METHOD FOR THE PROCUREMENT OF FAT-SOLUBLE AND WATER-SOLUBLE COMPOUNDS FROM MICROALGAE BY MODULATION OF THE POLARITY OF VEGETABLE OR ANIMAL OILS

Description

[0001] The present invention relates to the field of algal biomass valorization. The present invention relates to a process for obtaining fat-soluble and water-soluble compounds from eukaryotic or prokaryotic microalgae biomass (cyanobacteria), as well as the oil obtained by this process and its applications, particularly in the food sector or as food supplements.

[0002] Cultivated for over 30 years, microalgae are a fully-fledged food product. In human and animal nutrition, two approaches to their use exist: the first focuses on consuming the whole microalga, while the second involves the extraction, processing, and packaging of bioactive molecules derived from them. In France, only three microalgae species are authorized for unprocessed human consumption and possess exceptional nutritional properties: spirulina (Arthrospira platensis), the green microalga Chlorella, and the diatom Odontella aurita. These microorganisms are poised to become key players in the global food challenge, thanks to their unique protein content and the low environmental impact of their production: minimal water and energy consumption.

[0003] Today, consumers are increasingly turning to healthier, more natural foods. They seek quality products for their daily meals. Alongside this trend, a growing number of people are turning to so-called alternative medicines, often incorporating traditional remedies, to treat themselves as naturally as possible. However, a balanced diet is sometimes insufficient to prevent deficiencies. This is where dietary supplements come in, becoming widely available since the 2014 "plants" decree, which allowed the use of many plants previously reserved for pharmaceuticals. Consumers now have access to a wide range of products for preventing various health problems, such as obesity, cardiovascular issues, and improving concentration.Omega-3 and omega-6 fatty acids are among the most popular molecules due to their health benefits and wide range of applications. They are notably used in the prevention of cardiovascular disease. They are found in oily fish and seafood, but are also present in significant quantities in microalgae. These fatty acids are considered essential because the human body cannot synthesize them; therefore, they must be obtained through our diet.

[0004] The mammalian body is capable of synthesizing fatty acids from oleic acid (C18:1). However, this is not the case for certain 34-carbon polyunsaturated fatty acids, which are synthesized only by bacteria or plants. Mammals can therefore only obtain these through their diet; this is why these fatty acids are called essential. The two main essential fatty acids for humans are linoleic acid and alpha-linolenic acid. Other long-chain fatty acids, essential for the proper functioning of the body, are synthesized from these fatty acids. Thus, by adding a double bond and lengthening the carbon chain, arachidonic acid is obtained from linoleic acid, and eicosapentaenoic acid (EPA) from alpha-linolenic acid.The fatty acids synthesized via this metabolism are involved in various processes such as signaling, inflammatory reactions, brain and retinal development... These are omega 3 (EPA, DHA).

[0005] Lipid extraction, particularly of essential fatty acids, is frequently carried out using organic solvents derived from petrochemicals, such as hexane (Comparison of solvents for extraction of krill oil from krill meal: Lipid yield, phospholipids content, fatty acids composition and minor components. Xie et al, 2017). However, these organic solvents have the major drawback of being unsuitable for consumption.

[0006] Manufacturers use vegetable fats as solvents to extract fat-soluble active ingredients from plants. They are the source of numerous patents, such as FR3002845 B1, which protects a process for preparing a cosmetic and / or dermatological product based on a cork extract, at least one part of the cork tree, and at least one natural fat, or FR2994840 B1, which protects an oily extract obtained by extraction, using an oily vehicle, of non-volatile compounds contained in propolis. Other patents in a similar field have emerged, notably FR2694300 B1, which covers a process for extracting and fixing aromatic compounds onto a non-aqueous substrate.

[0007] However, none of these documents describes a modular process for extracting both fat-soluble and water-soluble compounds, particularly from microalgae biomass, the product of which can be used directly for human or animal consumption.

[0008] The Applicants have now developed a process that enables the industrial-scale production, from a starting biomass of microalgae, of an oil enriched in all the fat-soluble and water-soluble compounds of microalgae, that is, not only fat-soluble compounds but also water-soluble compounds. The process according to the invention, by modulating the extraction parameters and the polarity of the oil, also allows for the production of nonpolar compounds, such as proteins, while controlling their presence. Furthermore, since the solvent used is a natural oil, without any residual trace of organic solvent, the extract is pure and suitable for direct consumption, and no evaporation step is necessary.

