WO2023052387A1

WO2023052387A1 - Method of extraction of carotenoids from dunaliella, compositions and use for treating carotenoid and pro-vitamin a deficiencies

Info

Publication number: WO2023052387A1
Application number: PCT/EP2022/076909
Authority: WO
Inventors: Michael LAGUERRE; Simona BIRTIC; Mathieu TENON; Sara MADJI; Farid Chemat; Anne Sylvie TIXIER-FABIANO
Original assignee: Givaudan Sa
Priority date: 2021-10-01
Filing date: 2022-09-28
Publication date: 2023-04-06
Also published as: GB202114072D0

Abstract

The present invention relates to a method for enrichment and extraction of carotenoids from a Dunaliella biomass comprising contacting the cells with 2-methyl oxolane (Me- THF) or a mixture of Me-THF with a second solvent, thereby obtaining a carotenoid extract. The invention also relates to a carotenoid composition obtained by the method of the invention and to a carotenoid composition characterized in that the content of cis-carotenoids is of at least about 30% in weight with respect to the total carotenoid content, the weight ratio of cis- to trans carotenoids is of at least 0.7 and/or, the weight ratio of cis- to trans-carotenoids is at least 5% higher than that found in a Dunaliella biomass. The invention also relates to uses of the composition of the invention.

Description

METHOD OF EXTRACTION OF CAROTENOIDS FROM DUNALIELLA, COMPOSITIONS AND USE FOR TREATING CAROTENOID AND PRO-VITAMIN A DEFICIENCIES

FIELD OF THE INVENTION

The present invention relates generally to methods for the extraction of carotenoids from a Dunaliella biomass.

BACKGROUND OF INVENTION

Single-celled Dunaliella is a photosynthetic marine organism (green alga) able to outcompete other organisms and thrive in hypersaline media. It is known to produce large amounts of p-carotene in harsh growth conditions such as high salt concentrations and light intensities, as well as limited oxygen and nitrogen levels. A species from this genus, Dunaliella salina (syn. D. bardawil and D. kone) is generally considered the best commercial source of natural p-carotene in the world.

Carotenes are photosynthetic terpenoid pigments important for photosynthesis. When extracted, they have a variety of uses, including as food additive colorants, antioxidants, nutritional supplements and/or cosmetic agents.

Carotenes such as p-carotene occur in two types of chemical isomers: trans-carotenes and cis-carotenes. Associated with the different geometric isomers are different properties and possible functions. In particular, cis-isomers are much less likely to form crystals and thus are much more soluble than the trans-isomers in oily solutions, in lipid dispersions in water, or in organic solvents. For example, cis-isomers of p-carotene have also been found to have greater efficiency of incorporation into micelles than all- trans p-carotene in vitro. Consequently, cis-carotene forms are easier to formulate in a diverse set of applications.

It is therefore of interest to enrich carotenoid composition in cis-isomers compared to the initial composition of the microalgae.

With regard to this matter, US005612485 A (Schlipalius, High cis p-carotene composition, 1993) describes a carotenoid composition derived from a natural source wherein at least 50% by weight of the carotenoid content is cis-p-carotene and preferably 9 cis-p-carotene. This document also describes a purification method using centrifugation or filtration to separate cis-isomer carotenoids from trans-isomer carotenoids contained in the preparation. However, this method of enrichment in cis-isomers is only applicable to carotenoid compositions that are already solubilized and/or dispersed in oils. In case the final formulation of this carotene-rich extract is not an oily solution, it would be advantageous to have an enrichment method applicable to the extract prior to its formulation in order to have a higher degree of freedom to operate downstream the extraction. Most importantly, all oils described in the document US005612485 A have a boiling point above 170 °C which makes difficult if not impossible to isolate by vacuum evaporation the carotenoid composition from the formulation oils.

More recently, WO2019/097219 A1 (Harvey and Xu, Production of Dunaliella, 2017) describes Dunaliella algae and extracts thereof, grown under specific wavelength (above 500 nm) comprising increased level of cis-isomers of carotene compared to Dunaliella algae and extracts thereof grown or cultivated under natural light or white conditions.

However, such enrichment method is only implementable in expensive closed photobioreactors that are internally illuminated and for which a proper control of the wavelengths is possible. Therefore, this enrichment method cannot be implemented in cost-effective open ponds and/or raceway, which are nevertheless the most adapted and widespread cultivation techniques to grow Dunaliella. Additionally, from an energy standpoint, because the enrichment and cultivation method cannot use natural light, the photons needed for the microalgae growth and for the enrichment in cis-isomers are artificially generated using electricity, which implies additional cost and equipment compared to conventional systems using sunlight.

A method that can be applied after the cultivation step but before the formulation step is thus still needed to enrich carotenoid compositions in cis-isomers compared to the composition initially present in the microalgae. Such a method must be applied after the cultivation step so that the enrichment method does not constrain nor limit the possible cultivation technique to be used upstream. Such a method must also be applied before the formulation step, preferentially at the extraction step, so that the enriched composition is totally free from formulation solvent thus allowing to properly standardize the composition before its incorporation into a final formulation.

Therefore, a method of enrichment applicable during the extraction of the carotenoids is needed wherein a significant part of trans-carotene is converted by isomerization into cis-carotene such as 9-cis-p-carotene. Such a trans-cis isomerization of beta-carotene is difficult to obtain in practice. For example, when all-trans p-carotene is solubilized in bovine serum albumin or lecithin solutions, no isomerization is generally observed (Marx, Stuparic, Schieber, Carle, Effects of thermal processing on trans-cis isomerization of p-carotene in carrot juices and carotene-containing preparations. Food Chem 2003, 83, 609-617). The only known case where such a conversion takes place is when trans-carotene is thermally processed in toluene, hexane or dichloromethane. However, these three solvents are toxic and derived from a fossil source (petroleum) which is nonrenewable.

An enrichment method applicable at the extraction step and using a nontoxic and biosourced solvent is thus highly desirable for the industry.

Finally, the extraction of p-carotene directly from wet algae (without dry process) may be advantageous. Dry process indeed results in a high energetic consumption and may alter thermos-sensitive molecules such as carotenoids.

SUMMARY OF THE INVENTION

The authors of the present invention have surprisingly found specific solvents with an increased capacity to solubilize carotenoids. More particularly, the inventors have shown that the solvent in which all-trans-p-carotene solubilizes best is 2-methyloxolane (Me-THF), followed by dichloromethane (DCM) and pure monoterpenes or mixture thereof such as R-limonene, turpentine oil and p-menthane. This finding has allowed the inventors to develop a highly efficient carotenoid enrichment method applicable to the Dunaliella algae cell extract. The method is based in a first extraction step performed in the presence of any of the newly characterized solvents and optionally consecutive steps of filtration and evaporation of the solvent under different conditions. The inventors have surprisingly found out that with this methodology, there is an enrichment in the proportion of cis-isomers in total carotenoids when compared to the freeze-dried powder of Dunaliella extract. Thus, these new methods allow a significant part of trans-carotene to be converted by isomerization into cis-carotene such as 9-cis beta-carotene.

Thus, in a first aspect, the invention relates to a method of conversion of transcarotenoids into cis-isomers of carotenoids from Dunaliella and extraction of a cis- carotenoid enriched extract, comprising a) contacting the cells with 2 methyloxolane (Me-THF) or a mixture of Me-THF with a second solvent, b) optionally removing the solvent, thereby obtaining a cis-carotenoid enriched extract In a second aspect, the invention relates to a method for enrichment and extraction of cis-carotenoids from a Dunaliella biomass comprising: a) contacting the cells with 2 methyloxolane (Me-THF) or a mixture of Me-THF with a second solvent, b) optionally removing the solvent, thereby obtaining a carotenoid extract enriched in cis-carotenoids.

In a third aspect, the invention relates to a method for the extraction of carotenoids from a Dunaliella biomass comprising contacting the cells with 2 methyloxolane (Me- THF) or a mixture of Me-THF with a second solvent, thereby obtaining a carotenoid extract.

In a fourth aspect, the invention relates to a carotenoid composition obtained by any one of the methods according to the invention.

In a fifth aspect, the invention relates to a carotenoid composition characterized in that: a) the content of cis-carotenoids is of at least about 30% in weight with respect to the total carotenoid content, b) the weight ratio of cis- to trans carotenoids is of at least 0.7 and/or c) the weight ratio of cis- to trans-carotenoids is at least 5% higher than that found in a Dunaliella biomass.

In a further aspect, the invention relates to a pharmaceutical composition, a foodstuff, a nutritional supplement, a cosmetic composition or a nutraceutical composition comprising a carotenoid composition as defined in any of the second and third aspects of the invention and a vehicle adequate for the formulation of the composition into said pharmaceutical composition, foodstuff, nutritional supplement, cosmetic composition or nutraceutical composition.

In a further aspect, the invention relates to a cosmetic method for the treatment in a subject of a condition resulting from deficient carotenoid levels which comprises the administration to the subject of a composition as defined in any of the second and third aspects of the invention.

In a further aspect, the invention relates to a carotenoid composition as defined in any of the fourth and fifth aspects of the invention for use in the treatment of a disease associated with reduced carotenoid levels in the body. In a last aspect, the invention relates to the use of a carotenoid composition according to any of the fourth and fifth aspects of the invention as antioxidant, food colorant or cosmetic colorant.

DESCRIPTION OF THE FIGURES

Figure 1. Solubility of all-trans-p-carotene in different solvents measured by HPLC.

Figure 2. Solubility of microalgal carotenoids in sunflower oil as a function of the cisisomer proportion. Analysis by HPLC and results of a duplicated experiment.

DETAILED DESCRIPTION OF THE INVENTION

Methods of the invention

In a first aspect, the invention relates to a method of conversion of trans-carotenoids into cis-isomers of carotenoids from Dunaliella and extraction of a cis-carotenoid enriched extract, comprising a) contacting the cells with 2 methyloxolane (Me-THF) or a mixture of Me-THF with a second solvent, b) optionally removing the solvent, thereby obtaining a cis-carotenoid enriched extract

The invention relates to a method for enrichment and extraction of cis-carotenoids from a Dunaliella biomass comprising: a) contacting the cells with 2 methyloxolane (Me-THF) or a mixture of Me-THF with a second solvent, b) optionally removing the solvent, thereby obtaining a carotenoid extract enriched in cis-carotenoids.

The invention also relates to a method for the extraction of carotenoids from a Dunaliella biomass comprising contacting the cells with 2-methyloxolane (Me-THF), or a mixture of Me-THF with a second solvent, thereby obtaining a carotenoid extract.

"Carotenoid" as used herein refers to naturally-occurring pigments of the terpenoid group that can be found in plants, algae (including macro- and microalgae), yeasts, bacteria, and certain animals, such as birds and shellfish. Carotenoids include but are not limited to carotenes, which are hydrocarbons (i.e., without oxygen), and their oxygenated derivatives (i.e., xanthophylls). Examples of carotenoids include lycopene; a-carotene; y-carotene; B-carotene; echinenone; isozeaxanthin; canthaxanthin; citranaxanthin; B-apo-8'-carotenic acid ethyl ester; hydroxy carotenoids, such as alloxanthin, apocarotenol, astacene, astaxanthin, capsanthin, capsorubm, carotenediols, carotenetriols, carotenols, cryptoxanthin, B-cryptoxanthin, decaprenoxanthin, epilutein, fucoxanthin, hydroxycarotenones, hydroxyechinenones, hydroxylycopene, lutein, lycoxanthin, neurosporine, phytoene, phytotluoene, rhodopin, spheroidene, torulene, violaxanthin, and zeaxanthin; and carboxylic carotenoids, such as apocarotenoic acid, B-apo-8'-carotenoic acid, bixin, carboxylcarotenes, crocetin, diapocarotenoic acid, neurosporaxanthin, norbixin, and lycopenoic acid. In a preferred embodiment, the carotenoid is phytoene, phytofluene, all-trans-p-carotene, a-carotene 9-cis-p-carotene, 13-cis-p-carotene, 15-cis-p-carotene, lutein, zeaxanthin or canthaxanthin.

The inventors have surprisingly found out that with the methods of the invention, there is an enrichment in the proportion of cis-isomers in total carotenoids when compared to the freeze-dried powder of Dunaliella extract. Thus, these new methods allow a significant part of trans-carotene to be converted by isomerization into cis-carotene such as 9-cis beta-carotene.

