between red in an acidic medium and brown-black in a basic medium.

Example 8: obtaining a red dye from date stones by acid hydrolysis

The chemical method of treating polyphenols is carried out by hydrolysis in an acidic medium using an acidified alcoholic solvent and proceeds as follows:

Steps (a) and (b): The fruits are selected and harvested, the stones are separated and constitute source fractions of components including polyphenols Step (d): Acid hydrolysis was carried out by using a solution of 4 liters of ethanol with 1% HCl per 1 kg of date stones.

One kilogram of stones was therefore finely ground. After grinding, one liter of hexane was used in order to release the oily part (lipids) from the matrix. After delipidation, the solution treated with hexane was then hydrolyzed by the use of a mixture of water / boiling ethanol. To do this, 4 liters of 96% ethanol and 1% HCl, previously brought to the boil, were added. After 8-10 hours of incubation at 4 ° C, the solution was filtered through sintered glass. The filtrate is then evaporated to remove the ethanol which was used for the extraction. The solution after filtration shows a red appearance.

The pH of the solution is close to 1.

From this concentrated solution (evaporated solution, phi) two solutions were produced by adjusting the ph to PH 3 and 6 with concentrated sodium hydroxide. The two solutions were then filtered before the various analyzes:

Solution 1: pH 3

Solution 2: pH 6

Absorbance reading:

- Preparation of diluted solutions and passage to UV.

- Wave logger scan between 380 and 720 nm.

Reading at a wavelength of 520nm to compare with carmine red and black carrot (example 1).

The results of the L * a * b measurements are shown in Table 38.

Table 38

Red dyes were obtained.

Example 9: obtaining dyes by enzymatic treatment of polyphenols

The goal here is to produce a dye using the enzyme polyphenol oxidase (PPO) contained in date fruits or purified from fruits. Indeed, during the loss of cell integrity, the oxidation of polyphenols takes place mainly under the enzymatic action of polyphenoloxidase (PPO). This enzyme, located in the plastid structures, can, in the presence of atmospheric oxygen, catalyze the oxidation of these vacuolar polyphenolic substrates. The oxidation reaction is thus effective as soon as the fruit is cut and crushed.

Example 9.1: enzymatic treatment with an enzyme contained in crushed fresh fruits.

This example describes the result of the oxidation of polyphenols by the PPO contained in the fruits and released during the crushing of these fruits.

This example requires obtaining a fraction of fresh fruit from dates as in Example 4.1, and a fraction of polyphenols extracted and purified from date fruit tissues, as in Example 2.1.

Step (d): 50 g of the edible part of the fruit were ground in 100 mL of phosphate buffer at pH 6.8. Two solutions were prepared, a control solution with PPO inhibited by sodium fluoride (NaF) solution (10g / L) and a solution with PPO capable of producing an oxidation reaction of polyphenols.

To the solutions prepared, phenolic extracts purified from date tissues (skin and pupa) were added at a concentration of 20mg / 100mL.

After stirring for 4 hours and incubation at a temperature of 28 ° C, the absorbance reading is taken with a scan between 350 and 800 nm. The l * a * b * c and h values were determined in order to compare between the prepared solutions and the caramel coloring E150d as standard.

The results are reported in Table 39.

Table 39

Fresh fruit - nOX extract: control solution with inhibited PPO

Fresh fruit - OX extract: solution with uninhibited PPO

In this example, a beige to light brown colorant is obtained.

Example 9.2: enzymatic treatment with an enzyme extracted and purified from fresh crushed fruits.

In this example, the result obtained on polyphenols purified from fruit tissue and oxidized by PPO purified from fruit is described. In order to extract the date PPO, the edible part of the date is ground in a phosphate buffer at pH 6.8 containing ascorbic acid (lg.L ¹ ) used as a quinone reducer. Two solutions were prepared, one is a control with the PPO inhibited by a solution of NaF (10g / L) (PPO nOX), the other is the solution with uninhibited PPO (purified PPO). A sieving operation followed by centrifugation is carried out for 10 min at 4500 rev / min. The PPO of the date is recovered in the pellet.

The efficiency of the purification of PPO is 1.5 to 1.9 g of PPO per 50 g of fresh fruit.

Once the PPO purification step is complete, mixtures of this PPO and phenolic extracts are prepared in order to start the oxidation cycle in the presence of atmospheric oxygen.

The absorbance reading of the solutions is taken in cuvettes. The spectrophotometer scan is set between 350nm and 800nm. The l * a * b, c and h values are determined using a macro.

The results are reported in Table 40.

Table 40

Also in this example, a beige to light brown color dye is obtained.