[0009] Thus, a first object of the present invention relates to a process for obtaining fat-soluble and water-soluble compounds from a biomass of eukaryotic or prokaryotic microalgae (cyanobacteria), characterized in that said process comprises 1. a step of mixing said biomass with oil, preferably vegetable oil, said oil comprising between 0.25% and 10% by mass of at least one amphiphilic adjuvant, chosen from a monoglyceride, a diglyceride, or a phospholipid, allowing modulation of the polarity of said oil, and 3. a step of extracting said liposoluble and water-soluble compounds by an ultrasonic treatment with application of an ultrasonic power (Pus) of between 1 and 1000 W / L applied to said biomass mixed with said oil obtained in step 1) at a temperature (T) of between 15 and 70°C for a duration of between 30 seconds and 1 hour, and / or 4.an extraction step of said fat-soluble and water-soluble compounds by treatment with electromagnetic microwaves with application of a power (W) between 1 and 1000 W / L applied to said biomass in mixture with said oil obtained in step 1) at a temperature (T) between 15 and 70°C for a duration between 30 seconds and 1 hour.

[0010] Preferably according to the invention, the process according to the invention further comprises a step 2. of macerating said biomass with said oil allowing to homogenize the mixture, said step 2. being carried out between step 1. and step 3 or between step 1. and step 4 when said process does not comprise a step 3.

[0011] For the sake of clarity, the method according to the invention may include: The mixing step 1) followed by an ultrasonic treatment step 3) The mixing step 1) followed by an electromagnetic microwave treatment step 4) The mixing step 1) followed by a maceration step 2) and an ultrasonic treatment step 3) The mixing step 1) followed by a maceration step 2) and an electromagnetic microwave treatment step 4) The mixing step 1) followed by an ultrasonic treatment step 3) and an electromagnetic microwave treatment step 4) The mixing step 1) followed by a maceration step 2) an ultrasonic treatment step 3), and an electromagnetic microwave treatment step 4)

[0012] More preferably according to the invention, the method according to the invention comprises: The mixing step 1) followed by a maceration step 2) and an ultrasonic treatment step 3), or The mixing step 1) followed by a maceration step 2) and an electromagnetic microwave treatment step 4), or The mixing step 1) followed by an ultrasonic treatment step 3) and an electromagnetic microwave treatment step 4), or The mixing step 1) followed by a maceration step 2) an ultrasonic treatment step 3), and an electromagnetic microwave treatment step 4).

[0013] According to the present invention, fat-soluble compounds are defined as compounds soluble in fats and contained in microalgae and / or cyanobacteria, preferably selected from short-chain saturated fatty acids such as butyric acid, caproic acid, or caprylic acid; long-chain saturated fatty acids such as capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristyl acid, pentadecanoic acid, palmitic acid, margaric acid, or stearic acid; very long-chain saturated fatty acids such as arachidic acid, docosanoic acid, tricosanoic acid, or tetracosanoic acid; and monounsaturated fatty acids such as undecenoic acid, lauroleic acid, pentadecenoic acid, palmitoleic acid, and other fatty acids. Heptadecenoic acid, oleic acid, eicosic acid, docosaenoic acid, or tetracosenoic acid,polyunsaturated fatty acids such as hexadecadienoic acid, linoleic acid (precursor of omega 3 and 6), alpha-linolenic acid (ALA, precursor of omega 3 and 6), gamma-linolenic acid (GLA), eicosadienoic acid, dihomolinolenic acid, eicosapentaenoic acid (EPA), fat-soluble vitamins such as, l' α-Tocopherol (vitamin E), fat-soluble pigments such as chlorophyll, or carotenoids such as β-carotene (vitamin A precursor), lutein, asthaxanthin, zeaxanthin, cryptoxanthin (vitamin A precursor), lycopene, sterols, alkaloids, phenolic compounds such as flavonoids, or terpenes such as phytol (vitamin E precursor).

[0014] Water-soluble compounds, according to the present invention, are meant compounds soluble in water and contained in microalgae and / or cyanobacteria, preferably selected from proteins and enzymes, vitamins of groups B and C, minerals such as zinc, calcium, potassium, magnesium and iron, pigments such as phycocyanin, allophycocyanin, or phycoerythrin, and essential amino acids.

[0015] For the purposes of this invention, microalgae are defined as eukaryotic microalgae, which are characterized by a cell wall and a nucleus, including chlorophytes, chrysophytes and pyrrophytes, said eukaryotic microalgae being commonly referred to as "microalgae", and prokaryotic microalgae, which do not possess a nucleus and a cell wall, including cyanophytes, hereinafter specifically referred to as "cyanobacteria".