In a preferred embodiment, the method of the invention relates to the extraction of cis carotenoids.

Cis-trans isomers are pairs of molecules which have the same formula but whose functional groups are in different orientations in three-dimensional space. Cis isomer indicates that the functional groups (substituents) are on the same side of some plane, while trans conveys that they are on opposing sides.

Illustrative non-limitative examples of cis carotenoids are 13-cis p-carotene, 15-cis p- carotene and 9-cis p-carotene. In a more particular embodiment, the carotenoid extract comprises 9-cis p-carotene.

The term “Dunaliella" as used herein to refer to unicellular green motile algae belonging to the class Chlorophyceae. It refers to the multiple strains of Dunaliella that produce carotenoids. Nomenclature of these strains has not historically been consistent. For example, Dunaliella bardawil is considered by some references to be a strain of Dunaliella salina, but is considered by others to be a different strain. Preferably, the term Dunahella algae, as used herein refers to any strain of Dunahella salina salina, Dunaliella salina rubeus, Dunaliella salina bardawil and Dunaliella tertiolecta.

Dunaliella may be cultivated under natural light or white light conditions in ponds, lakes, lagoons, raceways or closed vessels under natural light or under artificial white light. The term ‘raceway’ as used herein, refers to a shallow pond that uses sunlight as the light source and paddlewheels to provide the flow to circulate algae, water and nutrients keeping the algae suspended in the water, and circulating them back to the surface on a regular frequency. The ponds are operated continuously with carbon dioxide or flue gas containing CO2 and nutrients are fed constantly or by batch to the ponds.

“Biomass” refers to material produced by growth and/or propagation of cells. Biomass may contain cells and/or intracellular contents as well as extracellular material, and includes, but is not limited to, compounds secreted by a cell.

The methods of the invention have a first step a) of contacting the cells with 2-methyloxolane (Me-THF) or a mixture of Me-THF with a second solvent. This step involves mixing a solvent with the cell paste or dried biomass, under conditions that cause the solvent to dissolve as much of the carotenoids as possible. Also, and without to be bound to any theory, the inventors believe that during this step of contacting the biomass with the 2-methyloxolane (Me-THF) or a mixture of Me-THF with a second solvent, a conversion of trans-carotenoids into cis-isomers of carotenoids from Dunaliella occurs. The extraction conditions of time and temperature will depend on the carried out process.

The method of the invention requires the contact of the Dunaliella biomass with the solvent.

The term "contacting”, as used herein refers to the process by which cells come into contact with the solvent. The contacting step includes any possible conventional method that allows the interaction of the cells with the solvent to obtain the cis- carotenoid enriched extract. The contacting of the algae with the solvent typically leads to the lysis of the cells and the generation of a lysate. The contacting of the algae with the solvent can also lead to passive diffusion of the solvent through the biological material, the solubilisation of the solutes of interest (here carotenoids) and finally the diffusion out of the material to come back to the bulk solvent phase.

“Lysis” refers to the breakage of the plasma membrane and optionally the cell wall of a biological organism sufficient to release at least some intracellular content, in this case by a physical chemical mechanisms that compromise its integrity.

“Lysate” and “extract” are used interchangeably and refer to a solution containing the contents of lysed cells.

In a particular embodiment, the method of the invention comprises contacting the Dunaliella biomass with 2-methyl oxolane (Me-THF).

2-Methyl oxolane, also known as 2-methyl tetrahydrofuran, 2-MeOx or 2-MeTHF refers to a cyclic ether with molecular formula C10H20O2, issued from carbohydrates derived from lignocellulosic biomass and corresponds to the compound having the CAS number 96-47-9.

In a preferred embodiment, the 2-MeTHF is provided in substantially pure form.

“Substantially pure" as used herein refers to the purity of the solvent which is at least about 90.0%, at least about 90.5%, at least about 91.0%, at least about 91.5%^ at least about 92.0%^ at least about 92.5% at least about 93.0%^ at least about 93.5%^ at least about 94.0%^ at least about 94.5% at least about 95.0%^ at least about 95.5%^ at least about 96.0%, at least about 96.5%, at least about 97.0%, at least about 97.5%^ at least about 98.0%^ at least about 98.5%, at least about 99.0%, at least about 99.1 %, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9% or 100% as measured for example by a liquid chromatography method.

In a preferred embodiment, the 2-MeTHF contains less than about 5% (v/v) water, more preferably 4.5% (v/v). In a preferred embodiment, the second solvent can be selected from the group consisting of hexane, a-pinene, p-pinene, D-limonene, p-cymene, p-myrcene, ethyl oleate, isopropyl palmitate, ethyl laurate, cyclopentenyl methyl ether (CPME), ethyl acetate, methyl acetate, dimethyl carbonate (DMC), levulinic acid and its esters such ethyl levulinate, and lactic acid and its esters such as ethyl lactate.

In a preferred embodiment of the method of the invention, the mixture is selected from the group consisting of 2-methyl oxolane (Me-THF) and hexane, 2-methyl oxolane and a-pinene, 2-methyl oxolane and p-pinene, 2-methyl oxolane and D-limonene, 2-methyl oxolane and p-cymene, 2-methyl oxolane and p-myrcene, 2-methyl oxolane and ethyl oleate, 2-methyl oxolane and isopropyl palmitate, 2-methyl oxolane and ethyl laurate, 2- methyl oxolane and CPME, 2-methyl oxolane and ethyl acetate, 2-methyl oxolane and methyl acetate, 2-methyl oxolane and levulinic acid and its esters such ethyl levulinate . 2-methyl oxolane and DMC, 2-methyl oxolane and lactic acid and its esters such as ethyl lactate. a-Pinene refers to the compound having the CAS number 80-56-8; p-pinene refers to the compound having the CAS number 127-91-3; p-cymene refers to the compound having the CAS number 99-87-6; p-myrcene refers to the compound having the CAS number 123-35-3; ethyl oleate refers to the compound having the CAS number 111-62- 6; isopropyl palmitate refers to the compound having the CAS number 142-91-6; ethyl laurate refers to the compound having the CAS number 106-33-2; CPME refers to cyclopentyl methyl ether refers to the compound having the CAS number 5614-37-9; ethyl acetate refers to the compound having the CAS number 141-78-6; methyl acetate refers to the compound having the CAS number 79-20-9.

In another preferred embodiment, the second solvent is selected from the group consisting of hexane, dichloromethane (DCM), a monoterpene or a mixture thereof.

The term “hexane” refers to an organic compound with CAS number 110-54-3 (n- hexane), a straight-chain alkane with six carbon atoms and has the molecular formula CeHi4.

“Dichloromethane” (DCM or methylene chloride) is an organochloride compound with the formula CH2CI2 and the CAS number 75-09-2. Monoterpenes are a class of terpenes that consist of two isoprene units and have the molecular formula C10H16. Monoterpenes may be linear (acyclic) or contain rings (monocyclic and bicyclic). The term also refers to modified terpenes, such as those containing oxygen functionality or missing a methyl group which are called monoterpenoids.

In a preferred embodiment, the monoterpene is selected from the group consisting of R-limonene (syn. D-limonene), turpentine oil and p-menthane. In a preferred embodiment of the method of the invention, the mixture is selected from the group consisting of 2- methyl oxolane (Me-THF) and R-limonene, 2-methyloxolane (Me-THF) and turpentine oil and 2-methyloxolane (Me-THF) and p-menthane.

The term “R-limonene”, also called 1-methyl-4-prop-1-en-2-ylcyclohexene is a monoterpene with molecular formula C10H16 that is cyclohex- 1-ene substituted by a methyl group at position 1 and a prop-1 -en-2-yl group at position 4 respectively, having the CAS number 5989-27-5.

The term “turpentine” (also called gum turpentine, terebenthene, terebinthine) refers to a fluid obtained by the distillation of resin harvested from living trees, mainly pines. Mainly used as a specialized solvent, it is also a source of material for organic syntheses. The CAS number of turpertine oil is 8006-64-2. Turpentine is composed of terpenes, primarily the monoterpenes a- and p-pinene, with lesser amounts of carene, camphene, dipentene, and terpinolene.

“p-Menthane”, as used herein, refers to the compound having the CAS number 99-82- 1. It is the product of the hydrogenation or hydrogenolysis of various terpenoids, including p-cymene, terpinolenes, phellandrene, and limonene.

The method of the invention can comprise additional down-stream steps.

In an embodiment, the method further comprises the step of removing the residual cell material from the carotenoid extract. The process of removing the residual cell material from the carotenoid extract may be performed using mechanical means such as centrifugation or filtering. The solids that remain after separation can be discarded, or used as feedstock for other processing steps (including repeated solvent extraction cycles, if desired). Separation may be perfomed by gravity or by suction, centrifugation, filtration, decantation, or the like or combinations thereof. Stirring or other alternate methods such as shaking, agitation, or the like, may also be employed for the isolation. In a particular embodiment, the removal of the residual cell material from the carotenoid extract is carried out by filtration.

In another embodiment, the method further comprises at least one additional cycle of contacting the residual cell material with fresh solvent thereby obtaining a second carotenoid extract and, optionally, removing the residual cell material from said second extract. More particularly, three extraction cycles are carried out.

The solvent-soluble liquid fraction may be then treated to remove the solvent, usually by evaporation at a suitable temperature and pressure. This leaves behind a viscous oil which contains solvent-extracted carotenoids along with other solvent-soluble components that were drawn out of the cell paste by the solvent.

Thus, in another embodiment, the solvent used in the extraction is removed thereby obtaining a dry carotenoid composition.

In a particular embodiment, the removal of the solvent is carried out by evaporation. In a more particular embodiment, the evaporation of the solvent is carried out under reduced pressure, more particularly under vacuum.

In a preferred embodiment, the solvent is provided in substantially pure form.

In a preferred embodiment, the solvent contains less than about 5% (v/v) water, more preferably 4.5% (v/v).

Temperature

The step a) and/or b) may be carried out at a temperature of about 20-130°C, such as of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, to about 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20°C such as about 30-90 °C, such as about 70 to 85, such as 78 °C.

In certain embodiments, when Me-THF is used in a purity of above 90 % w/w, the temperature may be of about 20-130°C, more preferably 30-80 °C, and more preferably the temperature is of about 70 to 85°C, such as 78 °C.

In certain embodiments, when Me-THF is used in a purity of less than 90 % w/w, the temperature may be of about 20-130°C, more preferably 50-130°C, and more preferably the temperature is of about 70-100°C, such as 90°C.

The step b) of optionally removing the solvent, may be done by evaporating the solvent. In certain embodiments the evaporation is done under reduced pressure. The temperature and/or pressure used for evaporating the solvent will be selected depending on the solvent used and will be the temperature where the solvent evaporates. The expert in the art would know and select the right temperature and pressure for evaporating the solvent depending on the composition of said solvent.

In another embodiment, the Dunaliella biomass is provided in dry form.

“Dry form” as used herein refers to less than 20% moisture, less than 19% moisture, less than 18% moisture, less than 17% moisture, less than 16% moisture, less than 15% moisture, less than 14% moisture, less than 13% moisture, less than 12% moisture, less than 11% moisture, less than 10% moisture, less than about 9% moisture, less than about 8% moisture, less than about 7% moisture, less than about 6% moisture, less than about 5% moisture, less than about 4% moisture less than 3% moisture, less than 2% moisture or less than 1% moisture.

The extraction, (here understood as including the method of conversion of cis- carotenoids and extraction of an enriched cis-carotenoids extract) according to the invention can be performed by several methods such as single stage reflux, two-stage reflux or the standard Soxhlet method. In a more preferred embodiment, the extraction is performed under magnetic stirring. In another preferred embodiment, the extraction is performed by mechanical stirring. In another preferred embodiment, the mechanical stirring is performed when the extraction is carried out at a starting dry biomass to solvent ratio of about 1 :5 to 1 :3.

According to the method of the invention, the contact of the cells with 2-methyl oxolane (Me-THF) or a mixture of Me-THF with a second solvent is carried out at single reflux. More particularly, single stage reflux heating.

