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    • 专利摘要:
    Procedure for the extraction of carotenoids using liquid nanostructured phases. The present invention relates to a process for obtaining and enriching carotenoids from biomass, based on the use of nanostructured liquid phases, and to the use of the carotenoids thus obtained as food additives and in the pharmaceutical industry. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2694600A1
    申请号:ES201730822
    申请日:2017-06-21
    公开日:2018-12-21
    发明作者:Soledad RUBIO BRAVO；Mª Dolores CRIADO SICILIA；Carmen CABALLO LINARES；Noelia CABALLERO CASERO；Graciela Pavon-Djavid；Virginie GUEGUEN；Jorge Eduardo BASTIAS VENEGAS
    申请人:Universidad de Cordoba；Universite Sorbonne Paris Nord Paris 13；
    IPC主号:
    专利说明:

    DESCRIPTION
    PROCEDURE FOR THE EXTRACTION OF CAROTENOIDS USING NANOSTRUCTURED LIQUID PHASES
    Sector of the technique
    The present invention falls within the general field of chemistry of natural products and in particular it relates to a process for obtaining carotenoids from biomass, and to the use of said products in the pharmaceutical and food industry where carotenoids are used. They use as nutritional supplements and additives.
    State of the art
    Carotenoids (carotenoids and xanthophylls) are pigments synthesized by photosynthetic organisms, some bacteria and fungi. They are used as food additives in aquaculture for coloring the meat of salmonids and as nutraceuticals and additives in food for human consumption. Among the benefits shown or attributed to carotenoids are its anti-tumor activity, anti-inflammatory and antidiabetic properties, and protective effect of the heart, nervous system, eyes and skin (Microalgae Biotechnology 15, E. Forján Lozano, C. Vilchez Lobato, JM Vega Piqueres , Cepsa 2014, ISBN 974-84-617-2314-0).
    The carotenes β-carotene and lycopene and the xanthophylls astaxanthin, lutein and canthaxanthin are the carotenoids of greatest commercial interest. It is estimated that the global carotenoid market will reach $ 1.4 trillion in 2019 with an annual growth rate of 3.5% since 2014 (http://www.bcresearch.com). Carotenoids are obtained by synthetic procedures or through natural sources. Although synthetic forms are cheaper, natural forms have better nutritional properties and the demand for these types of sources has experienced a great increase in recent years. The main natural sources of carotenoids are 25 microalgae (eg Dunaliella salina for β-carotene and Haematococcus pluvialis for astaxanthin), yeasts (eg Phaffia rhodozyma for astaxanthin) and flowers (eg petals of Tagetes erecta for lutein). Xanthophylls are esterified in some sources (eg astaxanthin in Haematococcus pluvialis and lutein in Tagetes erecta).
    Many methods have been described for obtaining carotenoids from 30 microalgae (EP1808483B1, 05.10.2011, Cognis IP Management GmbH / University of Seville, Mar. Drugs, 2011, 9, 625-644). In general, the biomass is separated from the
    culture by centrifugation, sedimentation or filtration and the concentrate (5-10% dry weight) is dehydrated by spraying or lyophilization. Breaking microalgae cells with homogenizers, ultrasounds, glass bead mill, freezing, osmotic shock, etc., is essential to release intracellular carotenoids, and thus increase their digestibility for humans and animals. This operation is carried out in the presence of antioxidants to prevent the degradation of carotenoids (WO0277105, 03.03.2002, Fuji Chemical Industry Co.). The dry biomass, packed in vacuum and stored at temperatures of -20ºC, is marketed for use in aquaculture and as an additive in foods for human consumption. Part of this biomass is subjected to processes of enrichment of the carotenoids present in it by extraction with organic solvents or supercritical fluids, for later use as a nutraceutical in the form of capsules, tablets, etc. (Current Anal. Chem., 2014, 10, 29-66; Anal. Methods, 2013, 5, 2916-2924, Recent Patents on Food, Nutrition and Agriculture, 2010, 2, 75-82).