[0016] According to the invention, oil is defined as a fatty substance, liquid at room temperature and insoluble in water, of vegetable, animal or mineral origin, and suitable for food use. Since this oil is essentially composed of triglycerides, it is completely nonpolar and initially only allows the extraction of compounds of the same nature as itself, that is to say, highly nonpolar compounds.

[0017] Preferably, the oil used according to the invention is a vegetable oil.

[0018] Preferably, the oil used according to the invention is selected from coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, canola oil, safflower oil, sesame oil, soybean oil, sunflower oil, almond oil, beech nut oil, Brazil nut oil, cashew nut oil, hazelnut oil, macadamia oil, mongongo nut oil, pecan nut oil, pine nut oil, pistachio oil, walnut oil, pumpkin seed oil, grapefruit seed oil, lemon oil, orange oil, bitter gourd oil, buffalo gourd oil, and other oils. butternut squash seeds, egusi seed oil, pumpkin seed oil, watermelon seed oil, acai oil, black seed oil, blackcurrant seed oil, borage seed oil, evening primrose oil, linseed oil,amaranth oil, apricot oil, apple seed oil, argan oil, avocado oil, babassu oil, ben oil, Borneo tallow nut oil, Cape chestnut oil, carob pod oil (algaroba oil), cocoa butter oil, cocklebur oil, cohune oil, coriander oil, camelina oil, grapeseed oil, hemp oil, kapok seed oil, kenaf seed oil, allemantia oil, mafura oil, marula oil, meadowfoam seed oil, mustard oil, niger seed oil, poppy seed oil, nut oil nutmeg, okra seed oil, papaya seed oil, perilla seed oil, persimmon seed oil, pequi oil, pili nut oil, pomegranate seed oil, poppy oil, pracaxi oil, virgin pracaxi oil, prune kernel oil, quinoa oil,Ramtil oil, rice bran oil, royal oil, shea nut oil, sacha inchi oil, sapote oil, seje oil, shea butter oil, taramira oil, tea seed oil, thistle oil, tiger nut oil, tobacco seed oil, tomato seed oil, and wheat germ oil.

[0019] Even more preferably, the oil used according to the invention is sunflower oil.

[0020] An amphiphilic adjuvant is defined as a compound possessing a lipid-soluble portion (long carbon chain) and a water-soluble portion (ionic or insoluble). These molecules have the characteristic of concentrating and aggregating at the interfaces between water and other substances that are poorly soluble in water, such as fats, primarily due to their free hydroxide groups. Surfactants are examples of amphiphilic adjuvants and are classified according to the HLB (Hydrophilic / Lipophilic Balance) index, with oleic acid corresponding to oleic acid and potassium oleate to 20. This scale allows for an assessment of whether an amphiphile is more lipid-soluble or, conversely, more hydrophilic, and for determining its most suitable application. A surfactant with an HLB <9 will be of a liposoluble nature (favoring water-in-oil emulsions) while a surfactant with an HLB > 11 will be of a hydrophilic nature (favoring oil-in-water emulsions).The use of amphiphilic adjuvants having different HLBs makes it possible to modulate the polarity of the oil used in the process according to the invention and to extract less nonpolar compounds.

[0021] Preferably, the amphiphilic adjuvant(s) according to the invention are selected from monoglycerides such as Monostearine (E471), Sorbitan Monooleate (Span 80, E494), Sorbitan Monolaurate (Tween 20, E432), Polyoxyethylene Sorbitan Monopalmitate (Tween 40, E434), Polyoxyethylene Sorbitan Monooleate (Tween 80 or polysorbate 80, E433), Glyceryl Monooleate (type 40, E491), mono- and diglycerides of fatty acids such as Glyceryl Palmitostearate (E471), and phosphatidylcholines such as Soy Lecithin (E322).

[0022] By modulating the polarity of the oil, we mean modifying the distribution of negative and positive charges in the oil, which allows, by reducing the non-polar character of the oil, to promote the extraction of polar (water-soluble) compounds, or by increasing it, to promote the extraction of non-polar (lipid-soluble) compounds.

[0023] Preferably according to the invention, the biomass of eukaryotic or prokaryotic microalgae (cyanobacteria) is in dry, wet or pre-extracted form.