“Reflux”, as used herein relates to the process which allows for a liquid to heat and condense, with the condensed liquid returning to the original flask. A reflux setup is analogous to a distillation, with the main difference being the vertical placement of the condenser. The liquid remains at the boiling point of the solvent (or solution) during active reflux.

The main purpose of refluxing a solution is to heat a solution in a controlled manner at a constant temperature. In a preferred embodiment, the temperature is of about 30-130 °C, such as 60-130 °C, such as 40-90, such as from 60-80°C, such as from 70- 80°Cmore preferably 77-80 °C, and more preferably the temperature is of about 78 °C. When a mixture of Me-THF and the second solvent is used, the temperature of the heating can be calculated as an average of the different boiling temperatures, as known in the art. When the two solvents form azeotropes there is a significant deviation of the boiling point of the mixture compared to the theoretical one calculated from the individual boiling point of the solvents, which is known in the art.

The extraction (as already mentioned, here understood as the method of conversion of cis-carotenoids and extraction of an enriched cis-carotenoids extract) can be performed during different time, for example during 6, 7, 8, 9, or 10 h, more preferably during about 8 hours. In another preferred embodiment, the extraction is performed during about 1, 2 or 3 hours. In a more preferred embodiment, the extraction by single reflux is carried out at a temperature of about 78 °C during at least 8 h.

In another preferred embodiment of the method of the invention, the method is carried out at a starting dry biomass to solvent ratio of about 1 :3 w/v to 1 :20 w/v, such as 1 :2 to 1:15 w/v. In a more preferred embodiment the starting dry biomass to solvent ratio is about 1:3 w/v, 1 :4 w/v, 1:5 w/v, 1 :6 w/v, 1:7 w/v, 1:8 w/v, 1 :9 w/v, 1 :10 w/v, 1 :11 w/v, 1:12 w/v, 1 :13 w/v, 1 :14 w/v or 1:15 w/v. In a more preferred embodiment, the starting dry biomass to solvent ratio is about 1 :3, 1 :5, 1:8, 1:10 or 1 :15 w/v.

In another preferred embodiment, the method is carried out at two extraction cycles at reflux. Said extraction can be carried out at different temperatures and during different time. In a preferred embodiment, the temperature is of about 68-82 °C, more preferably 70-80 °C, more preferably 74-78 °C, more preferably the temperature is of about 78 °C. In another preferred embodiment, the temperature is 68 °C. The extraction can be performed during different time, for example each extraction cycle at least 2 h, at least 3 h, at least 4 h or at least 5 h. In another preferred embodiment, each extraction cycle is carried out during at least 2 h. In another preferred embodiment, each extraction cycle is carried out during at least 4 h.

In another preferred embodiment, the extraction under two-stage reflux is carried out at a starting dry biomass to solvent ratio of about from 1:3 w/v to 1 :20 w/v, such as 1:2 to 1:15 w/v, such as 1 :3, 1 :5, 1 :8, 1:10 or 1:15, more preferably 1:5 w/v

In another preferred embodiment, the extraction is carried out at three extraction cycles at reflux. Said extraction can be carried out at different temperatures and during different time. In a preferred embodiment, the temperature is of about 60-130 °C, such as of about 75-82 °C, more preferably 77-80 °C, more preferably the temperature is of about 78 °C. The extraction can be performed during different time, for example each extraction cycle at least 2 h, at least 3 h, at least 4 h or at least 5 h. In another preferred embodiment, each extraction cycle is carried out during at least 2 h. In another preferred embodiment of the method of the invention, the extraction is carried out using the Soxhlet technique.

Soxhlet technique as used herein relates to a procedure for extracting nonvolatile and semivolatile organic compounds from solids such as soils, sludges, and wastes. The Soxhlet extraction process ensures intimate contact of the sample matrix with the extraction solvent. This method is applicable to the isolation and concentration of water-insoluble and slightly water-soluble organics in preparation for a variety of chromatographic procedures. In a preferred embodiment, the extractions is carried out by the standard method of Soxhlet (F. Soxhlet 1879 Dinglers Polytechnisches Journal, 232, 461-465.)

Said extraction can be carried out at different temperatures and during different time. In a preferred embodiment, the extraction using the Soxhlet technique is carried out at a temperature of about 25-45 °C, more preferably 28-40 °C, more preferably 28-32 °C. In a more preferred embodiment, the temperature is of about 30 °C. The extraction can be performed during different times, for example, at least 1 h, at least 4 h, at least 5 h, at least 6 h, at least 7 h, at least 8 h or at least 9 h. In a preferred embodiment, the extraction using the Soxhlet technique is performed during at least 8 h.

In another preferred embodiment, the extraction using the Soxhlet technique is carried out at a starting dry biomass to solvent ratio of about 1:10 w/v, such as of about from 1 :3 w/v to 1 :5 w/v.

Alternatively, to the provision of the Dunaliella biomass as previously mentioned in dry form, it can be provided as a wet paste and wherein the biomass is used without a prior drying or with a prior drying, wherein said prior drying is partial drying which results in a biomass having a water content of at least about 80% (w/v).

In another preferred embodiment, Dunaliella biomass is provided as a wet paste and wherein the biomass is used without a prior drying or with a prior drying, wherein said prior drying is partial drying which results in a biomass having a water content of at least about 20% (w/v), at least about 25% (w/v), at least about 30% (w/v), at least about 35% (w/v) at least about 40% (w/v)₃ at least about 45% (w/v) at least about 50% (w/v)₃ at least about 55% (w/v) at least about 60% (w/v)₃ at least about 65% (w/v) at least about 70% (w/v) at least about 75% (w/v)₃ at least about 80% (w/v), at least about 85% (w/v), at least about 90% (w/v). In a more preferred embodiment, the water content of the biomass is of at least 80% (w/v).

In the case that the biomass is provided without a prior drying or with a prior drying as previously described, the extraction is carried out by shearing the wet paste with the solvent, treating the mixture so as to separate the residual cell material, the aqueous phase and the organic phase containing the solvent and collecting the solvent. Shearing the wet paste with a solvent can be performed by any method known in the art, for example using a colloid mill.

In the case the biomass is provided without a prior drying, the starting biomass to solvent ratio may be of about from 1:3 w/v to 1 :20 w/v, such as 1 :2 to 1:15 w/v, such as 1:3, 1:5, 1:8, 1:10 or 1:15, more preferably 1:5 w/v (weight of dried biomass to v of solvent).

A further step of separation of the residual cell material can be performed. The separation of the residual cell material can be performed by any method known in the art, as a way of illustrative non-limitative example sedimentation, decantation, membrane separation such as micro or ultra-filtration, flocculation, floatation, filtration, and centrifugation. In a preferred embodiment, the separation is carried out by centrifugation.

The method for the conversion of trans-carotenoids into cis-isomers of carotenoids from Dunaliella, for enrichment and extraction of cis-carotenoids from a Dunaliella biomass according to the invention can comprise additional steps. A preferred embodiment of the methods of the invention comprises a further step b) of removing the solvent. For example, step b may be performed by evaporating the solvent so as to obtain a dry carotenoid composition (cis-carotenoid enriched extract).

“Dry carotenoid composition” or ” dry cis-carotenoid enriched extract”, as used herein relates to a carotenoid composition comprising less than 20% moisture, less than 19% moisture, less than 18% moisture, less than 17% moisture, less than 16% moisture, less than 15% moisture, less than 14% moisture, less than 13% moisture, less than

12% moisture, less than 11% moisture, less than 10% moisture, less than about 9% moisture, less than about 8% moisture, less than about 7% moisture, less than about 6% moisture, less than about 5% moisture, less than about 4% moisture, less than 3% moisture, less than 2% moisture or less than 1% moisture.

The methods can further comprise dissolving or dispersing the dry carotenoid composition in an acceptable vehicle.

In another preferred embodiment, the solvent is provided as a mixture with an acceptable vehicle, which is adequate for forming a solution or a dispersion of carotenoids. In a more preferred embodiment, the extract is processed so as to remove the solvent thereby obtaining a carotenoid composition dispersed or dissolved in the acceptable vehicle.

In a preferred embodiment, the acceptable vehicle is an oil. Suitable oils that can be used in the present invention are olive oil, sunflower oil, oil comprising medium chain triglycerides, peanut oil, cottonseed oil, flaxseed oil, sesame oil, corn oil, castor oil, camellia oil, rapeseed oil, canola oil and soybean oil. In a more preferred embodiment, the oil is sunflower oil. In another preferred embodiment, the oil is olive oil. In another preferred embodiment, the oil is and oil comprising medium chain triglycerides.

“Medium chain triglycerides” as used herein relates to a class of lipids in which three intermediate carbon length saturated fats are bound to a glycerol backbone wherein fatty acid molecules are six to twelve carbon lengths. Illustrative, non-limitative examples of suitable medium chain triglycerides are C6: caproic acid or hexanoic acid, C8: caprylic acid or octanoic acid, C10: capric acid or decanoic acid or C12: lauric acid or dodecanoic acid. Illustrative non-limitative examples of oils comprising medium chain triglycerides are coconut oil and palm oil.

The inventors have shown that in the resulting cis-carotenoid enriched extract there is an enrichment in the proportion of cis-isomers in total carotenoids when compared to the freeze-dried powder of Dunaliella extract.

Thus in a further embodiment of the methods of the invention, the weight ratio of cis- to trans-carotenoids in the cis-carotenoid enriched extract is at least about 5% higher than that found in a Dunaliella biomass, such as at least about 6, 7, 8, 9, 10, 15, 20, 25 or at least about 30% in weight higher than that found in a Dunahella biomass (i.e the originating biomass before extraction and conversion).

In another preferred embodiment of the methods of the invention, the extract is further processed to separate the cis and the trans isomers of the carotenoids. Different methods can be used to separate said isomers.

In a preferred embodiment, the separation of the cis and trans isomers can be performed by drying the extract, adding to the dry extract an isomer-specific solvent which preferentially solubilizes one type of isomers and separating the isomer typespecific solvent from the insoluble fraction. In a preferred embodiment, the isomer typespecific solvent is ethanol, methanol and/or other polar alcohols, and the isomer type which is solubilized is the cis isomer.

To obtain a Dunaliella biomass, Dunaliella cells can be cultured by any method known in the art. It can be required to add some nutrients and carbon sources such as CO2, and to adjust some limiting physiological factors for optimal growth and carotenogenesis, for example adjusting light and salinity.

In a preferred embodiment of the method of the invention, the Dunaliella biomass has been grown under natural light or white conditions.

Natural light conditions, relates to conditions similar to sunlight in terms of photoperiod, and light intensity. Natural as used herein relates to a color temperature of 5,000 K - 6,500K. White conditions, as used herein relates to a color temperature 3,500 K - 4,100 K.

Carotenoid composition

In another aspect, the invention relates to a cis-carotenoid enriched extract or composition obtained by any one of the methods according to the invention.

The inventors have shown that in the resulting cis-carotenoid enriched extract obtained by the methods of the invention, there is an enrichment in the proportion of cis-isomers in total carotenoids when compared to the freeze-dried powder of Dunaliella extract. For Table XIX of the examples, shows that an extract of the invention obtained as described in Example 18, the cis-trans ratio is 1.4, while the corresponding proportion of c/s-isomers in total carotenoids is 58.0% in average. For comparison, the freeze- dried powder of Dunaliella salina microalgae has a proportion of c/s-isomers in total carotenoids of 40.9%, which corresponds to a cis-trans ratio of 0.7. The enrichment factor in c/s-isomers is thus equal to 41.8%. This shows that starting from 40.9% cis- carotenoids in the starting biomass, using the method of the invention, there is a transformation to cis-isomers so as to obtain 58%.

In another aspect, the invention relates to a carotenoid composition characterized in that: a. the content of cis-carotenoids is of at least about 30% in weight with respect to the total carotenoid content, b. the weight ratio of cis- to trans carotenoids is of at least 0.4, such as of at least 0.7 and/or c. the weight ratio of cis- to trans-carotenoids is at least 5% higher than that found in a Dunaliella biomass.

