METHOD FOR AUTHENTICATION AND DETECTION OF ADULTERATION OF OILS OF MARINE ORIGIN

Abstract

METHOD FOR AUTHENTICATING AND DETECTING ADULTERATION OF OIL OF MARINE ORIGIN The present invention relates to a method for authenticating and detecting adulteration of an oil of marine origin, characterized in that the method comprises: Obtaining molecular data including: - Determining the composition of fatty acids and / or so-called unsaponifiable compounds of said oil, Obtaining isotopic data including: - Determining the ratio of stable carbon isotopes (13C / 12C) of fatty acids and / or unsaponifiables, - Obtaining a profile of said oil based on the data, - Comparing the profile obtained with reference profiles of oils of determined marine origin, - Concluding that the oil to be identified has a profile substantially equal to one of the reference profiles and therefore the indication of origin, if, at the end of the comparison, the profile corresponds to a recorded profile and, if not,the conclusion that the said oil is a mixture or of another origin than pure fish oil or pure microorganism oil, and an estimate of the proportions of this mixture. [Fig.1],

Description

Title of the invention: METHOD FOR AUTHENTICATING AND DETECTING ADULTERATION OF OILS OF MARINE ORIGIN

[0001] The present invention belongs to the technical field of oils of marine origin, in particular microalgae oils, intended for nutraceutics, pharmacy, human or animal food and cosmetics.

[0002] More specifically, the invention relates to a method for authenticating the nature of an oil of marine origin, in particular microalgae oils, and for detecting any adulteration of this oil, i.e. for detecting any mixture of several oils of different natures.

[0003] The invention also relates to a method for creating a library of profiles of marine oils of different natures for the implementation of the authentication method, as well as the library obtained. STATE OF THE ART

[0004] Long-chain n-3 polyunsaturated fatty acids (omega-3) play important roles in human and animal health, particularly in the prevention of various disorders and diseases. The main source of omega-3 has long been fish consumption. Since then, dietary supplements rich in omega-3, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have appeared on the market, representing an environmentally friendly alternative source compared to fishing. These supplements were initially formulated from fish oils, and more recently from microalgae oils. The cultivation of microalgae, very rich in omega-3, is becoming the main source of omega-3 in the dietary supplement market, which represents several billion dollars per year.

[0005] Omega 3 oils from microalgae are sought after by consumers because of their environmentally friendly origin, or their vegan, halal or kosher certification for example, and are sold at a higher price than those from fish. Such a market attracts fraud which mainly consists of diluting microalgae oils with fish oils to resell them at the price of pure microalgae oil. Such practices require the development of a methodology to trace the origin of the oils and in particular to be able to detect the presence of fish oil in microalgae oils.

[0006] To date, studies on fraud mainly consist of identifying the presence of vegetable oils (sunflower, grape, palm, corn, etc.) or animal oils (pork, chicken etc.) in fish oils (cod, salmon, shark etc.) or to identify their origin (geographical and aquaculture or fishing). These studies are generally based on individual analyses of the composition and / or form of fatty acids present in the oils or organisms concerned (triacylglycerols or ethyl esters) by chromatographic or spectroscopic analyses combined with multivariate statistical analyses to compare reference signatures.

[0007] These analyses remain limited in reliably identifying frauds concerning refined products.

[0008] PURPOSE OF THE INVENTION

[0009] The aim of the present invention is to propose a reliable solution for authenticating the nature of an oil of marine origin, in particular a heterotrophic microalgal oil, and for detecting a possible adulteration of this oil, that is to say for detecting a possible mixture of several oils of different natures, in particular a mixture of microalgal oil and fish oil.

[0010] OBJECTS OF THE INVENTION

[0011] According to a first aspect, the invention relates to a method for authenticating and detecting adulteration of an oil of marine origin, in particular a heterotrophic microalgae oil.

[0012] According to a second aspect, the invention relates to a method for creating a library, or database, of reference marine oil profiles.

[0013] According to a third aspect, the invention relates to a library of reference marine oil profiles.

[0014] Thus, the invention relates to a method for authenticating and detecting adulteration of an oil of marine origin, characterized in that the method comprises:

[0015] obtaining molecular data including:

[0016] - the determination of the composition in fatty acids and / or in compounds called unsaponifiables of said oil,

[0017] obtaining isotopic data including:

[0018] - determination of the ratio of stable carbon isotopes (13C / 12C) of fatty acids and / or unsaponifiables of said oil,

[0019] - obtaining a profile of said oil based on the data,

[0020] Comparison of the profile obtained with reference profiles of oils of determined marine origin,

[0021] The conclusion that the oil to be identified has a profile substantially equal to one of the reference profiles and therefore the indication of origin, if, at Tissue of the comparison, the profile corresponds to a recorded profile and, otherwise, the conclusion that said oil is a mixture or of another origin than pure fish oil or pure microorganism oil, and an estimate of the proportions of this mixture.