[0024] By "dry biomass," we mean, according to the invention, biomass that has undergone drying or freeze-drying. Traditionally, dry biomass has a moisture content of approximately 10% water by weight, but the moisture content can be higher if the drying process is shorter. The drying stage can be carried out using any method known to those skilled in the art, such as thermal drying, which involves drying the biomass with heat generated by the combustion of natural gas (conduction, convection, etc.), or solar drying, which uses sunlight to heat and dry the biomass, for example, in outdoor basins. Examples of conditions for carrying out these drying stages can be found in the document "Different Techniques for Harvesting Microalgae: Technical, Economic, and Environmental Aspects."» by Florian Delrue published as part of the Algo'Réso seminar on October 22, 2013 and available on https: / / www.mio.univ-amu.fr / IMG / pdf / 20131022_02_presa_CEA.pdf.

[0025] According to the invention, biomass in wet form means biomass that has not undergone a drying stage and therefore contains a high moisture content, on the order of 75% water by weight.

[0026] According to the invention, pre-extracted biomass refers to biomass that has undergone prior extraction, particularly of water-soluble compounds, for example by a mechanical extraction step. This pre-extracted biomass may be in the form of a freeze-dried pellet.

[0027] Preferably according to the invention, in the case where the process includes an ultrasonic treatment step 3), the ultrasonic power and temperature are modulated to vary the nature and quantity of compounds extracted from said biomass.

[0028] In the present invention, ultrasonic treatment with the application of ultrasonic power (Up) refers to a physical treatment using an ultrasonic reactor. Preferably, according to the invention, the ultrasonic power applied to the microalgae or cyanobacteria mixed with oil and at least one amphiphilic adjuvant is between 1 and 1000 W / L, and even more preferably between 50 and 80 W / L. Preferably, the energy delivered by the ultrasonic reactor varies between 500 and 5000 J, the feed rate of the ultrasonic reactor varies from 1 L / hour to 1000 L / hour, preferably 1 L / hour, and the ultrasonic treatment is carried out for a duration ranging from 30 seconds to 1 hour.

[0029] Preferably according to the invention, in the case where the process includes a step 4) of treatment with electromagnetic microwaves, the power, the number of exposures and the temperature are modulated to vary the nature and quantity of the compounds extracted from said biomass.

[0030] Preferably, according to the invention, modulating the power of electromagnetic microwaves allows increasing or decreasing the quantity of fat-soluble and water-soluble compounds extracted from said biomass, each of said compounds having an optimal power at which a maximum of it is extracted, for example, 850 W for chlorophyll a and 1000 W for carotenoids. Using other power levels for these compounds will thus reduce the quantity extracted.

[0031] Electromagnetic microwave treatment, according to the present invention, refers to the treatment of biomass with electromagnetic waves imposed by a microwave generator.

[0032] The number of exposures refers to the number of repetitions of the microwave treatment.

[0033] Preferably, according to the invention, the treatment takes the form of at least one exposure lasting from 30 seconds to 30 minutes. Even more preferably, according to the invention, the treatment takes the form of several exposures, each lasting from 30 seconds to 30 minutes.

[0034] Preferably according to the invention, the form of the original biomass, the nature and quantity of the oil and of said at least one amphiphilic adjuvant, and the steps 3) of ultrasonic treatment and / or 4) of electromagnetic microwave treatment are modulated to vary the nature and quantity of the fat-soluble and water-soluble compounds extracted from said eukaryotic microalgae or cyanobacteria.

[0035] Modulating the form of the source biomass allows for increasing or decreasing the amount of fat-soluble and water-soluble compounds extracted from it. Using pre-extracted biomass allows for the extraction of more compounds than using dry biomass, while using dry biomass allows for the extraction of more compounds than using wet biomass.

[0036] Modulation by the type and quantity of amphiphilic adjuvant affects the extraction yields of both fat-soluble and water-soluble compounds, regardless of the technique used. For example, the addition of Tween 80 or Span 80, when treated with ultrasound or microwaves, increases the quantity of extracted compounds. Maceration treatment has also shown that the addition of Span 80 increases the extraction yield of carotenoids and chlorophyll a, whereas the addition of Tween 80 only increases the chlorophyll a extraction yield, with the carotenoid extraction yield decreasing.

[0037] The modulation of step 3) of ultrasonic treatment allows increasing or decreasing the quantity of fat-soluble and water-soluble compounds extracted from said biomass, each of said compounds having an optimal ultrasonic power where a maximum of it is extracted.