Example 10: Determination of total phenolic compounds by Folin-Ciocalteu of the dyes of Examples 2.2, 3.2 and 4.2, obtained after basic oxidation and purification by chromatography

The determination of total phenolic compounds by the Folin-Ciocalteu reagent was described as early as 1965 (Singleton and Rossi). This is a biochemical analytical method requiring the extraction of phenolic compounds from these samples and then a spectrocolorimetric measurement of the extracts.

The objective is to estimate the amount of polyphenols and polyphenol derivatives in the purified dyes, these representing the active principles of the coloring agents according to the invention. The content of total polyphenols (TPC) extracted from the fractions of Examples 2.2, 3.2 and 4.2 was estimated from the linear calibration curve for gallic acid. The results of the determination of the phenolic compounds of different coloring extracts have been listed in Table 4L The analyzes show that the extracts of the purified dyes have high contents of polyphenols.

Table 41

Polyphenols are known as compounds of medical interest. They contribute, for example, to the prevention of degenerative and cardiovascular diseases (Manach et al. 2004). They participate in the generation of certain antioxidants such as vitamin E. They are also able to scavenge free radicals (Doukani et al. 2014).

The coloring agents obtained in the context of the invention thus have a dual advantage: their coloring activity and their health activity, in particular by virtue of their antioxidant property.

Example 11 Analysis and structural characterization by liquid chromatography coupled to mass (LC-MS and LC-MSn) of the dyes of Examples 2.2, 3.2 and 4.2, obtained after oxidation with NaOH and purification by chromatography.

In order to characterize the neoformed products after the oxidation or hydrolysis of polyphenols and for each coloring product obtained in powder form, an aliquot (of the order of 1 mg, weighed precisely to 0.01 mg) is placed in an Eppendorf. lmL of 1% acetic acid in methanol is added. The mixture is placed in an ultrasonic bath for about 15 min and the supernatant is filtered using a Teflon filter (0.45 μm porosity and 13 mm in diameter). Under these conditions, the extracts are soluble and can be injected directly by HPLC.

Principle: In simple mass spectrometry (MS), the ions introduced into the source are desolvated more or less rapidly depending on the voltages applied to the lenses. Then, they are analyzed at the level of the ion trap. Tandem MS allows a particular ion to be trapped and fragmented by collision with helium in the ion trap. This mode makes it possible to obtain information on the structure of the newly formed products. Under the effect of an electric field, the liquid is vaporized into a fine mist of charged droplets (nebulization). The solvent of the latter is gradually evaporated under the effect of the heated nitrogen which circulates against the current. As the solvent evaporates, the droplet size decreases until the Rayleigh limit is reached. A series of lenses placed under vacuum then makes it possible to focus the ion beam at the entrance of the analyzer.

Apparatus: The separation of the compounds is carried out on an HPLC line consisting of an SCM1000 degassing system (ThermoQuest, San José, CA, USA), an automatic injection system (ThermoFinnigan, San José, CA, USA), a pump 1100 Series binary (Agilent Technologies, Palo Alto, CA, USA), and a Spectra System UV6000LP diode array detector (ThermoFinnigan, San José, CA, USA). The LCQ Deçà ion trap mass spectrometer (ThermoFinnigan San José, CA, USA) is equipped with an Electrospray ionization source (EIS). All of the effluents from the diode array detector are injected into the mass spectrometer. The injected samples (2 µL) are separated on a Zorbax Eclipse XDB-C18 column (2.1 mm x 150 mm, 3.5 µm, Agilent Technologies) where the temperature is maintained at 30 ° C.

Analysis conditions: The analysis conditions are as follows: eluent A is formic acid (Fischer Scientific, Loughborough, UK) diluted to 0.1% in ultrapure water, and eluent B is 0.1% formic acid diluted in acetonitrile. The gradient applied with a flow rate of 0.2 mL / min is: initial state, 3% of B; 0-5 min, 9% B, linear; 5-15 min, 16% B, linear; 15-45 min, 50% B, linear; then a rinsing and a return to the initial conditions.

SIE, MS and MS parameters n: The nitrogen (N2) is used to vaporize the sample in the form of droplets and to dry them in order to obtain the pseudo-molecular ions.

The analyzes are carried out in negative mode with as parameters: voltage 5 kV, the main N2 gas is at 67 a.u. the auxiliary N2 gas at 5 a.u. and the capillary temperature is set at 240 ° C. Helium is used as a stabilizing, focusing and collision gas to fragment ions into MSn. These ions are then converted into peaks of different intensities that can be observed on the chromatograms. The collision energy is fixed at 35% for the fragmentation in MSn.

The HPLC-MS spectra are acquired in "Full Scan", over the whole range of masses (m / z) going from 50 to 2000. All the data is then collected and processed by the Xcalibur version software. 1.2.