In a preferred embodiment, the content of cis-carotenoids in the carotenoid composition is of at least about 32%, at least about 34%, at least about 36%, at least about 38%, at least about 40%_s at least about 42%_s at least about 44%_s at least about 45%, of at least about 45.5%, at least about 46%, at least about 47%, at least about 47.1 %, at least about 47.9%, at least about 48%, at least about 49%, at least about 50%, at least about 50.1%, at least about 50.3%, at least about 51%, at least about 52%, at least about 53%, at least about 54%_s at least about 55%_s at least about 56%_s at least about 56.9%, at least about 57%_s at least about 57.5%, at least about 57.9% at least about 58%_s at least about 58.7%^ at least about 58.9%^ at least about 59%_s at least about 59.5%^ at least about 60%, at least about 60.7%, at least about 61 %₃ at least about 62%_s at least about 62.4%^ at least about 63%_s at least about 64%, at least about 64.4%^ at least about 64.6%^ at least about 70% or of at least about 75% with respect to the total carotenoid content.

In another preferred embodiment, the content of cis-carotenoids in the carotenoid composition of the invention is 30-50% with respect to the total carotenoid content. In another preferred embodiment, the weight ratio of cis- to trans carotenoids is of at least 0.4, of at least 0.45, of at least 0.5, of at least 0.55, of at least 0.6, of at least 0.65 of at least 0.7, of at least 0.72, of at least 0.73, of at least 0.74, of at least 0.75_s of at least 0.76, of at least 0.77_s of at least 0.78_s of at least 0.79_s of at least 0.80_s of at least 0.82, of at least 0.83, of at least 0.84, of at least 0.86_s of at least 0.88_s of at least 0.89, of at least 0.90_s of at least 0.92 of at least 0.94_s of at least 0.96 of at least 0.98, of at least 1 , of at least 1.1₃ of at least 1.2, of at least 1.3, of at least 1.4, of at least 1.5, of at least 1 .6, of at least 1.7 or of at least 1 .8.

In another preferred embodiment, the weight ratio of cis- to trans-carotenoids is at least 5.5% higher than that found in a Dunaliella biomass, at least 6% higher, at least 6.5% higher, at least 7% higher, at least 7.5% higher^ at least 8% higher^ at least 8.5% higher, at least 9% higher, at least 9.5% higher, at least 10% higher^ at least 10.5% higher, at least 11 % higher^ at least 11.5 % higher^ at least 12% higher, at least 15%, higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 45% higher, at least 50% higher or at least 55% higher than that found in a Dunaliella biomass.

In another preferred embodiment of the carotenoid composition, the content of trans- carotenoids is of less than about 40% in weight with respect to the total carotenoid content. In a more preferred the content of trans-carotenoids is of less than about 38% in weight, less than about 36% in weight, less than about 34% in weight, less than about 32% in weight, less than about 30% in weight, less than about 25% in weight, less than about 20% in weight, less than about 15% in weight, less than about 10% in weight, less than about 5% in weight with respect to the total carotenoid content.

In another preferred embodiment, the carotenoid composition of the invention comprises at least 10% of carotenoids, more preferably about 10-60%.

In a more preferred embodiment, the carotenoid composition characterized as previously described has been obtained by a method of the invention.

In another preferred embodiment, the cis-carotenoid present in the composition is selected from the group consisting of 13-cis p-carotene, 15-cis p-carotene and/or 9-cis P-carotene and wherein the trans-carotenoids are lutein, zeaxanthme, a-carotene and all-trans p-carotene.

13-cis p-Carotene relates to the compound having the CAS number 6811-73-0, 15-cis P-carotene refers to the compound having the CAS number 19361-58-1, 9-cis p- carotene refers to the compound having the CAS number 13312-52-2, lutein relates to the compound having the CAS number 127-40-2, zeaxanthine relates to the compound having the CAS number 144-68-3, a-carotene relates to the compound having the CAS number 7488-99-5, and p-carotene relates to the compound having the CAS number 99-82-1.

In another preferred embodiment, the concentration of 13-cis p-carotene in the composition is of at least about 0.5 % (w:w), the concentration of 15-cis p-carotene in the composition is of at least about 2.8 % (w/w) and/or the concentration of 9-cis p- carotene in the composition is of at least about 7.8 %(/w/w).

In another preferred embodiment, the concentration of 13-cis p-carotene in the composition is of at least about 0.6% (w:w), at least about 0.7% (w:w), at least about 0.8% (w:w), at least about 0.9% (w:w) at least about 1% (w:w), at least about 1.1 % (w:w), at least about 1.2% (w:w) at least about 1.3% (w:w), at least about 1.4%(w:w), at least about 1.5% (w:w), at least about 1.6% (w:w), at least about 2%(w:w), ), at least about 3% (w:w), at least about 4% (w:w), at least about 5% (w:w), at least about 10% (w:w), at least about 15% (w:w), at least about 20% (w:w) or at least about 25% (w:w).

In another preferred embodiment, the concentration of 15-cis p-carotene in the composition is of at least about 2.9% (w/w), at least about 3% (w/w), at least about 3.2% (w/w), at least about 3.4% (w/w), at least about 3.5% (w/w), at least about 3.8% (w/w), at least about 4% (w/w), at least about 3.3% (w/w), at least about 3.6% (w/w), at least about 3.9% (w/w), at least about 4.1% (w/w), at least about 4.2% (w/w), at least about 4.3% (w/w)₃ at least about 4.4% (w/w)₃ at least about 4.9% (w/w)₃ at least about 5% (w/w), at least about 5.1% (w/w)₃ at least about 7.6% (w/w)₃ at least about 9.2% (w/w), at least about 10 % (w/w), at least about 15% (w/w), at least about 20% (w/w), at least about 25% (w/w) or at least about 30% (w/w). In another preferred embodiment, the concentration of 9-cis p-carotene in the composition is of at least about 7.8% (w/w), at least about 8 % (w/w), at least about 8.9 %(w/w), at least about 9% (w/w), at least about 9.2% (w/w), at least about 9.4% (w/w), at least about 9.5% (w/w), at least about 9.6% (w/w), at least about 9.8% (w/w), at least about 10% (w/w), at least about 10.5% (w/w), at least about 10.6% (w/w), at least about 11% (w/w), at least about 11.5% (w/w), at least about 12% (w/w), at least about 12.5% (w/w), at least about 13% (w/w), at least about 13.5% (w/w), at least about 14% (w/w), at least about 14.5% (w/w), at least about 14.8% (w/w), at least about 14.9% (w/w), at least about 15% (w/w), at least about 15.1% (w/w), at least about 15.5% (w/w), at least about 17.5% (w/w) or at least about 18.2% (w/w).

In another preferred embodiment, the concentration of lutein in the composition is of less than 1% (w/w), the concentration of zeaxanthine in the composition is of less than about 0.5% (w/w), the concentration of a-carotene is of less than about 6% and/or the concentration of all-trans p-carotene in the composition is of less than about 24% (w/w).

In a more preferred embodiment, the concentration of lutein in the composition is of less than 0.9% (w/w), less than 0.8% (w/w), less than 0.7% (w/w), less than 0.6% (w/w), less than 0.5% (w/w), less than 0.4% (w/w), less than 0.3% (w/w), less than 0.2% (w/w), less than 0.1% (w/w), less than 0.05% (w/w) or less than 0.10% (w/w).

In another more preferred embodiment, the concentration of zeaxanthine in the composition is of less than about 0.4% (w/w), less than about 0.3% (w/w), less than about 0.2% (w/w), less than about 0.1% (w/w), less than about 0.05% (w/w), less than about 0.01% (w/w), less than about 0.015% (w/w) or less than about 0.02% (w/w),

In another more preferred embodiment, the concentration of a-carotene is of less than about 6.1%, less than about 5.7, less than about 5.5%, less than about 5.1%, less than about 5%, less than about 4.9% less than about 4.5%, less than about 4.4%, less than about 4.3%, less than about 4.2%, less than about 4.1%, less than about 4%, less than about 3.9%, less than about 3.5%, less than about 3.3%, less than about 3.1%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, less than about 0.5%, less than about 0.10%, less than about 0.05%, less than about 0.01%, less than about 0.015% or less than about 0.02%, In another more preferred embodiment, the concentration of all-trans p-carotene in the composition is less than about 23.7% (w/w), less than about 23% (w/w), less than about 22% (w/w), less than about 21% (w/w), less than about 21.5% (w/w), less than about 20% (w/w), less than about 19.2% (w/w), less than about 16.1% (w/w), less than about 15% (w/w), less than about 13.6% (w/w), less than about 12.5% (w/w), less than about 12.3% (w/w) or less than about 11.8% (w/w), less than about 11.4% (w/w), less than about 11.3% (w/w), less than about 11.2% (w/w), less than about 10% (w/w), less than about 10.3% (w/w), less than about 10.2% (w/w), less than about 10.1% (w/w), less than about 7.9% (w/w), less than about 7.0% (w/w), less than about 6.3% (w/w), less than about 5% (w/w) or less than about 1% (w/w).

In a particular case, the carotenoid composition of the invention can be mixed with an oil forming an oily solution of the carotenoid composition so that the final carotenoid content is equal or superior to 30% w/w. In a preferred embodiment, the final carotenoid content in the oily solution is at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% (w/w).

This oily solution of carotenoid could be optionally emulsified as oil-in-water emulsion containing between 1 and 20% of oil (preferably olive oil, sunflower oil, oil comprising medium chain triglycerides, or mix of them). In a particular embodiment, the final concentration of carotenoids in the oil-in-water emulsion is between 0.1 to 10%, more preferably 0.5 to 8%.

Foodstuff, nutritional supplement or nutraceutical composition

In another aspect, the invention relates to a foodstuff, a nutritional supplement or a nutraceutical composition comprising a carotenoid composition as defined in the present document and a vehicle adequate for the formulation of the composition into said foodstuff, nutritional supplement or nutraceutical composition.

In certain embodiments, the vehicle is an oil. The composition of the invention can be present in a foodstuff. Foodstuff as used herein relates to a substance that can be used or prepared for use as food. The term also relates to any substance or product, whether processed, partially processed or unprocessed, intended to be, or reasonably expected to be ingested by humans. These compositions can also be named as terms “food product”, “food composition”, food ingredient or food additive. These compositions can also relate to a food that beneficially affects one or more functions of the body, so as to provide better health and wellness. Accordingly, such a food product may be intended for the prevention and/or treatment of a disease or a disease-causing factor. Therefore, these compositions can also be named as functional food for particular nutritional purposes.

The foodstuff, nutritional supplement or nutraceutical of the invention may be the oil solution or the oil-in-water solution of the carotenoid composition.

The composition of the invention can be added to a food to achieve a desired effect such as imparting a particular colour. Non-limiting examples of suitable foodstuffs in the present invention are beverage, confectionary, dairy products, margarine and spreads, bread, cakes, semi- or synthetic diet formulations, infant formulae, clinical nutrition formulae, flours.

In a preferred embodiment, the foodstuff is an edible oil. Edible oil as used herein includes any oil used for human consumption and includes hydrogenated vegetable oils. Illustrative non-limitative examples of oils are olive oil, sunflower oil, oil comprising medium chain triglycerides, peanut oil, cottonseed oil, flaxseed oil, sesame oil, corn oil, castor oil, camellia oil, rapeseed oil, canola oil and soybean oil. In a more preferred embodiment, the oil is sunflower oil. In another preferred embodiment, the oil is olive oil. In another preferred embodiment, the oil is an oil comprising medium chain triglycerides or a mixture thereof. Illustrative non-limitative examples of oils comprising medium chain triglycerides are coconut oil and palm oil.

“Nutritional supplement”, as used herein relates to a compound that beneficially affects one or more functions of the body, so as to provide better health and wellness. Accordingly, such a nutritional supplement may be intended for the prevention and/or treatment of a disease or a disease-causing factor. Therefore, the term "nutritional composition" of the present invention can be used as a synonym for functional food or foods for particular nutritional purposes, or medical food. A nutritional composition is similar to that of a conventional food and consumed as part of a normal diet appearance.