[0038] Modulating the electromagnetic microwave treatment step (step 4) allows for increasing or decreasing the amount of fat-soluble and water-soluble compounds extracted from the biomass. Each compound has an optimal power level at which maximum extraction is achieved; for example, 850 W for chlorophyll a and 1000 W for carotenoids. Using power levels other than these optimal levels will reduce the amount extracted.

[0039] Preferably according to the invention, the duration of step 3) and / or step 4) is each between 30 seconds and 1 hour.

[0040] Even more preferably according to the invention, the duration of step 3) and step 4) is each between 30 seconds and 30 minutes.

[0041] Preferably according to the invention, the fat-soluble compounds are selected from short-chain saturated fatty acids such as butyric acid, caproic acid, or caprylic acid; long-chain saturated fatty acids such as capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristyl acid, pentadecanoic acid, palmitic acid, margaric acid, or stearic acid; very long-chain saturated fatty acids such as arachidic acid, docosanoic acid, tricosanoic acid, or tetracosanoic acid; monounsaturated fatty acids such as undecenoic acid, lauroleic acid, pentadecenoic acid, palmitoleic acid, heptadecenoic acid, oleic acid, eicosic acid, Docosaenoic acid, or Tetracosenoic acid, polyunsaturated fatty acids such as Hexadecadienoic acid, Linoleic acid (precursor of omega 3 and 6), Alpha-linolenic acid (ALA, precursor of omega 3 and 6),Gamma-linolenic acid (GLA), eicosadienoic acid, dihomolinolenic acid, eicosapentaenoic acid (EPA), fat-soluble vitamins such as α-tocopherol (vitamin E), or fat-soluble pigments such as chlorophyll, or carotenoids such as β-carotene (vitamin A precursor), lutein, asthaxanthin, zeaxanthin, cryptoxanthin (vitamin A precursor), or lycopene.

[0042] Preferably according to the invention, the water-soluble compounds are selected from proteins and enzymes, vitamins of the B and C groups, minerals such as zinc, calcium, potassium, magnesium and iron, pigments such as phycocyanin, allophycocyanin, or phycoerythrin, essential amino acids, polysaccharides and fibers.

[0043] Preferably according to the invention, eukaryotic microalgae are selected from Chlorella, Nannocloropsis, Dunaliella and Euglena, and cyanobacteria are selected from spirulina (Arthrospira platensis or Spirulina maxima) and AFA (Aphanizomenon Floes-aquae).

[0044] Preferably according to the invention, the process includes an additional step 5) of solid-liquid separation, preferably by centrifugation, of the extract obtained after step 2), 3) or 4).

[0045] Preferably according to the invention, the process includes an additional step 6) of filtering the liquid portion obtained by separating the extract obtained after step 5).

[0046] Preferably according to the invention, the microalgae or cyanobacteria / oil ratio is between 1 / 5th and 1 / 50th of the volume, preferably 1 / 20th of the volume.

[0047] According to a second aspect, the invention relates to an oil enriched in fat-soluble and water-soluble compounds of eukaryotic or prokaryotic microalgae (cyanobacteria) as obtained by the process according to the invention.

[0048] Oil enriched with fat-soluble and water-soluble compounds from eukaryotic or prokaryotic microalgae (cyanobacteria) means an oil containing water-soluble and fat-soluble compounds not initially present in the oil, and / or an increase in the concentration of water-soluble and fat-soluble compounds initially present in the oil in extremely small quantities (measurement less than a microgram).

[0049] Preferably, the invention relates to an oil enriched with water-soluble and fat-soluble compounds not initially present in the oil.

[0050] More specifically, this process allows for the extraction of fat-soluble and water-soluble compounds from microalgae and cyanobacteria. Preferably, the resulting extract will possess optimal organoleptic qualities and a nutritional composition suitable for human consumption.

[0051] According to a third aspect, the invention relates to the use of according to the invention in chemical, food, cosmetic or pharmaceutical compositions, preferably food.