[0022] In particular, the reference profiles of oils of determined marine origin, that is to say whose nature is known, include the profiles of a pure fish oil, of a pure photosynthetic microalgae oil, of a pure heterotrophic microalgae oil having been fermented from a carbon substrate originating from a plant with the C3 or C4 Calvin cycle, of a terrestrial plant oil having been genetically modified to produce long carbon chain omega 3 fatty acids such as DHA.

[0023] Advantageously, the reference profiles are available in the form of a library, or database, obtained according to a method as described below.

[0024] The combination of isotopic and molecular analyses makes it possible to improve fraud detection, i.e. to determine whether an oil of marine origin, in particular a microalgae oil, assumed to be pure, has been mixed with an oil of another origin, for example a fish oil. The invention therefore makes it possible to determine whether an oil is an oil produced by heterotrophic or photoautotrophic microalgae, fish, or a mixture of the two sources, or a refined oil, by combining different analytical techniques making it possible to determine the molecular and isotopic composition of the lipids in these oils.

[0025] The invention presented here is a molecular and isotopic diagnostic tool for certifying the purity of an oil of marine origin, in particular a microalgae oil.

[0026] It should be noted that for the purposes of the invention, the term "microalga" means a unicellular microorganism of marine origin and more precisely a heterotrophic microalga such as a microalga of the genus Thraustochytrium, Schizochytrium, Nannochloropsis, Isochrysis, Phaeodactylum, Nitzchia, Staurosira, Crypthecodinium or Ulkenia, preferably of the genus Schizochytrium.

[0027] According to one embodiment, obtaining isotopic data further comprises determining the ratio of stable isotopes of total hydrogen (2H / 'H) and / or fatty acid hydrogen and / or unsaponifiables.

[0028] Advantageously, the determination of the molecular composition of the fatty acids includes the quantification and calculation of the relative abundance of omega-6 docosapentaenoic acid (DPA n-6 or Osbond acid), omega-3 docosapentaenoic acid (DPA n-3), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) as well as calculations of the ratios between DPA n-6 / DPA n-3 and DHA / EPA.

[0029] Advantageously, the determination of the molecular composition of the unsaponifiable compounds includes the quantification and calculation of the relative abundance of sterols, in particular cholesterol, as well as squalene or squalane.

[0030] Advantageously, the determination of the ratio of stable carbon isotopes of fatty acids includes the determination, by gas chromatography coupled with a combustion furnace and an isotope ratio mass spectrometer, of the ratio of stable carbon isotopes (13C / 12C) of DHA, EPA, DPA n-3, and DPA n-6.

[0031] Advantageously, the determination of the ratio of stable carbon isotopes of the unsaponifiables includes the determination, by gas chromatography coupled with a combustion furnace and an isotope ratio mass spectrometer, of the ratio of stable carbon isotopes (13C / 12C) of cholesterol, 24-methylenecholesterol, stigmasterol, squalene and squalane.

[0032] According to one embodiment, the determination of the ratio of stable isotopes of total hydrogen (2H / 'H) is obtained by an elemental analyzer coupled to an isotope ratio mass spectrometer.

[0033] According to one embodiment, the determination of the ratio of stable hydrogen isotopes of fatty acids includes the determination, by gas chromatography coupled with a pyrolysis oven and an isotope ratio mass spectrometer, of the ratio of stable hydrogen isotopes (2H / 'H) of DHA, EPA, DPA n-3, and DPA n-6.

[0034] According to one embodiment, the determination of the ratio of stable hydrogen isotopes of the unsaponifiables includes the determination, by gas chromatography coupled with a pyrolysis oven and an isotope ratio mass spectrometer, of the ratio of stable hydrogen isotopes (2H / 'H) of cholesterol and squalene.

[0035] Advantageously, obtaining isotopic data includes: - determination of the difference in carbon isotope ratio between DHA, EPA, DPA n-3, or DPA n-6,

[0036] and / or

[0037] - the determination of the difference in the carbon isotopic ratio between the cholesterol, squalene, EPA or DHA.

[0038] and / or

[0039] - determination of the difference in the hydrogen isotopic ratio between DHA and the EPA, DPA3, or DPA6,

[0040] and / or

[0041] - the determination of the difference in the hydrogen isotopic ratio between the cholesterol, squalene, EPA or DHA.