    The enrichment with organic solvents is carried out using polar solvents (eg acetone, methanol, ethanol, ethyl acetate, etc., US14420136, 04.19.2016, Roquette Freres, 15 US20030087335 A1, 08.05.2003, Jacobson Holman PLLC, US8097761B2, 17.01 .2012, JX Nippon Oil & Energy Corporation), non-polar (eg hexane, pentane, benzene, etc., US20070196383 A1, 08.23.2007, Yamaha Hatsukoki Kabushiki Kaisha), or mixtures thereof to adjust polarity and increase efficiency extraction (eg isopropanol: hexane, ethanol: hexane, water: ethanol: hexane, alkane: methanol, etc. WO2009063100A1, 22.05.2009, 20 University of Almeria, US20070196894 A1, 08.23.2007, Sungkyunkwan University, Chin. Chem. Eng. 2013, 21, 776-780). The main disadvantage of the use of organic solvents is that multiple extraction stages are required to obtain acceptable yields, high ratios of biomass to solvent (generally 1:10), high temperatures and extraction times between 24 and 48 h. In addition, it is necessary to evaporate high volumes of solvent in the extract, a process in which carotenoids can degrade. On the other hand, the legislation regarding nutraceuticals and food additives is very restrictive with respect to solvent residues, so the use of solvents included in category 3 is recommended by the US Food and Drug Administration, FDA (eg ethanol) , acetone, ethyl acetate, etc.) and that the final product 30 contains an amount of solvent lower than 0.5% (Guidance for Industry Q3C, FDA, 2012). These restrictions, together with the pressure of legal regulations on the control, prevention and remediation of environmental pollution, have promoted the use of less toxic solvents such as vegetable oils, although the extraction with the
    It requires 48 hours to achieve an extraction yield of 88% and no industrial applications have been developed (Biotechnol Letter 2008, 30, 441-444).
    Enrichment with supercritical fluids (EFS) is carried out mainly with carbon dioxide, either alone or in the presence of polar modifiers, since it is harmless, non-flammable and relatively inert (Int. J. Mol. Sci. 2014, 15, 6725-6740). The main advantage 5 of the use of EFS is that it reduces the consumption of organic solvents and the supercritical fluids used can be recycled. However, the operating costs are very high and a high initial investment is needed, in addition to low equipment availability and reduced design development. On the other hand, xanthophils, relatively polar and high molecular weight compounds, have low solubility in carbon dioxide, so it is required to work at very high pressures (over 50 MPa) to obtain acceptable yields and extraction times, which considerably increases the costs. The addition of ethanol as a modifier (10-70%) allows to obtain yields of approximately 85% because it improves the humidification of the pores of the biomass and establishes hydrogen bonds with the xanthophylls (J. Supercritical 15 Fluids, 2014, 92 , 75-83, Eur. Food Res. Technol. 2006, 223, 787-790), however, it is necessary to eliminate it from the extract. The extraction yield can be increased using supercritical fluids that establish hydrogen bonds with xanthophylls, such as dimethyl ether (US 7696396B2, 06.196.2008, Phasex Corporation, Lawrence, MA, US). The product obtained is generally an oleoresin containing the carotenoids and fatty acids present in the biomass while the polar components such as proteins and carbohydrates remain in the residue.
    Given the limitations of the above processes, there is a need to develop extraction and enrichment methods for carotenoids from natural sources using fast, efficient, economical and safe processes that do not require special installations or complicated operations and provide products that do not contain toxic waste and, therefore, can be used in pharmaceutical and food applications.
    BRIEF DESCRIPTION OF THE INVENTION
    The present invention solves the problems described in the state of the art since it provides a method for the extraction and enrichment of carotenoids from biomass, based on the use of nanostructured liquid phases obtained by spontaneous processes of self-assembly and coacervation of amphiphilic molecules .