[0052] Given the advantages of these filtrates / extracts mentioned above, their applications in the food, medical, or cosmetic fields, particularly as dietary supplements, are especially promising. The invention also relates to the retentates obtained after the filtration step as novel products. These retentates are also usable in the food, medical, or cosmetic fields, or as dietary supplements. Description of the Figures

[0053] Figure 1: Absorption spectra of the oil alone (A) and of the oil enriched with spirulina compounds (B) after the Arthrospira platensis biomass was mixed with the extraction solvent (1% Tween 80 sunflower vegetable oil) in the ratio 1 / 20 (0.5 g of spirulina in 10 g of oil) and the mixture was introduced into a ball mill with ceramic balls and subjected to rotations at a speed of 4000 rpm for 1 hour at room temperature, then centrifuged for 10 min at 9000 rpm. Figure 2Quantitative analysis was performed using spectrophotometric measurements to evaluate chlorophyll a and carotenoid content as a function of the biomass type (wet, dry, or pre-extracted) and the type of amphiphilic additive (none, Tween 80, or Span 80) after the mixture had been incubated for 1 hour in a ball mill at 4000 rpm at room temperature. The first line presents a control value. The following three lines present the data after using wet biomass. The next three lines present the data after using dry biomass. The following three lines present the data after using pre-extracted biomass, specifically a freeze-dried pellet. Figure 3Qualitative analysis of neutral lipids was performed by high-performance thin-layer chromatography (HPTLC). 20 µL of a 1 mg / mL sample were loaded into chloroform. The presence of monoglycerides (MAG), free fatty acids (FFA), triglycerides (TAG), diglycerides (DAG), and sterols (STE) was investigated. Figure 4Quantitative analysis was performed using spectrophotometric measurements to evaluate chlorophyll a and carotenoid content according to the extraction technique (ball mill, maceration, microwave, or ultrasound) and the nature of the amphiphilic additive (none, Tween 80, or Span 80) using moist biomass (80% moisture). The first row presents a control value. The next three rows present the data after maceration for 24 hours. The following three rows present the data after treatment with electromagnetic microwaves for four one-minute intervals. The next three rows present the data after treatment with ultrasound, applying ultrasonic power for 30 minutes. The last three rows present the data after treatment with a ball mill for one hour. Figure 5: Schematic representation of microwave application cycles. The rising curves represent the microwave treatment times. Figure 6 : Photos characterizing the extractions carried out by microwave treatment at powers of 300, 600, 850 and 1000 W and according to the nature of the amphiphilic adjuvant (none, Tween 80, or Span 80). Figure 7 Quantitative analysis was performed using spectrophotometric measurements to evaluate chlorophyll a and carotenoid content as a function of microwave treatment power (300, 600, 850, and 1000 W). The first three rows present the data for 1000 W treatment. The next three rows present the data for 850 W treatment. The next three rows present the data for 600 W treatment. The last three rows present the data for 300 W treatment. Examples Example 1: Study of the modularity of the biomass state and the addition of an amphiphilic adjuvant by enriching sunflower oil using a ball mill :

[0054] Biomass ( Arthrospira platensis)The spirulina, whether wet, dry, or pre-extracted, is mixed with the extraction solvent (sunflower oil, sunflower oil with 1% Tween 80, and sunflower oil with 1% Span 80) in a 1:20 ratio; that is, 1 g of biomass to 20 g of solvent. In this case, 0.5 g of spirulina is introduced into 10 g of oil. The mixtures are then fed into a ball mill with ceramic balls and rotated at 4000 rpm for 1 hour at room temperature. The extracts are then centrifuged for 10 minutes at 9000 rpm to obtain a clear oil.

[0055] The oil collected after these extractions has a green color, different from the initial color of the starting sunflower oil (light yellow). Comparison of the absorption spectra of the oil alone ( Figure 1A ) and 1% Tween 80 sunflower vegetable oil enriched with spirulina compounds ( Figure 1B) reveals the presence of characteristic peaks of plant pigments: carotenoids (λ max = 416 nm) and chlorophyll (λ max = 668 nm). According to these initial qualitative observations, the sunflower oil would therefore have been enriched with spirulina pigments by the process according to the invention.

[0056] A quantitative analysis was then carried out using spectrophotometric measurements to evaluate the content of each of its pigments as a function of the nature of the biomass and the amphiphilic adjuvant. The results obtained ( Figure 2These findings highlight the fact that the nature of the biomass modulates the pigment content. Indeed, maximum pigment concentrations are achieved with pre-extracted biomass (lyophilized pellet: having already undergone extraction). Modulating polarity by adding an amphiphilic adjuvant to the oil also affects pigment yields. Thus, with the surfactant Tween 80, the extract contains 238.61 µg of chlorophyll a per gram of oil and 61.46 µg of carotenoids per gram of oil.