[0042] According to a characteristic of the invention, obtaining a profile of said oil includes a principal component analysis of the molecular data obtained, making it possible to determine a proportion of purity or mixture of said oil.

[0043] According to another characteristic of the invention, obtaining a profile of said oil includes assigning an adulteration score of the oil between 0 and 14 from the isotopic data obtained.

[0044] The invention also relates to a method for creating a library of oil profiles, the method comprising the determination of reference profiles, each profile corresponding to an oil of identified origin or to a mixture of oils of identified origin, characterized in that the method comprises, for each profile:

[0045] Obtaining molecular data including:

[0046] - the determination of the composition in fatty acids and / or in so-called compounds unsaponifiables of said oil,

[0047] Obtaining isotopic data including:

[0048] - determination of the ratio of stable carbon isotopes (13C / 12C) of fatty acids and / or so-called unsaponifiable compounds,

[0049] Obtaining a reference profile, for each oil, or each mixture, depending on the data.

[0050] Thus, according to the invention, a database containing molecular and isotopic data of known oil is created in order to provide a source of information which will make it possible to identify an oil of unknown or doubtful origin by comparing the profile of such an oil with known profiles serving as references.

[0051] Advantageously, obtaining isotopic data further comprises determining the ratio of stable isotopes of total hydrogen (2H / 'H) and / or of hydrogen of fatty acids and / or of unsaponifiables.

[0052] Advantageously, the determination of the molecular composition of the fatty acids includes the quantification and calculation of the relative abundance of DPA n-6, DPA n-3, DHA and EPA and the calculations of the ratios between DPA n-6 / DPA n-3 and DHA / EPA.

[0053] Advantageously, the determination of the molecular composition of the unsaponifiables includes the quantification and calculation of the relative abundance of sterols, in particular cholesterol, as well as squalene or squalane.

[0054] According to one embodiment, the determination of the stable carbon isotope ratio of fatty acids includes the determination, by gas chromatography coupled with an oxidation furnace and an isotope ratio mass spectrometer, of the carbon isotope ratio of DHA, EPA, n-3 DPA, and n-6 ​​DPA.

[0055] According to one embodiment, the determination of the ratio of stable carbon isotopes of the unsaponifiables includes the determination, by phase chromatography gaseous coupled to a combustion furnace and an isotope ratio mass spectrometer, of the carbon isotope ratio of cholesterol, of 24-methylenecholesterol king, of stigmasterol of squalene and squalane.

[0056] According to one embodiment, the determination of the ratio of stable isotopes of total hydrogen is obtained by an elemental analyzer coupled to an isotope ratio mass spectrometer.

[0057] According to one embodiment, the determination of the ratio of stable hydrogen isotopes of fatty acids includes the determination, by gas chromatography coupled with a pyrolysis oven and an isotope ratio mass spectrometer, of the ratio of stable hydrogen isotopes of DHA, EPA, DPA n-3, and DPA n-6.

[0058] According to one embodiment, the determination of the ratio of stable hydrogen isotopes of the unsaponifiables includes the determination, by gas chromatography coupled with a pyrolysis oven and an isotope ratio mass spectrometer, of the ratio of stable hydrogen isotopes of cholesterol and squalene.

[0059] Advantageously, obtaining isotopic data includes: - determination of the difference in carbon isotope ratio between DHA, EPA, DPA n-3, or DPA n-6,

[0060] and / or - determining the difference in carbon isotope ratio between cholesterol, squalene, EPA or DHA,

[0061] and / or - determination of the difference in hydrogen isotope ratio between DHA and EPA, DPA n-3, or DPA n-6,

[0062] and / or - determination of the difference in hydrogen isotope ratio between cholesterol, squalene, EPA or DHA.

[0063] Advantageously, obtaining a profile of said oil includes a principal component analysis of the molecular data obtained.

[0064] The invention also relates to a library, or database, of profiles of marine oils obtained by a method as described above. The invention is thus based on different techniques:

[0065] - Molecular and isotopic profiling of oils, preferably of marine, by combined analysis:

[0066] fatty acids,

[0067] of unsaponifiables, in particular of sterols,

[0068] of the overall composition in stable isotopes of hydrogen (ô2H-IRMS bulk),

[0069] of the stable carbon isotope composition of fatty acids (ô13C-CSIA-AG),

[0070] of the stable carbon isotope composition of sterols (ô13C-CSIA-STEROL),

[0071] of the stable hydrogen isotope composition of fatty acids (ô2H-CSIA- AG),

[0072] - The creation of a database of these molecular and isotopic profiles on various oils or reference aquaculture feeds (fish, microalgae, mixtures, genetically modified terrestrial plants),