As used herein, the term “nutraceutical product” refers to a product suitable for use in human beings or animals, comprising one or more natural products with therapeutic action which provide a health benefit or have been associated with disease prevention or reduction, and it includes dietary supplements presented in a non-food matrix (e.g., capsules, powder, etc.) of a concentrated natural bioactive product usually present (or not) in the foods and which, when taken in a dose higher than that existing in those foods, exerts a favorable effect on health which is greater than effect which the normal food may have. Therefore, the term “nutraceutical product” includes isolated or purified food products as well as additives or food supplements which are generally presented in dosage forms normally used orally, for example, capsules, tablets, sachets, drinkable phials, etc.; such products provide a physiological benefit or protection against diseases, generally against chronic diseases. If desired, the nutraceutical product provided by the invention can contain, in addition to the composition of the invention, one or more nutraceuticals (products or substances associated with disease prevention or reduction), for example, flavonoids, omega-3 fatty acids, etc., and/or one or more prebiotics (non-digestible food ingredients which stimulate probiotic activity and/or growth), for example, oligofructose, pectin, inulin, galacto-oligosaccharides, lactulose, human milk oligosaccharides, dietary fiber, etc.

In the case of a foodstuff, nutritional supplement or nutraceutical composition, the vehicle adequate for the formulation can be any usual excipients and adjuvants for oral compositions or food supplements, such as and non-limited, fatty components, aqueous components, humectants, preservatives, texturizing agents, flavours, antioxidants and common pigments in the food sector.

In a more preferred embodiment of the foodstuff, nutritional supplement or nutraceutical composition, the vehicle is an oil. Suitable oils that can be used in the present invention are olive oil, sunflower oil, oil comprising medium chain triglycerides, peanut oil, cottonseed oil, flaxseed oil, sesame oil, corn oil, castor oil, camellia oil, rapeseed oil, canola oil and soybean oil. In a more preferred embodiment, the oil is sunflower oil. In another preferred embodiment, the oil is olive oil. In another preferred embodiment, the oil is and oil comprising medium chain triglycerides. Illustrative non- limitative examples of oils comprising medium chain triglycerides are coconut oil and palm oil.

In a more preferred embodiment, the final carotenoid content in the composition comprising the composition of the invention and an oil is at least about 30% (w/w).

In a preferred embodiment, the foodstuff is in the form of powdered, paste, liquid or gel. In another aspect, the invention relates to the use of a carotenoid according to the invention as antioxidant. In another preferred embodiment, the invention relates to the use of the carotenoid composition of the invention as food colorant or cosmetic colorant.

Food coloring, or color additive, is any dye, pigment, or substance that imparts color when it is added to food or drink. They come in many forms consisting of liquids, powders, gels, and pastes.

Cosmetic colorant or pigment relates to a substance that imparts color in cosmetic application for coloring of skin, lips, hair, eyebrows, nails, eyelashes or another body surface.

An antioxidant is a compound which inhibits reactions promoted by oxygen, thus avoiding oxidation reactions. These compounds also avoid rancidity of the compositions.

Antioxidants, preferably alpha-tocopherol or mixed tocopherols or more preferably carnosic acid or rosemary extract containing carnosic acid, may be added to the oil phase in order to protect carotenoids during heating and subsequently stabilize the final color formulation against oxidative degradation.

In certain embodiments, the nutritional supplement is to provide provitamin A.

Pharmaceutical composition and cosmetic composition of the invention In another aspect, the invention relates to a pharmaceutical composition, cosmeceutical or to a cosmetic composition comprising a carotenoid composition as defined in the present document and a vehicle adequate for the formulation of the composition into said pharmaceutical composition, cosmeceutical or cosmetic composition.

“Pharmaceutical composition”, as used herein, relates to compositions and molecular entities that are physiologically tolerable. Preferably, the term "pharmaceutically acceptable" means it is approved by a regulatory agency of a state or federal government or is included in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

“Cosmetic composition”, as used herein refers to a composition suitable for use in personal hygiene of human beings or animals, or in order to enhance the natural beauty or change the body appearance without affecting the structure or functions of the human or animal body, comprising one or more products providing such effects. If desired, the cosmetic composition provided by the invention can contain, in addition to the composition of the invention, one or more cosmetics or cosmetic products, i.e., substances or mixtures intended to be placed in contact with the external parts of the human or animal body (e.g., epidermis, hair system, nails, lips, etc.) or with the teeth and the buccal mucosa, for the exclusive or main purpose of cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition or correcting body odors. Illustrative examples of cosmetically acceptable vehicles include the products contained in the INCI (International Nomenclature of Cosmetic Ingredients) list. The composition of the present invention may be added to a wide variety of products for cosmetic application, including makeup, creams for cleansing, protecting, treating, or caring for the skin, in particular, the face, hands, and feet (e.g., day and night creams, makeup removal creams, foundation creams and sunscreens), liquid foundations, makeup removal lotions, protective or skin-care body lotions, sunscreen lotions, skin care lotions, gels, or foams, such as cleansing, sunscreen, and artificial tanning lotions, bath preparations, deodorant compositions, after-shave gels or lotions, depilatory creams, and compositions used for insect stings and against pain. The composition of the invention may take any of a wide variety of forms, and include, for example dressings, lotions, solutions, sprays, creams, gels, ointments, or the like. As used herein, the term cosmeceutical product refers to a product suitable for use in the body or animal body comprising one or more cosmeceutical products (functional cosmetics, dermaceuticals or active cosmetics), i.e., topical hybrid products with cosmetic-pharmaceutical characteristics containing active ingredients having effect on user’s skin, hair and/or nails, at higher and more effective concentrations, therefore they are located in an intermediate level between cosmetic and drug. Illustrative examples of cosmeceutical products include essential oils, ceramides, enzymes, minerals, peptides, vitamins, etc.

In a particular embodiment of the cosmetic composition, cosmetic for application to the skin is in the form of a cream emulsion, gel, milk, suspension, O/W emulsion, W/O emulsion, liposome foam, aqueous or emulsion lotion, spray or a waxy stick.

In another particular embodiment, the composition of the invention is a pharmaceutical composition comprising the composition of the invention together with a pharmaceutically acceptable vehicle, especially suitable for oral, topical, rectal or vaginal administration; to that end, said composition comprises a pharmaceutically acceptable vehicle comprising one or more excipients suitable for oral administration, for example, in the form of capsule, powder, granulate, tablet (coated or non-coated), sachet, matrix, suspension, etc., or a pharmaceutically acceptable vehicle comprising one or more excipients suitable for topical administration, for example, in the form of cream, ointment, salve, etc., or a pharmaceutically acceptable vehicle comprising one or more excipients suitable for rectal administration, for example, in the form of suppository, etc., or a pharmaceutically acceptable vehicle comprising one or more excipients suitable for vaginal administration, for example, in the form of bolus, suppository, etc. Information about excipients suitable for the formulation of pharmaceutical compositions intended for oral, topical, rectal or vaginal administration, as well as about the production of said pharmaceutical compositions can be found in the book “Tratado de Farmacia Galenica”, by C. Fault i Trillo, 10^th Edition, 1993, Luzan 5, S.A. de Ediciones.

If desired, the pharmaceutical or cosmetic composition of the invention is incorporated in a fabric, a non-woven fabric or a medical device. Illustrative examples of said fabric, non-woven fabric or medical device include but are not limited to bandages, gauzes, t- shirts, panty hose, socks, underwear, girdles, gloves, diapers, sanitary napkins, dressings, bedspreads, towelettes, adhesive patches, non-adhesive patches, occlusive patches, microelectric patches and face masks.

Where necessary, the pharmaceutical or cosmetic composition is comprised in a composition also including a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the combination, the pharmaceutical or cosmetic composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

In cases other than intravenous administration, the pharmaceutical or cosmetic composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, gel, polymer, or sustained release formulation. The composition can be formulated with traditional binders and carriers, as would be known in the art. Formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc., inert carriers having well established functionality in the manufacture of pharmaceuticals. Various delivery systems are known and can be used to administer a combination or pharmaceutical composition of the present invention including encapsulation in liposomes, microparticles, microcapsules and the like.

Solid dosage forms for oral administration may include conventional capsules, sustained release capsules, conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. At these solid dosage forms, the active compounds can be mixed with at least one inert excipient such as sucrose, lactose or starch. Such dosage forms can also comprise, as in normal practice, additional substances other than inert diluents, e.g. lubricating agents such as magnesium stearate. In the case of capsules, tablets, effervescent tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can be prepared with enteric coatings. Liquid dosage forms for oral administration may include emulsions, solutions, suspensions, syrups and elixirs pharmaceutically acceptable containing inert diluents commonly used in the technique, such as water. Those compositions may also comprise adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening agents, flavoring and perfuming agents.

Injectable preparations, for example, aqueous or oleaginous suspensions, sterile injectable may be formulated according with the technique known using suitable dispersing agents, wetting agents and/or suspending agents. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. Sterile oils are also conventionally used as solvents or suspending media.

For topical administration, the pharmaceutical or cosmetic composition of the invention can be formulated as creams, gels, lotions, liquids, pomades, spray solutions, dispersions, solid bars, emulsions, microemulsions and similars which may be formulated according to conventional methods that use suitable excipients, such as, for example, emulsifiers, surfactants, thickening agents, coloring agents and combinations of two or more thereof.

Additionally, the pharmaceutical or cosmetic composition of the invention may be administered in the form of transdermal patches or iontophoresis devices. In one embodiment, the combination of the invention is administered as a transdermal patch, for example, in the form of sustained-release transdermal patch. Suitable transdermal patches are known in the art.

In a preferred embodiment, the acceptable vehicle is an oil. Suitable oils that can be used in the present invention are olive oil, sunflower oil, oil comprising medium chain triglycerides, peanut oil, cottonseed oil, flaxseed oil, sesame oil, corn oil, castor oil, camellia oil, rapeseed oil, canola oil and soybean oil. In a more preferred embodiment, the oil is sunflower oil. In another preferred embodiment, the oil is olive oil. In another preferred embodiment, the oil is and oil comprising medium chain triglycerides. Illustrative non-limitative examples of oils comprising medium chain triglycerides are coconut oil and palm kernel oil.

All the terms and embodiments previously described are equally applicable to this aspect of the invention. Cosmetic method and uses

The invention also relates to a cosmetic method for the treatment in a subject of a condition resulting from deficient carotenoid levels which comprises the administration to the subject of a composition as defined in the invention.

The cosmetic composition can be administered at any route, for example, by systemic (e.g. intravenous, subcutaneous, intramuscular injection), oral, parenteral or topical administration. Additionally, it is also possible to administer the cosmetic compositions of the invention as defined above intranasally or sublingually which allows systemic administration by a non-aggressive mode of administration. In a particular embodiment, the cosmetic composition is topically administered. In another particular embodiment, it is intradermically administered.

The cosmetic composition of the invention is administered in a cosmetic effective amount.

The term “cosmetic effective amount”, as used herein, relates to the sufficient amount of a compound (i.e. of the composition of the invention) to provide the desired effect and it will generally be determined, among other causes, by the characteristics of the compound itself and the cosmetic effect to be achieved. The dosage for obtaining a cosmetic effective amount will also depend on a range of factors, such as, for example, age, weight, sex or tolerance of the animal, preferably a mammal and more preferably human.

In a preferred embodiment, the cosmetic composition of the invention is used for coloring of skin, lips, hair, eyebrows, nails, eyelashes or another body surface.

The cosmetic composition of the invention can be in the form of a cosmetic powder for application to skin. Examples of such cosmetic powders include but are not limited to: eye shadow, blusher, powder makeup, lip powder, face powder, body powder, bronzing powder. Aside from powders, it is contemplated that the powders can be incorporated in various systems so as to form formulations such as for example foundation (liquid and stick), face makeup such as cream-to-powder, eye highlighter, eye pencil, bronzing stick, etc.

Therapeutic compositions and uses thereof

In another aspect, the invention relates to a carotenoid composition as defined in the invention for use in the treatment of a disease associated with reduced carotenoid levels in the body.

Alternatively, the invention relates to a method for treating a disease associated with reduced carotenoid levels and/or pro-vitamin A levels in the body comprising administering a carotenoid composition according to the invention to a subject in need thereof.

Alternatively, the invention relates to the use of a carotenoid composition according to the invention for the preparation of a medicament for the treatment of a disease associated with reduced carotenoid levels and/or pro-vitamin A levels in the body.

As used herein the terms “treat”, “treatment”, or “treatment of” refers to reducing the potential for a certain disease or disorder, reducing the occurrence of a certain disease or disorder, and/or a reduction in the severity of a certain disease or disorder, preferably, to an extent that the subject no longer suffers discomfort and/or altered function due to it. It also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.