[0057] Furthermore, if we focus on the lipid classes, we also observe the enrichment of the vegetable oil in neutral lipids from spirulina after extraction. A qualitative analysis of the neutral lipids was carried out ( Figure 3) via high-performance thin-layer chromatography (HPTLC) where a 20 µL sample at 1 mg / mL was loaded into chloroform. The results obtained show the presence of monoglycerides (MAGs) in extracts 1 to 19 but not in the control, and the presence of free fatty acids (FFAs) in extracts 12 to 19 but not in the control. These results highlight the presence of neutral lipids such as free fatty acids (FFAs) and monoglycerides (MAGs) in the vegetable oil after extraction. Therefore, there was an enrichment of the vegetable oil, which did not initially contain these compounds. Example 2: Study of the modularity of the extraction technique - enrichment of sunflower oil using different extraction methods

[0058] For a given biomass (here, wet biomass (80% moisture) of Arthrospira platensis ), different extraction methods have been used to enrich sunflower oil: Ball mill: 4000 rpm, for 1 hour, using 20 g of ceramic balls. Ultrasonics: 25 kHz, 150 W, for 30 min. Microwave: tested with different powers ranging from 300 to 1000 W, by treatments in 1 minute cycles. Maceration.

[0059] The spectrophotometric measurements performed ( Figure 4 The results show that the yields of chlorophyll a and carotenoids are modulated by the extraction technique chosen. Indeed, maximum yields are obtained with ultrasonic extraction: 209.92 µg of chlorophyll a per g of oil and 35.60 µg of carotenoids per g of oil (with oil containing 1% span 80). In this case, the addition of span 80 to the oil modulates the polarity in favor of the extraction of chlorophyll and carotenoid pigments. Example 3: Study of the modularity of extraction parameters - enrichment of sunflower oil by microwaves

[0060] To highlight the modularity of the extraction technique, various parameters were tested. The biomass chosen for these extractions was a spirulina paste with 79.87% moisture content. The biomass / oil mixture was prepared at a dry matter ratio of 1 / 20th. Several 1-minute microwave exposure cycles were applied to the solvent / biomass mixture, interspersed with cooling in an ice bath (illustrated in the Figure 5 ).

[0061] Extractions were performed using microwave treatment at powers of 300, 600, 850, and 1000 W. figure 6This shows that the oil becomes increasingly dark as the microwave power increases, indicating that pigment extraction is therefore favored by high microwave power. Furthermore, polarity modulation also plays an important role in pigment extraction, as a color difference is observed depending on the amphiphilic additive used at the same power level. Thus, at 600 W, the oil containing 1% Tween 80 exhibits a more intense color than the extracts made with oil alone and with oil containing 1% Span 80.

[0062] The results presented in Figure 7confirm the observations of the previous paragraph. The higher the microwave power, the greater the pigment yield; similarly, the addition of Tween 80 to the oil allows a higher pigment yield than that obtained with the oil alone or with the oil supplemented with Span 80: 11.37 µg of chlorophyll a per g of oil and 9.76 µg of carotenoids per g of oil with the oil supplemented with 1% Tween 80 at 850 and 1000 W respectively. Conclusions

[0063] The results obtained in these three examples highlight that the modularity of the biomass composition influences the quantity of fat-soluble and water-soluble compounds extracted. Indeed, using pre-extracted biomass allows for better extraction of fat-soluble and water-soluble compounds than with dry biomass, while dry biomass, however, allows for better extraction of fat-soluble and water-soluble compounds than extraction from wet biomass.

[0064] The results obtained in these three examples also highlight that the extraction technique used influences the extraction yields of fat-soluble and water-soluble compounds. Indeed, the use of ultrasound yields higher extraction rates of carotenoids and chlorophyll a compared to maceration, microwave treatment, or the use of a ball mill.

[0065] The results obtained in these three examples also highlight that modulating the microwave-based technique influences the extraction yields of both fat-soluble and water-soluble compounds. Indeed, a maximum amount of carotenoids is extracted at a microwave power of 1000 W, while a maximum of chlorophyll a is extracted at 850 W.

[0066] Finally, the results obtained in these three examples highlight that the addition of amphiphilic adjuvants affects the extraction yields of both fat-soluble and water-soluble compounds, regardless of the technique used. For example, the addition of Tween 80 or Span 80, when treated with ultrasound or microwaves, increases the quantity of compounds extracted. Maceration treatment also showed that the addition of Span 80 increases the extraction yield of carotenoids and chlorophyll a, while the addition of Tween 80 only increases the chlorophyll a extraction yield, with the carotenoid extraction yield decreasing.