[0073] - The selection of molecular and isotopic indices allowing to distinguish the different sources of oils using multivariate statistical approaches such as PCA or other,

[0074] - The distinction between different carbon substrates used to cultivate certain microalgae strains by ô13C-CSIA-AG / STEROL,

[0075] - The identification of refined or blended oils based on different criteria chosen above,

[0076] - The assignment of a purity score to an oil relative to its origin. DETAILED DESCRIPTION

[0077] The characteristics of the invention mentioned above, as well as others, will appear more clearly on reading the examples which follow, in connection with the figures:

[0078] [Fig.l] illustrates a score scale obtained after isotopic analyses of different oils,

[0079] [Fig.2] illustrates the score plot of the PCA of fatty acid profiles of marine oils from different sources,

[0080] [Fig.3] illustrates the abundance of cholesterol in marine oils of different origins,

[0081] [Fig.4] illustrates the 13C isotopic signatures of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in marine oils of different origins,

[0082] [Fig.5] illustrates the 13C isotopic signatures of cholesterol, squalene, DHA and EAP in marine oils of different origins,

[0083] [Fig.6] illustrates the global 2H and 13C isotopic signatures of DHA in marine oils of different origins,

[0084] [Fig.7] illustrates the hydrogen isotopic ratio of EPA and DHA in marine oils of different origins.

[0085] EXAMPLE 1: Preparation of a database of oils of determined marine origin

[0086] A database was created on crude or refined oils, microalgae culture extracts, feeds intended for aquaculture and manually made oil mixtures:

[0087] • 30 fish oils

[0088] • 34 Thraustochytrid oils

[0089] • 5 photoautotrophic microalgae oils

[0090] • 4 fish-based aquaculture feeds

[0091] • 16 blends of microalgae and fish oils.

[0092] 1.1 Extractions of fatty acids and unsaponifiable compounds

[0093] The fatty acids and unsaponifiable compounds are extracted, for each oil or food, by methods known to those skilled in the art.

[0094] Extraction of fatty acids.:

[0095] In this example, the fatty acids are extracted according to the following method:

[0096] - Heat the crude oils to 50°C for 10 to 15 minutes to homogenize them,

[0097] - Weigh 40 pl of heated crude oil (between 20 and 40 mg) into a glass tube borosilicate and dissolve in a chloroform-methanol mixture in proportions 2:1 (by volume), dilute to obtain a concentration of approximately 100 mg / l,

[0098] - Take 2 ml of this solution, transfer it into a glass tube and add 2 µg tricosanoic acid (C23:0) as an internal standard,

[0099] - For each analysis batch, prepare an analytical blank that does not contain of oil but only the chloroform-methanol mixture and the internal standard. It will be treated like all other samples for the rest of the procedure,

[0100] - Evaporate under vacuum the 2 ml of diluted oil,

[0101] - Add 1 ml of a 0.5 mol / l potassium hydroxide-methanol mixture,

[0102] - Purge the air from each tube with nitrogen gas,

[0103] - Heat the tubes at 80°C for 30 min to saponify the bound fatty acids (ester and triacylglycerols),

[0104] - Let cool,

[0105] - Add 1600 pL of a mixture of sulfuric acid-methanol (3.4%, v / v),

[0106] - Purge the air from each tube with nitrogen gas,

[0107] - Heat the tubes to 100°C for 10 min to break the residual ester bonds, protonate carboxylate ions and transmethylate the released fatty acids into fatty acid methyl esters (FAME),

[0108] - Let cool,

[0109] - Add 800 μl of hexane and 1.5 ml of distilled water saturated with hexane,

[0110] -Mix,

[0111] - Separate the organic (upper) and aqueous (lower) phases by centrifugation at 1000 rpm for 1 min,

[0112] - Take and discard the lower aqueous phase using a Pasteur pipette,

[0113] - Add 1.5 ml of distilled water to the organic phase,

[0114] -Mix,

[0115] - Centrifuge,

[0116] - Take the lower aqueous phase and discard it,

[0117] - Repeat the washing of the organic phase a second time with 1.5 ml of water distilled,

[0118] -Mix,

[0119] -Centrifuge,

[0120] - Place the tube in the freezer for at least 3 hours so that the aqueous phase is completely frozen,

[0121] - Transfer only the unfrozen organic phase (FAME) into a vial for chromatography,

[0122] - Drive the air out of the bottle with nitrogen gas and store the bottle at -20°C before analysis.