    The type of nanostructures and components of these liquid phases maximizes the interaction energies with the carotenoids, avoids the extraction of the macromolecules present in the plant biomass through chemical and physical exclusion mechanisms and provides extracts enriched in carotenoids that can be used directly, or dilution with a vegetable oil, for the formulation of nutraceuticals and food additives. The method is fast, simple and of low cost, develops at atmospheric pressure and room temperature, using non-toxic solvents and providing a quantitative extraction of carotenoids in a single stage of equilibrium between the plant biomass and the solvent. The composition of the extracts enriched in carotenoids is similar to the oleoresins obtained by extraction with supercritical fluids without the requirement of special and expensive facilities.
    Thus, in a first aspect, the present invention relates to a process (hereinafter, method of the present invention) for obtaining carotenoids from biomass, comprising the following steps:
    a) dissolving an amphiphilic surfactant in an organic solvent and adding water, in a ratio comprised between 1: 4: 2.6 and 1: 7: 13 (g: mL: mL).
    b) centrifuge the mixture obtained in a), at least 3000 rpm for 10 min, obtaining a hydro-organic phase and a nanostructured liquid phase,
    c) separate the two phases obtained in stage b)
    d) mixing the biomass with the hydro-organic phase and the liquid phase obtained in step b) in a ratio comprised between 1: 0: 2 and 1: 10: 1 (g: mL: mL)
    e) centrifuge the mixture obtained in d) for at least 10 minutes at least 2500 rpm, obtaining 3 differentiated phases: liquid phase, solid phase and an intermediate phase,
    f) purifying the liquid phase obtained in e) to obtain the carotene extract.
    In a particular aspect of the present invention, the amphiphilic surfactant comprises a hydrocarbon chain of between 6 and 18 carbon atoms and at least one polar group selected from carboxylic groups, alcohols, aldehydes, ketones, phosphates.
    In another particular aspect of the present invention, the amphiphilic surfactant is selected from glycolipids, fatty acids, phospholipids, lipopeptides, neutral lipids, or any surfactant of natural origin. 30
    In another particular aspect of the present invention, the organic solvent is selected from tetrahydrofuran, ethylene glycol, dioxane, acetone, propanol, ethanol, acetonitrile and methanol.
    In another particular aspect of the present invention, the purification stage f) is carried out by eliminating the organic solvent. More particularly, the removal of the organic solvent is carried out by evaporation of the solvent by nitrogen flow.
    In another particular aspect, the process of the present invention comprises an additional step of diluting the extract obtained in step f) with a vegetable oil.
    In a particular aspect of the present invention, the biomass is vegetable, fungal or yeast biomass.
    In another particular aspect of the present invention, the biomass comprises a pretreatment of drying and grinding.
    In a second aspect, the present invention relates to the use of the extract obtained by the process of the present invention as a food additive. fifteen
    In another aspect, the present invention relates to the use of the extract obtained by the process of the present invention in pharmaceutical formulations.
    Brief description of the figures
    Figure 1 shows the chromatograms obtained by analyzing the extract of Haematococcus pluvialis samples produced by (A) extraction with supercritical fluids and (B) extraction with nanostructured liquid phases according to the procedure described in the invention. The total content and distribution of carotenoids present in the plant biomass depends on the culture conditions and strains used. Thus, the total content of astaxanthin generally varies between 3 and 5% of the weight of the biomass while the distribution of free astaxanthin, monoesters and diesters in Haematococcus pluvialis can vary in the ranges 1-5%, 46-79% and 10-39%, respectively. The distribution of free astaxanthin, monosters and diesters in the analyzed extracts were: (A) 1.7%, 76.1% and 22.2% and (B) 1.7%, 78.8% and 19.5%. The column used for the chromatographic analysis of the extracts was an Ultrabase C18 (5 μm, 250 mm × 4.6 mm internal diameter) supplied by Vínicos Analysis (Tomelloso, 30 Spain) and thermostatted at 20 ° C. For the elution a mobile phase constituted by acetone and water with an elution gradient from 83:17 to 98: 2 in 80 min at a speed was used.
    flow rate of 0.8 mL / min. The wavelength range measured in the diode detector in rows was 200 to 800 nm.