The term “prevention”, “preventing” or “prevent”, as used herein, refers to avoiding the appearance of a certain disease or disorder. The prevention can be complete (e.g. the total absence of a disease). The prevention can also be partial, such that for example the occurrence of a disease in a subject is less than that which would have occurred without the administration of the combination or composition of the present invention. Prevention also refers to reduced susceptibility to a clinical condition. The prevention also includes reducing the risk of suffering the disease. In a preferred embodiment, the disease is selected from the group consisting of diabetes, low plasma HDL and/or high plasma TG and/or atherosclerosis.

In a preferred embodiment, a disease associated with reduced carotenoid levels in the body is a disease related with increased production of reactive oxygen species (ROS).

In a preferred embodiment, since beta carotene is a pro-vitamin A, the beta-carotene of the invention may be used to prevent or treat low levels of Vitamin A.

All the terms an embodiment previously described are equally applicable to this aspect of the invention.

The invention will be described by way of the following examples which are to be considered as merely illustrative and not limitative of the scope of the invention.

EXAMPLES

Materials and methods

Soxhlet extraction

100 g of dry biomass was mixed with 100 g of sand and put into a 37 mm x 130 mm cellulose cartridge, itself, placed in the extraction chamber of the Soxhlet device. This is positioned above a 2,000 mL flask containing 1 ,000 mL of hexane or 2- methyloxolane and topped with a condenser. Extractions were carried out for 8 hours. After extraction, the liquid extract is filtered through a Buchner filter or glass 45-pm diameter frit to separate small particles from the liquid and thus recover the extract. This filtration step is accompanied by rinsing to avoid loss of the extracted substances. The solution obtained is then adjusted into a 1 ,000 mL volumetric flask. After adjustment, 15 mL of this solution are concentrated to determine the mass of the extract and 100 mL of extract are stored in an amber flask at -80 °C for further analyses. The standard Soxhlet method is done in several cycles or stages. In the present case, 96 cycles for 8 h for hexane and 98 cycles for 8 h for 2-methyloxolane have been achieved.

Single reflux

To extract the total carotenoids, 50 g of microalgae powder were heated to reflux with 250 mL of solvent (n-hexane or 2-methyloxolane) with magnetic stirring and boiled for 8 hours. After extraction, the whole was filtered through a Buchner filter before being fitted into a volumetric flask. A certain quantity of the extract was concentrated using a thermoblock, and the remaining amount was stored at -80 ° C for further analyses. The experiment was repeated in triplicate.

Two-stage extractions

The two-stage extraction is done with the same reflux set-up, described previously. The only change comes from its co-current (or cross-flow) flow. The same biomass, already extracted, is recovered after filtration and re-extracted again with a fresh solvent. 50 g of biomass powder were heated to reflux with 250 mL of solvent (n-hexane or 2- methyloxolane) with magnetic stirring. The biomass was extracted for 4 hours before being filtered, collected and reintroduced into the flask with 250 mL of fresh solvent to undergo a new extraction cycle for 4 hours under the same conditions. Liquid extracts from both stages are collected separately and sent to HPLC analysis. The experiment was repeated in triplicate

Reflux extraction with mechanical stirring

The extraction is carried out with the same reflux set-up, described previously, except that it was carried out with a mechanical stirring, using a two-necked flask to pass the stirring rod. The operating conditions are the same as those used for heating at reflux with magnetic stirring. The same amounts of biomass and solvent were used. After extraction, the whole was filtered through a Buchner filter before being fitted into a volumetric flask. A certain quantity of the extract was concentrated using a thermoblock and the remaining amount was stored at -80 °C for further analysis. The experiment was repeated in triplicate. Cis-trans mixture solubilization study

A Dunaliella salina dry extract containing 15.8% (w:w) carotenoids (of which 41.1% were trans-isomers and 58.9% were cis-isomers) was mixed with a beta-carotene standard containing 99% trans beta-carotene at different weight ratios of the two components (see Table I). These were added to a 20-mL IKA shaker tube with 10 g of ceramic beads and 10 mL of oil (sunflower oil or olive oil). The mixture was stirred mechanically by the effect of the beads at 4,000 rpm for 30 min at 25 °C with no control of temperature. The residue was removed by filtration, the resulting filtrate was centrifuged at 12,000 rpm for 15 min. After centrifugation the supernatant was collected and adjusted to 20 mL in a volumetric flask and analyzed by UV visible spectrophotometry and HPLC.

Table I. The different weight ratios (between the extract and the beta-carotene standard) used for the cis-trans mixture solubilization study.

Extraction on algal paste

The extraction of total carotenoids from the algal paste was performed using an llltra- Turrax rotor-stator (T-18 digital ULTRA-TURRAX®) with 2-methyloxolane used as solvent. A hundred grams of algae paste was added to 100 mL of 2-methyloxolane then sheared for 10 min at 25 °C with no control of temperature. The solution obtained was centrifuged for 15 min at 8,000 rpm then recovered and analyzed by HPLC.

Separation of the cis-isomer

A hundred grams of algal paste was added to 100 mL of 2-methyloxolane, then sheared for 10 min using an Ultra-Turrax rotor-stator at 25 °C with no control of temperature. The suspension obtained was centrifuged for 15 min at 8,000 rpm, then filtered, and the recovered supernatant was evaporated.

The dry extract obtained was dissolved in 100 mL of methanol and then filtered through a Buchner filter. During the filtration step, the trans-beta-carotene remained on the filter as crystals, while the cis-beta-carotene was solubilized in methanol.

Example 1. Solubilisation of all-trans-B-carotene in different solvents.

Results of HPLC analysis show that the solvent in which all-trans-p-carotene solubilizes best is 2-methyloxolane (Me-THF), followed by dichloromethane (DCM) and pure monoterpenes or mixture thereof such as R-limonene (syn. D-limonene), turpentine oil and p-menthane, which is hydrogenated R-limonene (Figure 1). Hexane capacity to solubilize this very hydrophobic carotenoid fall behind these five solvents, although hexane is commonly considered as a good extraction solvent for this type of molecules. Furthermore, the inventors verified that this compound is poorly soluble in acetone and ethanol.

Example 2. Use of pure 2-methyloxolane at a 1:3 microalgae:solvent (w:v) ratio in a three-pass extraction procedure for 2 hours each pass at reflux at 78 °C under magnetic stirring.

Fifty-three grams of a freeze-dried powder of Dunaliella salina microalgae (containing 11.7% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2- methyloxolane for 2 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate corresponding to the first pass was analyzed by HPLC.

These extraction and filtration steps were reiterated (second pass) on plant residues resulting from the first extraction pass. Fresh solvent was used here in the same conditions and quantities as in the first pass. The filtrate corresponding to the second pass was analyzed by HPLC.

This sequence of step was repeated a third time to generate a filtrate corresponding to the third extraction pass, which was then analyzed by HPLC.

After evaporation of the solvent under reduced pressure, mass yields of 15.1 , 4.5, and 0.9% were obtained for passes 1 , 2, and 3, respectively. Concentration of total carotenoids in the extracts obtained for passes 1 , 2, and 3 was found to be 37.4, 63.7, and 85.0% (w:w), respectively, which corresponds to a recovery rate of 48.4, 24.6, and 6.3% (w:w), respectively. The global mass yield, carotenoid content and carotenoid recovery rate of the pooled extracts collected through the three passes were 20.5, 45.2, and 79.2% (w:w), respectively.

Table II shows the carotenoid composition of the different extracts obtained in Example 2. Cis-and trans-isomers were identified based on the presence or absence of a UV cis-band around 350 nm on the diode array detector (DAD) spectrum of the chromatograms. In the native freeze-dried powder of Dunaliella salina microalgae the proportion of cis-carotenoids in total carotenoids is of 35.6% w/w and the ratio of cistrans is of 0.55. Applying the method of the invention enables to transform trans isomers in cis isomers: starting form an algae material with 35.6% w/w of cis isomer, the final extract obtained using the transformation method of the invention has 45.5% w/w of cis isomers. Thus there is an increase of the proportion of cis-isomers in the final extract (being the cis-trans ratio of the final extract of between 0.76 and 0.84) versus a ratio of cis-trans of 0.55 in the starting biomass. The enrichment factor in cis- isomers is thus equal to 27.8%.

Table II. Detailed composition in % (w:w) of the extracts obtained in Example 2.

Example 3. Use of pure 2-methyl oxolane at a 1:3 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux at 78 °C under magnetic stirring.

Fifty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 11.7% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2- methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure a mass yield of 19.3% were obtained. Content of total carotenoids in the extracts was found to be 59.2% (w:w), which corresponds to a recovery rate of 98.2% (w:w).

Table III shows the carotenoid composition of the extract obtained in Example 3. In the native freeze-dried powder of Dunaliella salina microalgae the proportion of cis- carotenoids in total carotenoids is of 35.6% w/w (cis-trans ratio = 0.55). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is of 47.9% and the cis-trans ratio = 0.92. The enrichment factor in cis-isomers is thus equal to 34.6%.

Table III. Detailed composition in % (w:w) of the extracts obtained in Example 3

Example 4. Use of pure 2-methyl oxolane at a 1:10 microalgae:solvent (w:v) ratio in a one-pass Soxhlet extraction procedure for 8 hours at 30 °C under magnetic stirring. Ninety nine grams of a freeze-dried powder of Dunahella salina microalgae (containing 11.7% (w:w) total carotenoids) were incubated with 1,000 mL of pure 2-methyloxolane for 8 hours in a Soxhlet apparatus (30 °C) under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure a mass yield of 17.9% were obtained. Content of total carotenoids in the extracts was found to be 52.0% (w:w), which corresponds to a recovery rate of 80.0% (w:w).

Table IV shows the carotenoid composition of the extract obtained in Example 4. In the native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers in total carotenoids is of 35.6% (cis-trans ratio = 0.55). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 47.1% (cis-trans ratio = 0.89). The enrichment factor in cis-isomers is thus equal to 32.3%.

Table IV. Detailed composition of the extracts obtained in Example 4.

Example 5. Use of pure 2-methyloxolane at a 1:10 microalgae:solvent (w:v) ratio in a one-pass Soxhlet extraction procedure for 8 hours at 30 °C under magnetic stirring.

One hundred grams of a freeze-dried powder of Dunaliella salina microalgae (containing 6.2% (w:w) total carotenoids) were incubated with 1.000 mL of pure 2- methyloxolane for 8 hours in a Soxhlet apparatus (30 °C) under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure a mass yield of 14.2% were obtained. Content of total carotenoids in the extracts was found to be 46.3 % (w:w), which corresponds to a recovery rate of 100% (w:w).

A repetition of this extraction gave a mass yield of 16.0%, a carotenoid content of 41.9%, and a carotenoid recovery rate of 100%.

Table V shows the carotenoid composition of the extract obtained in Example 5. In the native freeze-dried powder of Dunaliella salina microalgae has a proportion is of cisisomers in total carotenoids of 47.0% (cis-trans ratio = 0.89). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 50.1% globally (cis-trans ratio = 1.0 in average of two repetitions). The enrichment factor in cis-isomers is thus equal to 6.6%.

Table V. Detailed composition of the extracts obtained in Example 5.

Example 6. Use of pure 2-methyl oxolane at a 1:3 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under magnetic stirring.

Fifty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 6.2% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2- methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure a mass yield of 15.9% were obtained. Content of total carotenoids in the extracts was found to be 39.6% (w:w), which corresponds to a recovery rate of 100% (w:w). A repetition of this extraction gave a mass yield of 13.7%, a carotenoid content of 36.9%, and a carotenoid recovery rate of 82.2%.

Table VI shows the carotenoid composition of the extract obtained in Example 6. In the native freeze-dried powder of Dunaliella salina microalgae the proportion of cisisomers is of 47.0% (cis-trans ratio = 0.89). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cisisomers. In the final extract, the proportion of cis-isomers in total carotenoids is 51.5% globally (cis-trans ratio = 1.06 in average of two repetitions). The enrichment factor in cis-isomers is thus equal to 9.5%.

Table VI. Detailed composition of the extracts obtained in Example 6.

Example 7. Use of pure 2-methyl oxolane at a 1:3 microalgae:solvent (w:v) ratio in a two-pass extraction procedure for 2 hours each pass at reflux (78 °C) under magnetic stirring.