[0123] Extraction of unsaponifiable compounds:

[0124] In this example, the unsaponifiable compounds are extracted according to the following method:

[0125] - Heat the crude oils to 50°C for 10 to 15 minutes to homogenize them,

[0126] - Weigh 40 pl of heated crude oil (between 20 and 40 mg) into a glass tube borosilicate and dissolve in a chloroform-methanol mixture in proportions 2:1 (by volume), dilute to obtain a concentration of approximately 100 mg / l,

[0127] - Take 2 ml of this solution, transfer it into a glass tube and add 2 µg of 5[3-cholan-24-ol as internal standard,

[0128] - For each analysis batch, prepare an analytical blank that does not contain of oil but only the chloroform-methanol mixture and the internal standard. It will be treated like all other samples for the rest of the procedure,

[0129] - Evaporate the 2 ml of diluted oil under vacuum,

[0130] - Add 3 ml of a 1 mol / l potassium hydroxide-methanol mixture,

[0131] - Purge the air from each tube with nitrogen gas,

[0132] - Heat the tubes at 100°C for 10 min to saponify the bound sterols (ester of sterols, steryl glycosides etc.),

[0133] - Let cool,

[0134] - Add 1600 μl of hexane and 1.5 ml of distilled water,

[0135] - Mix,

[0136] - Separate the organic (upper) and aqueous (lower) phases by centrifugation at 1000 rpm for 1 min,

[0137] - Collect and discard the lower aqueous phase using a Pasteur pipette,

[0138] - Add 1.5 ml of distilled water to the organic phase,

[0139] - Mix,

[0140] - Centrifuge,

[0141] - Take the lower aqueous phase and discard it,

[0142] - Repeat the washing of the organic phase a second time with 1.5 ml of water distilled,

[0143] - Mix,

[0144] - Centrifuge,

[0145] - Place the tube in the freezer for at least 3 hours so that the aqueous phase is completely frozen,

[0146] - Transfer only the unfrozen organic phase into a vial for chromatography,

[0147] - Add to the tube 100 μl of a mixture of bis(trimethylsilyl)trifluoroacetamide- chlorotrimethylsilane (BSTFA-TMCS, 99:1, v / v),

[0148] - Add 100 pl of pyridine,

[0149] - Purge the air from each tube with nitrogen gas,

[0150] - Heat the tubes at 70°C for 30 min to trimethylsilylate the free sterols and other unsaponifiable lipids having a hydroxyl group,

[0151] - Let it cool,

[0152] - Add 800 μl of hexane and 1.5 ml of distilled water saturated with hexane,

[0153] - Mix,

[0154] - Separate the organic (upper) and aqueous (lower) phases by centrifugation at 1000 rpm for 1 min,

[0155] - Collect and discard the lower aqueous phase using a Pasteur pipette,

[0156] - Add 1.5 ml of distilled water to the organic phase,

[0157] - Mix,

[0158] - Centrifuge,

[0159] - Take the lower aqueous phase and discard it,

[0160] - Repeat the washing of the organic phase a second time with 1.5 ml of water distilled,

[0161] - Mix,

[0162] - Centrifuge,

[0163] - Place the tube in the freezer for at least 3 hours so that the aqueous phase is completely frozen,

[0164] - Transfer only the unfrozen organic phase into a vial for chromatography,

[0165] - Add 2 pg to 100 ppm of 5a-cholestane as an injection standard.

[0166] 1.2 Obtaining isotopic data

[0167] 1.2.1 Analysis of the stable carbon isotope composition of methyl esters of fatty acids by gas chromatography coupled with combustion furnace and isotope ratio mass spectrometer (GC-c-IRMS) - Thermo Fisher Scientific ISOLINK Trace ULTRA GC gas chromatograph. - DB-WAX polar column (30 mx 0.25 mm and a phase thickness of 0.25 pm, Agilent). - 1 pl of the FAME fraction is injected in splitless mode at 250°C. - The helium flow is set at 1.5 ml / min. The analysis is carried out in continuous flow. - The initial temperature of the chromatograph oven is 60°C, then increases to 150°C at 50°C / min, then to 170°C at 3.5°C / min, then to 185°C at 1.5°C / min, then to 225°C at 2.4°C / min and finally to 250°C at 5.5°C / min and maintained at 250°C for 15 min. - The flow at the column outlet goes to an ISOLINK combustion reactor (Thermo Fisher) where the molecules are converted into CO2(g) by combustion at 1000°C and oxidation on nickel and copper oxide wires. - The CO2 from the combustion of each fatty acid is sent to a Delta V Plus isotope ratio mass spectrometer (Thermo Fisher Scientific) where these three main isotopologues (m / z 44, 45 and 46) are detected. - The 13C / 12C isotopic ratios are expressed using the delta (ô) notation relative to international standards (Vienna Pee Dee Belemnite for ô13C), according to the equation:

[0168] [Math.l] <513d%4 = [ ( ) -11 x 103 \ / L \ ^standard / J * Sample is the 13C / 12C isotopic ratio measured in the sample, here for each methylated fatty acid, * Rstandard is the 13C / 12C isotopic ratio measured in a reference, here the co-injected CO2(g) having a known ô13C value. • Each sample / fraction is analyzed three times (analytical triplicates). The average value of the three replicates is used as the 13C value of the same sample. • The ô13C values ​​are calibrated by external calibration with the Schimmelmann Research (Indiana University Stable Isotope Reference Materials) F8-3 certified ô13C standard mixture consisting of four methyl esters and four ethyl esters of fatty acids (C14:0, C16:0, C18:0, and C20:0). The mixture is injected before and after the three analytical replicates of each sample and the average linear regression of the two calibrations is used to calibrate the ô13C values ​​of the fatty acids in the sample. • Correction: The methyl group of methanol grafted to fatty acids during transmethylation is corrected according to the following equation:

[0169] [Math.2] $ * = x [ ( n + 1 ) x <51 CFAME - ô13CMeOH ]

[0170] ô13CFA is the 13C / 12C isotopic ratio of the free, unmethylated fatty acid, analyzed from a CPG-c-SMRI standard.

[0171] ô13CFame is the 13C / 12C isotopic ratio of fatty acid methyl ester, the form analyzed in CPG-c-SMRI.

[0172] ô13CMeoH is the 13C / 12C isotopic ratio of methanol used during transmethylation determined by the difference between the ô13CFA and ô13CFA values ​​of the fatty acid standard.

[0173] n is the number of carbon atoms of the free fatty acid considered.

[0174] 1.2.2 Analysis of the stable hydrogen isotope composition of esters of fatty acid methylation by gas chromatography coupled with isotope ratio mass spectrometry (GC-p-IRMS)

[0175] - Thermo Fisher ISOLINK Trace ULTRA GC Gas Chromatograph Scientific

[0176] DB-WAX polar column (30 mx 0.25 mm and a phase thickness of 0.25 pm, Agilent). - 2 pl of the FAME fraction are injected in splitless mode at 250°C. - The helium flow is set at 3 ml / min during injection then 1 ml / min during the run. The analysis is done in continuous flow. - The initial temperature of the chromatograph oven is 60°C, then increases to 150°C at 50°C / min, then to 170°C at 3.5°C / min, then to 185°C at 1.5°C / min, then to 225°C at 2.4°C / min and finally to 250°C at 5.5°C / min and maintained at 250°C for 15 min. - The flow at the column outlet goes to an ISOLINK reduction reactor (Thermo Fisher) where the molecules are reduced and converted into H2 (g) by pyrolysis at 1450°C in a ceramic tube. - The H2 from the combustion of each fatty acid is sent to a Delta V Plus isotope ratio mass spectrometer (Thermo Fisher Scientific) where these three main isotopologues (m / z 2, 3 and 4) are detected. - The 2H / 'H isotopic ratios are expressed using the delta notation (ô) relative to international standards (Vienna StandardMean Ocean Water for ô2H), according to the equation:

[0177] [Math.3] ) -11 x 103 \ / L \ Standard * J

[0178] Sample is the 2H / 'H isotopic ratio measured in the sample, here for each methylated fatty acid.

[0179] Rstandard is the 2H / 'H isotopic ratio measured in an international reference, the VSMOW (Vienna Standard Mean Ocean Water).

[0180] Each sample / fraction is analyzed three times (analytical triplicates). The average value of the three replicates is used as the 2H value of the same sample.

[0181] The ô2H values ​​are calibrated by an external calibration with the Schimmelmann Research (Indiana University Stable Isotope Reference Materials) F8-3 certified ô2H standard mixture composed of four methyl esters and four ethyl esters of fatty acids (C14:0, C16:0, C18:0, and C20:0). The mixture is injected before and after the three analytical replicates of each sample and the average linear regression of the two calibrations is used to calibrate the ô2H values ​​of the fatty acids in the sample.