    Detailed description of the invention
    The nanostructured liquid phases were synthesized from amphiphilic molecules through spontaneous self-assembly and coacervation processes under experimental conditions in which the aggregation between the amphiphiles is more favorable than the interaction of the same with the solvent (The colloidal domain, where physics , chemistry, biology and technology meet, F. Evans, H. Wennnerströn, Wiley, BCH, 1999, 2nd edition). The most important differential characteristic of these liquid phases with respect to other solvents (eg organic solvents, ionic liquids and supercritical fluids) is that the components that make it form nanostructures sensitive to environmental stimuli and these can be designed to fulfill specific functions. There are a multitude of amphiphilic substances of natural and synthetic origin for the development of nanostructured liquid phases, although up to now they have not been used in industrial extraction processes. fifteen
    By means of the process of the present invention, nanostructured liquid phases are obtained with suitable characteristics for the extraction of carotenoids and the development of a process for obtaining extracts enriched from biomass that can be used directly, or after dilution with a vegetable oil, for the formulation of nutraceuticals and food additives. twenty
    Design and synthesis of nanostructured liquid phases
    The amphiphilic substances used in the present invention for the synthesis of liquid phases were preferably amphiphiles that allowed the formation of hydrogen bonds, and the establishment of polar interactions, in addition to dispersion interactions, with xanthophylls. Examples of amphiphilic substances for the synthesis of the nanostructured liquid phases are those containing polar groups consisting of carboxylic groups, alcohols, aldehydes, ketones, phosphates, etc.
    The amphiphilic substance used in the present invention must be compatible and legally accepted for use in pharmaceutical formulations and food additives. In addition, it must have low solubility in water and high solubility in organic solvents miscible in water. Examples of organic solvents that can be used for the synthesis of the nanostructured liquid phases are tetrahydrofuran, ethylene glycol, dioxane, acetone, propanol,
    ethanol, acetonitrile, methanol, etc. Solvents included in category 3 are preferred by the US Food and Drug Administration, FDA (eg ethanol, acetone, etc.).
    The amphiphilic substance must produce, in the selected hydro-organic medium, at room temperature and through spontaneous self-assembly and coacervation processes, the nanostructured liquid phase. 5
    The percentage of organic solvent in the organic medium was between 5 and 40% (v / v).
    The spatial arrangement of the amphiphilic substances in the nanostructures maximizes the interactions by hydrogen, polar and dispersion bridges with the carotenoids.
    The nanostructures were reversible and adapted to the environment, that is to say the organic hydro-10 medium in which they self-assemble and coacervate, in such a way that they allow the easy modification of them according to the extraction requirements.
    The formed nanostructures effectively solubilize the carotenoids excluding the extraction of polar macromolecules (proteins, carbohydrates, etc.) through chemical and physical mechanisms. fifteen
    Process of extraction of carotenoids in vegetable biomass
    The process developed in the present invention is applicable to the extraction of carotenoids and xanthophylls from vegetable biomass, including microalgae (eg Dunaliella salina, Haematococcus pluvialis, Chlorella, Scenedemus almeriensis, etc.) and plants (eg Tagetes erecta and Tagetes patula ). It can also be extended to other sources of carotenoids such as yeasts (eg Phaffia rhodozyma), after optimization of the experimental extraction conditions.
    The plant biomass is preferably dry and crushed.
    The nanostructured liquid phase was synthesized and separated from the hydro-organic solution in equilibrium with it, by centrifugation. 25
    A volume of nanostructured liquid phase and equilibrium solution was added to the dried and ground vegetable biomass. The function of the equilibrium solution is to rehydrate the plant biomass, thus decreasing the need to use the nanostructured liquid phase for this purpose. The ratio biomass: equilibrium solution: nanostructured liquid phase (p / v / v; g / mL / mL) varies in the range 1: 0: 2 and 1: 10: 1 and preferably in the interval 1: 2: 1 and 30 1: 3: 1.
    The mixture was stirred in the range 2-15 min and preferably for 5 min.