Claims

1. Process for obtaining liposoluble and hydrosoluble compounds from a biomass of eukaryotic or prokaryotic microalgae, characterized in that said process comprises:

1. a step of mixing said biomass with oil, preferably vegetable oil, said oil comprising between 0.25% and 10% by mass of at least one amphiphilic adjuvant selected from a monoglyceride, a diglyceride, and a phospholipid, allowing the polarity of said oil to be modulated, and 3. a step of extracting said liposoluble and hydrosoluble compounds by means of an ultrasound treatment with the application of an ultrasonic power (Pus) of between 1 and 1000 W / L applied to said biomass mixed with said oil obtained in step 1) at a temperature (T) of between 15 and 70°C for a time of between 30 seconds and 1 hour, and / or 4. a step of extracting said liposoluble and hydrosoluble compounds by means of electromagnetic microwave treatment with the application of a power (W) of between 1 and 1000 W / L applied to said biomass mixed with said oil obtained in step 1) at a temperature (T) of between 15 and 70°C for a time of between 30 seconds and 1 hour.

2. Process according to the preceding claim, characterized in that the biomass of eukaryotic or prokaryotic microalgae is in dry, moist or pre-extracted form.

3. Process according to either one of the preceding claims, where said at least one amphiphilic adjuvant is selected from monostearin, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monooleate, glyceryl monooleate, glyceryl palmitostearate, and soy lecithin.

4. Process according to the preceding claim, where said at least one amphiphilic adjuvant is selected from sorbitan monooleate and polyoxyethylene sorbitan monooleate.

5. Process according to any one of the preceding claims, characterized in that the liposoluble compounds are selected from short-chain saturated fatty acids such as butyric acid, caproic acid or caprylic acid, long-chain saturated fatty acids such as capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristyic acid, pentadecanoic acid, palmitic acid, margaric acid or stearic acid, very-long-chain saturated fatty acids such as arachidic acid, docosanoic acid, tricosanoic acid or tetracosanoic acid, monounsaturated fatty acids such as undecenoic acid, lauroleic acid, pentadecenoic acid, palmitoleic acid, heptadecenoic acid, oleic acid, eicosic acid, docosaenoic acid or tetracosenoic acid, polyunsaturated fatty acids such as hexadecadienoic acid, linoleic acid (precursor of omega-3 and omega-6), alpha-linolenic acid (ALA, precursor of omega-3 and omega-6), gamma-linolenic acid (GLA), eicosadienoic acid, dihomolinolenic acid or eicosapentaenoic acid (EPA), liposoluble vitamins such as α-tocopherol (vitamin E), liposoluble pigments such as chlorophyll, carotenoids such as β-carotene (vitamin A precursor), lutein, asthaxanthin, zeaxanthin, cryptoxanthin (vitamin A precursor) or lycopene, sterols, alkaloids, phenolic compounds such as flavonoids, and terpenes such as phytol (vitamin E precursor).

6. Process according to any one of the preceding claims, characterized in that the eukaryotic microalgae are selected from Chlorella, Nannochloropsis, Dunaliella and Euglena, and in that the prokaryotic microalgae are selected from Arthrospira platensis spirulina, Spirulina maxima spirulina, and Aphanizomenon flos-aquae.

7. Process according to any one of the preceding claims, characterized in that it comprises an additional step, step 5), of solid-liquid separation, preferably by means of centrifugation, of the extract obtained after step 2), 3) or 4).

8. Process according to the preceding claim, characterized in that it comprises an additional step, step 6), of filtering the liquid part obtained by separating the extract obtained after step 5).

9. Process according to any one of the preceding claims, characterized in that the ratio of microalgae or cyanobacteria to oil is between 1 / 5th and 1 / 50th of the volume, preferably 1 / 20th of the volume.

10. Use of the process according to any one of the preceding claims, where if the process comprises an ultrasound treatment step, step 3), the ultrasonic power and the temperature are modulated so as to vary the nature and the quantity of the compounds extracted from said biomass.

11. Use of the process according to any one of claims 1 to 9, where if the process comprises an electromagnetic microwave treatment step, step 4), the power, the number of exposures and the temperature are modulated so as to vary the nature and quantity of the compounds extracted from said biomass.

12. Use of the process according to any one of claims 1 to 9, where the form of the original biomass, the nature and quantity of the oil and the nature and quantity of said at least one amphiphilic adjuvant, and step 3) of ultrasound treatment and / or step 4) of electromagnetic microwave treatment are modulated so as to vary the liposoluble and hydrosoluble compounds extracted from said eukaryotic microalgae or cyanobacteria.