[0182] Correction: The methyl group of methanol grafted to fatty acids during transmethylation is corrected according to the following equation:

[0183] [Math.4] X [(^n + 2) ^52Hfame- 3 ^ô2HMeOH]

[0184] ô2Hfa is the 2H / 'H isotopic ratio of the free, unmethylated fatty acid

[0185] ô2HFame is the 2H / 'H isotopic ratio of fatty acid methyl ester, the form analyzed in CPG-p-SMRI.

[0186] ô2HMe0H is the 2H / 'H isotopic ratio of methanol used during transmethylation determined by the difference between the ô2HFA and ô2HFA values ​​of the fatty acid standard.

[0187] n is the number of hydrogen atoms of the free fatty acid considered.

[0188] 1.2.3 Analysis of the stable carbon isotope composition of silylated esters of sterols and other unsaponifiables by gas chromatography coupled with an isotope ratio mass spectrometer (GC-c-SMRI):

[0189] - Thermo Fisher ISOLINK Trace ULTRA GC Gas Chromatograph Scientific.

[0190] - RTX-65 column (30 mx 0.25 mm and a phase thickness of 0.25 pm, Restek).

[0191] 1 μl of the unsaponifiable fraction is injected in splitless mode at 250°C.

[0192] The helium flow is set at 1.5 ml / min. The analysis is carried out in continuous flow.

[0193] The initial temperature of the chromatograph oven is 60°C, then increases to 250°C at 60°C / min, then to 280°C at 2°C / min, and maintained at 280°C for 5 min.

[0194] The flow at the column outlet goes to an ISOLINK combustion reactor (Thermo Fisher) where the molecules are converted into CO2(g) by combustion at 1000°C and oxidation on nickel and copper oxide wires.

[0195] The CO2 from the combustion of each unsaponifiable is sent to a Delta V Plus isotope ratio mass spectrometer (Thermo Fisher Scientific) where these three main isotopologues (m / z 44, 45 and 46) are detected.

[0196] The 13C / 12C isotopic ratios are expressed using the delta (ô) notation relative to international standards (Vienna Pee Dee Belemnite for ô13C), according to the equation:

[0197] [Math.5] ô13cW= [ ( ) - Ü x 103 \ / L \ ^standard / J

[0198] Sample is the 13C / 12C isotopic ratio measured in the sample, here for each trimethylsilylated sterol

[0199] Rstandard is the 13C / 12C isotopic ratio measured in a reference, here the injected CO2(g) having a certified ô13C value.

[0200] Each sample / fraction is analyzed three times (analytical triplicates). The average value of the three replicates is used as the 13C value of the same sample.

[0201] The ô13C values ​​are calibrated by an external calibration with the Schimmelmann Research (Indiana University Stable Isotope Reference Materials) F8-3 certified ô13C standard mixture composed of four methyl esters and four ethyl esters of fatty acids (C14:0, C16:0, C18:0, and C20:0). The mixture is injected before and after the three analytical replicates of each sample and the average linear regression of the two calibrations is used to calibrate the ô13C values ​​of the fatty acids in the sample.

[0202] The combustion reactor is re-oxidized 1h after the three analyses of the same sample (triplicate).

[0203] 1.2.4 Analysis of the stable hydrogen isotope composition of silylated esters of sterols and other unsaponifiables by gas chromatography coupled with an isotope ratio mass spectrometer (GC-p-SMRI):

[0204] - Thermo Fisher ISOLINK Trace ULTRA GC Gas Chromatograph Scientific.

[0205] - RTX-65 column (30 mx 0.25 mm and a phase thickness of 0.25 pm, Restek).

[0206] 1 μl of the unsaponifiable fraction is injected in splitless mode at 250°C.

[0207] The helium flow is set at 1.5 ml / min. The analysis is carried out in continuous flow.

[0208] The initial temperature of the chromatograph oven is 60°C, then increases to 250°C at 60°C / min, then to 280°C at 2°C / min, and maintained at 280°C for 5 min.

[0209] The flow at the column outlet goes to an ISOLINK reduction reactor (Thermo Fisher) where the molecules are reduced and converted into H2 (g) by pyrolysis at 1400°C in a ceramic tube.

[0210] The H2 resulting from the combustion of each unsaponifiable is sent to a Delta V Plus isotope ratio mass spectrometer (Thermo Fisher Scientific) where these three main isotopologues (m / z 2, 3 and 4) are detected.

[0211] The 2H / ' H isotopic ratios are expressed according to the delta notation (ô) relative to international standards (Vienna StandardMean Ocean Water for Ô2H), according to the equation:

[0212] [Math.6] Ô2h(cÛ= [ () -1 ] x 103 X / L \ -standard / J

[0213] Sample is the 2H / 'H isotopic ratio measured in the sample, here for each methylated unsaponifiable.