Fifty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 6.2% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2- methyloxolane for 2 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate corresponding to the first pass was analyzed by HPLC. After evaporation of the solvent under reduced pressure a mass yield of 13.8% were obtained. Content of total carotenoids in the extract was found to be 35.7% (w:w) which corresponds to a recovery rate of 79.7% (w:w). These extraction and filtration steps were reiterated (second pass) on plant residues resulting from the first extraction pass. Fresh solvent was used here in the same conditions and quantities as in the first pass. The filtrate corresponding to the second pass was analyzed by HPLC. After evaporation of the solvent under reduced pressure a mass yield of 3.3% were obtained. Content of total carotenoids in the extract was found to be 38.1% (w:w) which corresponds to a recovery rate of 20.3% (w:w).

The global mass yield carotenoid content and carotenoid recovery rate of the pooled extracts collected through the two passes were 17.1. 36.1 and 100.0% (w:w) respectively.

A repetition of this two-pass extraction procedure gave a mass yield of 13.5 and 3.5%, a carotenoid content of 35.3 and 34.8%, and a carotenoid recovery rate of 77.3 and 19.7% for the first and second passes respectively. The global mass yield carotenoid content and carotenoid recovery rate of the pooled extracts collected through the two passes were 17.0, 35.2 and 97.0% (w:w) respectively.

Table VII shows the carotenoid composition of the different extracts obtained in Example 7. In the native freeze-dried powder of Dunaliella salina microalgae the proportion of cis-isomers is of 47.0 % (cis-trans ratio = 0.89). Applying the method of the invention enables to increase, in the final extract, the proportion of cis-isomers in total carotenoids to 51.7% globally (cis-trans ratio = 1.07 in average of two repetitions). The enrichment factor in cis-isomers is thus equal to 10.0%.

Table VII. Detailed composition in % (w:w) of the extracts obtained in Example 7.

Example 8. Use of pure 2-methyl oxolane at a 1:5 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under magnetic stirring.

Fifty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 6.4% (w:w) total carotenoids) were incubated at reflux (78 °C) with 250 mL of pure 2- methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure a mass yield of 14.8% were obtained. Content of total carotenoids in the extracts was found to be 23.6% (w:w), which corresponds to a recovery rate of 54.1% (w:w).

Two repetitions of this extraction gave a mass yield of 16.4 and 16.1%, a carotenoid content of 23.9 and 22.6% and a carotenoid recovery rate of 61.0 and 56.6%.

For comparison purposes, the inventors performed the extraction in the exact same conditions except that they used a reflux at 68 °C and n-hexane, a petroleum-based solvent currently used for the extraction of carotenoids. The three repetitions gave a mass yield of 12.4, 11.2. and 10.2%, a carotenoid content of 28.4, 24.2, and 27.4%, and a carotenoid recovery rate of 54.8, 42.0, and 43.2%.

Table VIII shows the carotenoid composition of the extract obtained in Example 8. In the native freeze-dried powder of Dunaliella salina microalgae the proportion of cisisomers is of 56.9% (cis-trans ratio = 1.32). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cisisomers. In the final extract, the proportion of cis-isomers in total carotenoids is 64.7% globally (cis-trans ratio = 1.83 in average of three repetitions). The enrichment factor in cis-isomers is thus equal to 13.7%.

Table VIII. Detailed composition of the extracts obtained in Example 8. Me-THF: 2- methyloxolane .

Example 9. Use of pure 2-methyl oxolane at a 1:10 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under magnetic stirring.

Twenty five grams of a freeze-dried powder of Dunaliella salina microalgae (containing 6.4% (w:w) total carotenoids) were incubated at reflux (78 °C) with 250 mL of pure 2- methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 18.0% were obtained. Content of total carotenoids in the extracts was found to be 21.7% (w:w), which corresponds to a recovery rate of 60.5% (w:w).

Two repetitions of this extraction gave a mass yield of 16.0 and 16.5%, a carotenoid content of 19.3 and 19.5%, and a carotenoid recovery rate of 47.9 and 50.2%.

For comparison purposes, the inventors performed the extraction in the exact same conditions except that we used a reflux at 68 °C and n-hexane, a petroleum-based solvent currently used for the extraction of carotenoids. The three repetitions gave a mass yield of 13.3, 13.6, and 13.1%, a carotenoid content of 28.3, 26.6, and 24.6%, and a carotenoid recovery rate of 58.3, 56.0, and 50.0%.

Table IX shows the carotenoid composition of the extract obtained in Example 9. In the native freeze-dried powder of Dunaliella salina microalgae the proportion of cisisomers is of 56.9% (cis-trans ratio = 1.32). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cisisomers. In the final extract, the proportion of cis-isomers in total carotenoids is 65.0% globally (cis-trans ratio = 1.86 in average of three repetitions). The enrichment factor in cis-isomers is thus equal to 14.2%.

Table IX. Detailed composition of the extracts obtained in Example 9 Me-THF: 2- methyloxolane.

Example 10. Use of pure 2-methyloxolane at a 1:5 microalgae:solvent (w:v) ratio in a two-pass extraction procedure for 4 hours each pass at reflux (78 °C) under magnetic stirring.

Fifty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 6.4% (w:w) total carotenoids) were incubated at reflux (78 °C) with 250 mL of pure 2- methyloxolane for 4 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate corresponding to the first pass was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 13.9% were obtained. Content of total carotenoids in the extract was found to be 23.3% (w:w), which corresponds to a recovery rate of 50.3% (w:w).

These extraction and filtration steps were reiterated (second pass) on plant residues resulting from the first extraction pass. Fresh solvent was used here in the same conditions and quantities as in the first pass. The filtrate corresponding to the second pass was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 3.4% were obtained. Content of total carotenoids in the extract was found to be 32.6% (w:w), which corresponds to a recovery rate of 17.4% (w:w). The global mass yield carotenoid content and carotenoid recovery rate of the pooled extracts collected through the two passes were 17.4, 25.1, and 67.7% (w:w), respectively.

A repetition (repetition 2) of this two-pass extraction procedure gave a mass yield of 13.5 and 4.3%, a carotenoid content of 24.7 and 33.6%, and a carotenoid recovery rate of 51.8 and 22.4% for the first and second passes, respectively. The global mass yield, carotenoid content and carotenoid recovery rate of the pooled extracts collected through the these two passes were 17.8, 26.9, and 74.2% (w:w), respectively.

Another repetition (repetition 3) of this two-pass extraction procedure gave a mass yield of 12.6 and 3.6%, a carotenoid content of 26.8 and 34.0%, and a carotenoid recovery rate of 52.4 and 19.1% for the first and second passes, respectively. The global mass yield, carotenoid content and carotenoid recovery rate of the pooled extracts collected through these two passes were 16.2, 28.4, and 71.6% (w:w), respectively.

Table X shows the carotenoid composition of the different extracts obtained in Example 10 using 2-methyloxolane as extraction solvent.

Table X. Detailed composition in % (w:w) of the extracts obtained in Example 10.

Example 11. Use of pure n-hexane at a 1:5 microalgae:solvent (w:v) ratio in a two pass extraction procedure for 4 hours each pass at reflux (68 °C) under magnetic stirring.

For comparison purposes, the inventors performed an extraction in the exact same conditions as in Example 10 except that here they used a reflux at 68 °C and n- hexane, a petroleum-based solvent currently used for the extraction of carotenoids.

A first repetition of the two-pass extraction procedure with n-hexane gave a mass yield of 11.1 and 2.5%, a carotenoid content of 36.5 and 35.3%, and a carotenoid recovery rate of 62.8 and 13.8% for the first and the second passes, respectively. When extracts resulting from the two passes are pooled together, the mass yield is of 13.6%, the carotenoid content is of 36.3%, and the carotenoid recovery rate is of 76.6%.

A second repetition of the two-pass extraction procedure with n-hexane gave a mass yield of 11.4 and 3.0%, a carotenoid content of 35.7 and 34.6%, and a carotenoid recovery rate of 63.4 and 16% for the first and the second passes, respectively. When extracts resulting from the two passes are pooled together, the mass yield is of 14.4%, the carotenoid content is of 35.5%, and the carotenoid recovery rate is of 79.4%.

Table XI shows the carotenoid composition of the hexanic extract obtained in Example 11 using hexane as extraction solvent. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cis-isomers of 56.9% (cis-trans ratio = 1.32). In the final extract, the proportion of cis-isomers in total carotenoids to 60.2% globally (cis-trans ratio = 1.5 in average for the two repetitions). There is a very low enrichment of cis-isomers using Hexane (enrichment factor in cis-isomers equal to 5.7%).

Table XI. Detailed composition of the extracts obtained in Example 11.

Example 12. Use of pure 2-methyloxolane at a 1 :3 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under magnetic stirring.

Fifty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 11.0% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2- methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 22.3% were obtained. Content of total carotenoids in the extracts was found to be 35.1% (w:w), which corresponds to a recovery rate of 71.4% (w:w).

Two repetitions of this extraction gave a mass yield of 23.7 and 22.2%, a carotenoid content of 29.5 and 26.3%, and a carotenoid recovery rate of 64.0 and 53.4%.

Table XII shows the carotenoid composition of the extract obtained in Example 12. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers of 48.5% (cis-trans ratio = 0.9). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 59.5% globally (cis-trans ratio = 1.5 in average of three repetitions). The enrichment factor in cis- isomers is thus equal to 22.7%.

Table XII. Detailed composition of the extracts obtained in Example 12. Me-THF: 2- methyloxolane

Example 13. Use of pure 2-methyloxolane at a 1:15 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under magnetic stirring.

Ten grams of a freeze-dried powder of Dunaliella salina microalgae (containing 11.0% (w:w) total carotenoids) were incubated at reflux (78 °C) with 250 mL of pure 2- methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 36.3% were obtained. Content of total carotenoids in the extracts was found to be 28.5% (w:w), which corresponds to a recovery rate of 94.5% (w:w).

Two repetitions of this extraction gave a mass yield of 36.2 and 37.4%, a carotenoid content of 29.1 and 28.5%, and a carotenoid recovery rate of 96.1 and 97.3%.

Table XIII shows the carotenoid composition of the extract obtained in Example 13. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers of 48.5% (cis-trans ratio = 0.9). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 57.2% globally (cis-trans ratio = 1.3 in average of three repetitions). The enrichment factor in cis- isomers is thus equal to 17.9%. Table XIII Detailed composition of the extracts obtained in Example 13. Me-THF: 2- methyloxolane.

Example 14. Use of pure 2-methyloxolane at a 1:5 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under magnetic stirring.

Thirty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 11.4% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2- methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 32.1% were obtained. Content of total carotenoids in the extracts was found to be 30.1% (w:w), which corresponds to a recovery rate of 84.5% (w:w).

Two repetitions of this extraction gave a mass yield of 35.2 and 34.1%, a carotenoid content of 28.8 and 28.9%, and a carotenoid recovery rate of 88.7 and 86.3%.

Table XIV shows the carotenoid composition of the extract obtained in Example 14. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers of 49.0% (cis-trans ratio = 1.0). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 59.5% globally (cis-trans ratio = 1.5 in average of three repetitions). The enrichment factor in cis- isomers is thus equal to 21.4%. Table XIV. Detailed composition of the extracts obtained in Example 14. Me-THF: 2- methyloxolane.

Example 15. Use of pure 2-methyloxolane at a 1:8 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under magnetic stirring.

Around nineteen grams of a freeze-dried powder of Dunaliella salina microalgae (containing 11.4% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2-methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 35.6% were obtained. Content of total carotenoids in the extracts was found to be 29.8% (w:w), which corresponds to a recovery rate of 93.0% (w:w).

Two repetitions of this extraction gave a mass yield of 35.3 and 36.6%, a carotenoid content of 29.8 and 28.9%, and a carotenoid recovery rate of 92.1 and 92.5%.

Table XV shows the carotenoid composition of the extract obtained in Example 15. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers of 49.0% (cis-trans ratio = 1.0). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 58.6% globally (cis-trans ratio = 1.4 in average of three repetitions). The enrichment factor in cis- isomers is thus equal to 19.6%. Table XV. Detailed composition of the extracts obtained in Example 15. Me-THF: 2- methyloxolane

Example 16. Use of pure 2-methyloxolane at a 1:10 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under magnetic stirring.