Polyphenols identified in the dye obtained from date fabric powders according to the example

3.2:

The results are shown in Fig. 1 and in Table 40. They show the presence of pseudo-molecular ions at m / z 335 and which have the characteristic UV spectrum of caffeoylshikimic acid. The rest of the phenolic compounds identified and quantified are flavonols and flavones. Mention may be made of chrysoriol, quercetin-hexosulfate, quercetin-3-O-retinoglycoside, two isomers of quercetin 3-O-glucoside, risorhamnetin-3-rutinoglycoside and isorhamnetine hexoside. The total concentration of the identified compounds is 0.98 g / kg. In Table 40, the compounds identified with the retention times (RT) and negative mode pseudo-molecular ions [MH] - as well as the l max and concentrations of each identified phenolic compound.

Table 42

Unidentified compounds in the dye obtained from date tissue powders according to Example 3.2:

The results are shown in Table 43.

Table 43

Polyphenols identified in the color derived from whole fresh date fruits according to Example 4.2:

The results are shown in Fig. 2 and in Table 44. They show the presence of pseudo-molecular ions in negative mode of phenolic compounds of flavonols and flavones. The analyzes show the presence of chrysoriol, quercetin-3-O-retinoglycoside, two isomers of quercetin 3-O-glucoside, Tisorhamnetine, risorhamnetin-3-rutinoside and Tisorhamnetine hexoside. The total concentration of the identified compounds is 0.36 g / kg. The following table shows the compounds identified with the retention times and the pseudo-molecular ions in negative mode [M-H] as well as the l max and the concentrations of each identified phenolic compound. Table 44:

Unidentified compounds in the dye obtained from date tissue powders according to Example 4.2:

The results are shown in Table 45. Table 45

The dyes obtained are thus rich in polyphenols, which gives them a dual functionality: coloring power and antioxidant power. This is proving to be very useful in the fields of agrifood, pharmaceuticals and cosmetics.

Claims

1) Coloring agent based on components derived from fruits of the Phoenix dactylifera date palm, said components including polyphenol derivatives.

2) Coloring agent according to claim 1, characterized in that the components are obtained from a whole fruit, stone, pulp, skin of the fruit, or a mixture of these tissues, the fruit being selected in Hababouk, Kimri, Besser, Tamar maturation stage, or a mixture of these stages.

3) Coloring agent according to the preceding claim, characterized in that it has values of a and b in the CIELAB space of between 0 and 100.

4) Coloring agent according to the preceding claim, characterized in that it has values in the CIELAB space such that a is between 5 and 60, more preferably between 9 and 40, and b is between 40 and 90, more preferably between 45 and 90.

5) Coloring agent according to one of the preceding claims, characterized in that it comprises at least one compound selected from caffeoylshikimic acid, chrysoriol, quercetin-hexo-sulfate, quercetin-3-O-rutinoside, quercetin 3-O-glucoside or one of its isomers, risorhamnetin-3-rutinoside, an isorhamnetine hexoside and isorhamnetine.

6) Process for obtaining a coloring agent based on fruit components of the date palm Phoenix dactylifera, characterized in that it comprises the following steps of:

b) obtaining from the fruits, one or more fractions of matter containing components including polyphenols,

d) chemical or enzymatic treatment of the polyphenols of one or a mixture of the fractions of step b,

7) Method according to claim 6, characterized in that step (b) comprises obtaining one or more of the following fractions:

(i) a fraction of core tissue,

(ii) a fraction of pulp tissue,

(iii) a fraction of skin tissue,

(iv) a whole fruit fraction.

8) Method according to claim 6 or 7, characterized in that it comprises a step (c) of obtaining a fraction of a purified extract of polyphenols (v) from fractions (i), (ii) , (iii) and / or (iv). 9) Method according to one of claims 6 to 8, characterized in that the chemical treatment comprises an oxidation reaction, preferably in a basic medium.

10) Method according to one of claims 6 to 8, characterized in that the chemical treatment comprises a hydrolysis reaction, preferably in an acidic medium.

11) Method according to one of claims 6 to 8, characterized in that the enzymatic treatment comprises a reaction in the presence of polyphenoloxidase.

12) Method according to one of claims 6 to 11, characterized in that it comprises a step (f) of concentration of the polyphenols and polyphenol derivatives contained in the coloring agents obtained in step (e).

13) Method according to one of claims 6 to 12, characterized in that it comprises adjusting the concentrations and adjusting the pH of the coloring agents to predetermined values.

14) Use of date palm fruits Phoenix dactylifera as coloring agents or sources of coloring agents.

15) Use of components derived from fruits of the Phoenix dactylifera date palm, said components including polyphenol derivatives as coloring agents.

16) Use according to claim 14 or 15 wherein the fruits of the Phoenix dactylifera date palm are used as coloring agents and antioxidants.

17) Composition comprising at least one coloring agent as defined in one of claims 1 to 5 or obtainable by a process as defined in one of claims 6 to 13, the composition being a food composition, cosmetic, pharmaceutical, nutraceutical, paint or dye.