    The mixture was centrifuged until obtaining three phases; a solid phase that contains the residue of the plant biomass constituted fundamentally by proteins, carbohydrates and mineral substances; an intermediate liquid phase which is the hydro-organic equilibrium solution and the nanostructured liquid phase containing the carotenoids and fatty acids of the biomass, in addition to the constituents of the nanostructured liquid phase.
    The process allowed the extraction of free and esterified carotenoids from the plant biomass with a percentage of recovery over 90% using a single stage of equilibrium between the biomass and the nanostructured liquid phase. The equilibrium was reached in a short time interval using a ratio of biomass to nanostructured liquid phase 10 of 1: 1 (m: v).
    The hydro-organic equilibrium solution can be recycled for the synthesis of new nanostructured phases while the residue of the plant biomass can find other applications related to the use of proteins and carbohydrates. Thus, the present invention could be part of the process flow diagram of a biorefinery. fifteen
    Obtaining oleoresins enriched in carotenoids
    The extract obtained in the process described above can be transformed into an oleoresin, similar to that obtained with EFS, after elimination of the organic solvent contained therein with a nitrogen stream or similar procedure that maintains the integrity of the carotenoids. twenty
    By eliminating the organic solvent, which can be recycled, the weight of the extract is reduced approximately 2.5 times, obtaining a carotenoid enrichment factor in the oleoresin equivalent to this weight reduction. This enrichment factor is similar to that obtained with EFS.
    The distribution of carotenoids in the extract obtained in the present invention is similar to that of the vegetable biomass and that obtained with EFS (Figure 1).
    As with oleoresin obtained with EFS, the extract has a higher concentration of lipids than plant biomass, but this is irrelevant since the oleoresin is diluted with vegetable oils for commercialization as a nutraceutical or for use as a food additive. 30
    The obtained extract, therefore, is similar to the products already existing in the market obtained by EFS, but its obtaining is done with a simple procedure that does not
    it requires large installations or high investments. The consumption of energy and materials is very low compared to current processes and, therefore, the process proposed in this invention is more economical.
    EXAMPLES OF EMBODIMENT OF THE INVENTION
    Example 1: Synthesis of nanostructured liquid phases 5
    The amphiphilic substance was dissolved in ethanol (12%, w / v) and the solution was diluted with water by a factor of 1.5. The mixture was homogenized by magnetic stirring for 5 min. The nanostructured liquid phase, produced spontaneously, was separated from the hydro-organic solution in equilibrium with it by centrifugation at 3500 rpm for 10 min and transferred to another vessel. Both phases were kept in hermetically sealed containers until use.
    Example 2. Extraction of astaxanthin from Haematococcus pluvialis
    The appropriate amount of Haematococcus pluvialis biomass, dry and crushed, was mixed with the hydro-organic equilibrium solution and the nanostructured liquid phase, synthesized according to the procedure described in the previous example, in a ratio of 1: 2.5: 1 15 ( p / v / v). The mixture was magnetically stirred for 5 min at 900 rpm and then centrifuged for 10 min at 3500 rpm for physical separation of three phases. In the nanostructured liquid phase, which contains the carotenoids, the organic solvent was evaporated by a stream of nitrogen to obtain an extract that can be used directly, or after dilution with vegetable oil, for pharmaceutical formulations or as a food additive. As an example, the recommended daily dose of astaxanthin intake for humans is around 4 mg, equivalent to the consumption of a 100 g serving of sockeye salmon. The recovery of carotenoids from Haematococcus pluvialis is greater than 90% and the enrichment factor in them is at least 2.5. The hydro-organic phase is recycled for the preparation of new nanostructured liquid phases, while the Haematococcus pluvialis residue, which contains proteins, carbohydrates and minerals, can be used for other applications.