Fifteen grams of a freeze-dried powder of Dunaliella salina microalgae (containing 11.4% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2- methyloxolane for 8 hours under magnetic stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 35.6% were obtained. Content of total carotenoids in the extracts was found to be 29.5% (w:w), which corresponds to a recovery rate of 91.9% (w:w).

Two repetitions of this extraction gave a mass yield of 32.8 and 35.7%, a carotenoid content of 32.4 and 28.6%, and a carotenoid recovery rate of 92.9 and 89.4%.

Table XVI shows the carotenoid composition of the extract obtained in Example 16. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers of 49.0% (cis-trans ratio = 1.0). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 58.1% globally (cis-trans ratio = 1.4 in average of three repetitions). The enrichment factor in cis- isomers is thus equal to 18.6%. Table XVI. Detailed composition of the extracts obtained in Example 16. Me-THF: 2- methyloxolane.

Example 17. Use of pure 2-methyloxolane at a 1:3 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under mechanical stirring.

Fifty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 11.6% (w:w) total carotenoids) were incubated at reflux (78 °C) with 150 mL of pure 2- methyloxolane for 8 hours under mechanical stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 33.4% were obtained. Content of total carotenoids in the extracts was found to be 31.3% (w:w), which corresponds to a recovery rate of 90.3% (w:w).

Two repetitions of this extraction gave a mass yield of 34.0 and 33.2%, a carotenoid content of 30.5 and 31.0%, and a carotenoid recovery rate of 89.4 and 88.9%.

Table XVII shows the carotenoid composition of the extract obtained in Example 17. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers of 49.1% (cis-trans ratio = 1.0). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 57.8% globally (cis-trans ratio = 1.4 in average of three repetitions). The enrichment factor in cis- isomers is thus equal to 17.7%. Table XVII. Detailed composition of the extracts obtained in Example 17. Me-THF: 2- methyloxolane.

Example 18. Use of pure 2-methyloxolane at a 1:5 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under mechanical stirring.

Forty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 12.5% (w:w) total carotenoids) were incubated at reflux (78 °C) with 200 mL of pure 2- methyloxolane for 8 hours under mechanical stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 35.6% were obtained. Content of total carotenoids in the extracts was found to be 31.9% (w:w), which corresponds to a recovery rate of 91.2% (w:w).

Three repetitions of this extraction gave a mass yield of 34.6. 34.7 and 36.5%, a carotenoid content of 35.0. 33.8 and 31.1%, and a carotenoid recovery rate of 97.2, 94.2 and 91.0%.

Table XVIII shows the carotenoid composition of the extract obtained in Example 18. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers in total carotenoids of 40.9% (cis-trans ratio = 0.7). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 58.0% globally (cis-trans ratio = 1.4 in average of four repetitions). The enrichment factor in cis-isomers is thus equal to 41.8%. Table XVIII. Detailed composition of the extracts obtained in Example 18. Me-THF: 2- methyloxolane.

Example 19. Use of pure 2-methyloxolane at a 1:5 microalgae:solvent (w:v) ratio in a one-pass extraction procedure for 8 hours at reflux (78 °C) under mechanical stirring.

Forty grams of a freeze-dried powder of Dunaliella salina microalgae (containing 12.4% (w:w) total carotenoids) were incubated at reflux (78 °C) with 200 mL of pure 2- methyloxolane for 8 hours under mechanical stirring. The enriched solution was separated from the residual microalgal materials by filtration. The filtrate was analyzed by HPLC. After evaporation of the solvent under reduced pressure, a mass yield of 37.0% were obtained. Content of total carotenoids in the extracts was found to be 32.2% (w:w), which corresponds to a recovery rate of 96.1% (w:w).

One repetition of this extraction gave a mass yield of 36.6%, a carotenoid content of 32.8%, and a carotenoid recovery rate of 96.8%.

Table XIX shows the carotenoid composition of the extract obtained in Example 19. The native freeze-dried powder of Dunaliella salina microalgae has a proportion of cisisomers of 41.1% (cis-trans ratio = 0.7). Applying the method of the invention enables to transform trans isomers in cis isomers, and thus increase the final % of cis-isomers. In the final extract, the proportion of cis-isomers in total carotenoids is 58.4% globally (cis-trans ratio = 1.4 in average of two repetitions). The enrichment factor in cis- isomers is thus equal to 42.0%. Table XIX. Detailed composition of the extracts obtained in Example 19. Me-THF: 2- methyloxolane.

Example 20. Solubility of microalgal carotenoids in sunflower oil as a function of the cis-isomer proportion.

A Dunaliella salina extract containing 15.8% (w:w) of carotenoids (of which 41.1% were trans-isomers and 58.9% were cis-isomers) was mixed with pure frans-beta-carotene (Sigma-Aldrich) at different weight ratios (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0; Table I). The mixture was then added in excess to 10 mL sunflower oil in a 20-mL I KA shaker tube containing 10 g of ceramic beads. The experiment was done in duplicate. The mixture was stirred mechanically by the effect of the beads at 4,000 rpm for 30 min at 25 °C. The residue was removed by filtration, and the resulting filtrate was centrifuged at 12,000 rpm for 15 min. After centrifugation, the supernatant was collected and adjusted to 20 mL in a volumetric flask and analyzed by HPLC.

Overall, the higher the c/s-isomer proportion in the carotenoid mixture, the higher the solubility of total carotenoids in sunflower oil (Figure 2), which is advantageous for formulation purpose.

Example 21. Direct extraction from wet algae, with optional cis-trans separation.

The extraction of total carotenoids from the algal paste (about 80% water) was performed using an Ultra-Turrax rotor-stator (T 18 digital ULTRA-TURRAX®) wherein 100 g of algal paste were suspended in 100 mL of 2-methyloxolane, and then sheared at 18,000 rpm for 10 min, at 25 °C with no control of temperature. The suspension obtained was centrifuged for 15 min at 8,000 rpm. The supernatant containing the methyloxolane phase rich in carotenoids was then recovered with a Pasteur pipette and analyzed by HPLC.

When procedure of Example 21 was repeated on dry pellets from algae that were rehydrated to 20% water (to artificially mimic authentic algal paste), the yield was much lower.

Example 22. Cis-trans separation.

The procedure of Example 21 was reproduced except that filtration was used instead of centrifugation after the Ultra-Turrax step. The resulting filtrate was concentrated to dryness and the obtained dry extract was mixed with 100 mL ethanol to further separate the cis-isomers from the trans-isomers. The mixture was filtered using a Buchner filter. During the filtration, the trans-carotenoids remained on the filter as crystals (solids), while the cis-carotenoids were mostly solubilised in the ethanol phase.

Dry pellets from algae that were rehydrated and the procedure of Example 21 was repeated without success.

Claims

1. A method of conversion of trans-carotenoids into cis-isomers of carotenoids from Dunaliella and extraction of a cis-carotenoid enriched extract, comprising a) contacting the cells with 2-methyloxolane (Me-THF) or a mixture of Me-THF with a second solvent, b) optionally removing the solvent, thereby obtaining a cis- carotenoid enriched extract.

2. A method for enrichment and extraction of cis-carotenoids from a Dunaliella biomass comprising: a) contacting the cells with 2-methyloxolane (Me-THF) or a mixture of Me-THF with a second solvent, b) optionally removing the solvent, thereby obtaining a carotenoid extract enriched in cis-carotenoids.

3. The method according to claim 1 or 2 wherein the second solvent is selected from the group consisting of hexane, dichloromethane (DCM), a monoterpene or a mixture thereof.

4. The method according to claim 3 wherein the monoterpene is selected from the group consisting of R-limonene (syn. D-limonene), turpentine oil and p-menthane.

5. The method according to any one of claims 1 to 4 wherein the solvent is provided in substantially pure form.

6. The method according to any one of claims 1 to 5 wherein the extraction is carried out using the Soxhlet technique.

7. The method according to any one of claims 1 to 6 wherein the step a) and/or b) is carried out at a temperature of about of about 20-130°C, such as of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, to about 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20°C such as about 30-90 °C, such as about 70 to 85, such as 78 °C.

8. The method according to any one of claims 1 to 7 wherein the Dunaliella biomass is provided in dry form.

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9. The method according to any one of claims 1 to 7 wherein the step b) is carried out at a starting dry biomass to solvent ratio of about 1 :3 w/v to 1 :20 w/v, such as 1 :3 to 1:15, such as about 1:3 to 1 :10, such as 1:3 to 1 :5, such as about 1 :5.

10. The method according to any one of claims 1 to 7 wherein the Dunaliella biomass is provided as a wet paste and wherein the biomass is used without a prior drying or with a prior drying, wherein said prior drying is partial drying which results in a biomass having a water content of at least about 80% (w/v).

11. The method according to claim 10, wherein the extraction (step a) is carried out by shearing the wet paste with the solvent.

12. The method according to any one of claims 1 to 11, wherein an additional step of treating the mixture so as to separate the residual cell material, the aqueous phase and the organic phase containing the solvent is performed after step a).

13. The method according to any one of claims 1 to 12 wherein the solvent is provided as a mixture with an acceptable vehicle which is adequate for forming a solution or a dispersion of carotenoids.

14. The method according to any one of claims 1 to 13 wherein the product is further processed to separate the cis and the trans isomers of the carotenoids.

15. The method according to claim 14 wherein the further processing is carried out by drying the extract, adding to the dry extract an isomer-specific solvent which preferentially solubilizes one type of isomers and separating the isomer typespecific solvent from the insoluble fraction.

16. The method according to claim 15 wherein the isomer type-specific solvent is ethanol and the isomer type which is solubilized is the cis isomer.

17. The method according to any one of claims 1 to 16 wherein the Dunaliella biomass has been grown under natural light or white conditions.

18. The method according to any one of claims 1 to 17 wherein the weight ratio of cis- to trans-carotenoids in the cis-carotenoid enriched extract is at least 5% higher than that found in a Dunaliella biomass.

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19. A carotenoid composition obtained by a method according to any one of claims 1 to 18.

20. A carotenoid composition characterized in that: a. the content of cis-carotenoids is of at least about 30% in weight with respect to the total carotenoid content, b. the weight ratio of cis- to trans carotenoids is of at least 0.4, such as at least 0.7, and/or c. the weight ratio of cis- to trans-carotenoids is at least 5% higher than that found in a Dunaliella biomass.

21. The carotenoid composition according to claim 20 which has been obtained by a method as defined in any of claims 1 to 18.

22. The carotenoid composition according to any one of claims 19 to 21 wherein the cis carotenoids present in the composition are 13-cis p-carotene, 15-cis p-carotene and/or 9-cis p-carotene and wherein the trans carotenoids are lutein, zeaxanthine, a-carotene and all-trans p-carotene.

23. The carotenoid composition according to any one of claims 19 to 22 wherein the concentration of 13-cis p-carotene in the composition is of at least about 0.6 % (w:w), the concentration of 15-cis p-carotene in the composition is of at least about 2.8 % (w/w) and/or the concentration of 9-cis p-carotene in the composition is of at least about 7.8% (w/w).

24. The carotenoid composition according to any of claims 19 to 23 wherein the concentration of lutein in the composition is of less than 1 % (w/w), the concentration of zeaxanthine in the composition is of less than about 0.5 % (w/w), the concentration of a-carotene is of less than about 6 % and/or the concentration of all-trans p-carotene in the composition is of less than about 24% (w/w).

25. A pharmaceutical composition, a foodstuff, a nutritional supplement, a cosmetic composition or a nutraceutical composition comprising a carotenoid composition as defined in any of claims 19 to 24 and a vehicle adequate for the formulation of the composition into said pharmaceutical composition, foodstuff, nutritional supplement, cosmetic composition or nutraceutical composition.

60 A cosmetic method for the treatment in a subject of a condition resulting from deficient carotenoid levels and/or deficient pro-vitamin A levels which comprises the administration to the subject of a composition as defined in any of claims 19 to 24. A carotenoid composition as defined in any of claims 19 to 24 for use in the treatment of a disease associated with reduced carotenoid and/or pro-vitamin A levels in the body. Use of a carotenoid composition according to any of claims 19 to 24 as antioxidant, food colorant or cosmetic colorant.

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