    Example 3. Extraction of lutein from Scenedesmus almeriensis
    The procedure for extracting lutein from Scenedesmus almeriensis, dried and crushed, as well as the products obtained, are similar to those described in example 2 for Haematococcus pluvialis. The ratio biomass: hydro-organic equilibrium solution: nanostructured liquid phase recommended is 1: 2: 1 (w / v / v). Lutein present in
    Scenedesmus almeriensis (approximately 0.5%, w / w, in free form), was extracted with recovery percentages higher than 90% and the extract, enriched by a factor of 2.5 times, contains the carotenoids in a matrix of fatty acids and liquid phase nanostructured The residue of the biomass is basically constituted by proteins and carbohydrates. 5
    Example 4. Extraction of β-carotene from Dunaliella salina
    The Dunaliella salina microalgae is one of the organisms with the highest production of β-carotene, being able to reach up to 10% in dry weight. It also contains lutein and zeaxanthin in smaller proportion. The extraction of the carotenoids present in it is carried out from the dry and crushed biomass, using the ratio biomass: solution 10 of hydro-organic equilibrium: liquid phase nanostructured 1: 2.5: 1 (p / v / v). The recovery percentage of β-carotene is greater than 98%. The extraction procedure, as well as the products obtained, are similar to those described in example 2 for Haematococcus pluvialis.
     fifteen
    权利要求:
    Claims (10)
    [1]

    1. Procedure for obtaining carotenoids from biomass comprising the following stages:
    a) dissolving an amphiphilic surfactant in an organic solvent and adding water, in a proportion of each component in the mixture comprised between 1: 4: 2.6 and 1: 7: 13 (g: mL: mL).
    b) centrifuge the mixture obtained in a), at least 3000 rpm for 10 min, obtaining a hydro-organic phase and a nanostructured liquid phase,
    c) separate the two phases obtained in stage b)
    d) mixing the biomass with the hydro-organic phase and the liquid phase obtained in step b) in a ratio between 1: 0: 2 and 1: 10: 1 (g: mL: mL),
    e) centrifuge the mixture obtained in d) for at least 10 minutes at least 2500 rpm, obtaining 3 differentiated phases: liquid phase, solid phase and an intermediate phase,
    f) purifying the liquid phase obtained in e) to obtain the carotene extract.
    [2]
    2. Process for obtaining carotenoids according to claim 1, wherein the amphiphilic surfactant comprises a hydrocarbon chain of between 6 and 18 carbon atoms and at least one polar group selected from carboxylic groups, alcohols, aldehydes, ketones, phosphates. ..
    [3]
    3. Process for obtaining carotenoids according to any of claims 1-2, wherein the amphiphilic surfactant is selected from among glycolipids, fatty acids, phospholipids, lipopeptides, neutral lipids, or any surfactant of natural origin
    [4]
    4. Process for obtaining carotenoids according to any of the preceding claims, wherein the organic solvent is selected from tetrahydrofuran, ethylene glycol, dioxane, acetone, propanol, ethanol, acetonitrile and methanol.
    [5]
    5. Process for obtaining carotenoids according to any of the preceding claims wherein step f) of purification is carried out by removing the organic solvent.
    [6]
    6. Process for obtaining carotenoids according to any of the preceding claims, comprising an additional step of diluting the extract obtained in step f) with a vegetable oil. 30
    [7]
    7. Process for obtaining carotenoids according to any of the preceding claims, wherein the biomass is vegetable, fungal or yeast biomass.
    [8]
    8. Process for obtaining carotenoids according to any of the preceding claims wherein the biomass comprises a pretreatment of drying and grinding.
    [9]
    9. Use of the extract obtained by the process according to any of claims 1-5 as a food additive.
    [10]
    10. Use of the extract obtained by the method according to any of claims 1-8 in pharmaceutical formulations.
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    同族专利:
    公开号 | 公开日
    ES2694600B2|2019-05-17|
    WO2018234603A1|2018-12-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2009063100A1|2007-11-13|2009-05-22|Universidad De Almería|Extraction of carotenoids using a single-phase ternary blend of ethanol:hexane:water.|
    CN112795485A|2019-10-28|2021-05-14|中国石油化工股份有限公司|Method for improving oil content of microalgae|
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    PCT/ES2018/070434| WO2018234603A1|2017-06-21|2018-06-20|Method for extracting carotenoids using nanostructured liquid phases|
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