[0214] Rstandard is the 2 H / 1 H isotopic ratio measured in an international reference, the VSMOW (Vienna StandardMean Ocean Water).

[0215] Each sample / fraction is analyzed three times (analytical triplicates). The average value of the three replicates is used as the 2H value of the same sample.

[0216] The ô2H values ​​are calibrated by an external calibration with the Schimmelmann Research (Indiana University Stable Isotope Reference Materials) F8-3 certified ô2H standard mixture composed of four methyl esters and four ethyl esters of fatty acids (C14:0, C16:0, C18:0, and C20:0). The mixture is injected before and after the three analytical replicates of each sample and the average linear regression of the two calibrations is used to calibrate the ô2H values ​​of the unsaponifiables in the sample.

[0217] 1.3 Obtaining molecular data

[0218] 1.3.1 Analysis of fatty acids by gas chromatography coupled with a flame ionization detector (GC-FID)

[0219] - Varian CP8400 Dual Column Hydrogen Gas Chromatograph as a carrier gas.

[0220] - DB-WAX polar column (30 mx 0.25 mm and a phase thickness of 0.25 qm, Agilent).

[0221] - DB-5 apolar column (30 mx 0.25 mm and a phase thickness of 0.25 qm, Agilent).

[0222] Injection into each of the two injectors in splitless mode maintained at 220°C.

[0223] The initial temperature of the chromatograph oven is 60°C, then increases to 150°C at 50°C / min, then to 170°C at 3.5°C / min, then to 185°C at 1.5°C / min, then to 225°C at 2.4°C / min and finally to 250°C at 5.5°C / min and maintained at 250°C for 15 min.

[0224] Both column outlets are equipped with a flame ionization detector maintained at 300°C.

[0225] The identification of fatty acid methyl esters is done by comparing the retention times on the two columns with standard mixtures from Merck (Supelco 37 Component FAME mixtures, PUFA No. 1, PUFA No. 3, Bacterial Acid Methyl Ester).

[0226] Each fatty acid methyl ester is quantified by comparing its integration area with that of the internal standard.

[0227] Fatty acid proportions are expressed relative to the total amount of fatty acid per oil.

[0228] 1.3.2 Analysis of unsaponifiables by gas chromatography coupled with a mass spectrometer

[0229] - Shimadzu GC-2010 Plus Gas Chromatograph with Helium as a carrier gas.

[0230] - RTX-65 polar column (30 mx 0.25 mm and a phase thickness of 0.25 pm, Restek).

[0231] 1 pl is injected in splitless mode with an injector temperature of 270°C.

[0232] The initial temperature of the chromatograph oven is 60°C, then increases to 250°C at 60°C / min, then to 280°C at 2°C / min, and maintained at 280°C for 5 min.

[0233] The temperature of the transfer line is 280°C.

[0234] The electron ionization mass spectrometer source has a collision energy of 70 eV and a temperature of 250°C.

[0235] The analysis is conducted in scanning mode (SCAN) from 50 to 1000 amu

[0236] The identification of silylated esters of sterols is done by comparison with the mass spectra of the injected standards, those found in the literature and by interpretation of the fragmentations.

[0237] 1.3.3 Analysis of unsaponifiables by gas chromatography coupled with a flame ionization detector (GC-FID)

[0238] - Shimadzu GC-2010 Plus Gas Chromatograph with Dihydrogen as a carrier gas.

[0239] - RTX-65 polar column (30 mx 0.25 mm and a phase thickness of 0.25 pm, Restek).

[0240] 1 pl is injected in splitless mode with an injector temperature of 270°C.

[0241] The initial temperature of the chromatograph oven is 60°C, then increases to 250°C at 60°C / min, then to 280°C at 2°C / min, and maintained at 280°C for 5 min.

[0242] The column outlet is equipped with a flame ionization detector maintained at 300°C.

[0243] Identification of sterol silyl esters is done by comparison of retention times with a mixture of individual standards and by identification by gas chromatography coupled with a mass spectrometer (see below).

[0244] Each sterol silyl ester is quantified by comparing its integration area with that of the internal standard.

[0245] The proportions of sterols are expressed relative to the total quantity of sterol in each oil.

[0246] 1.4 Processing of isotopic data

[0247] From the data acquired for each reference oil, certain data are selected because they are discriminating.

[0248] 1.4.1 In particular:

[0249] - A microorganism oil having been fermented from a carbon substrate (glucose, dextrose, etc.) from a plant with the C4 Calvin cycle (corn, cane, etc.) must have carbon isotopic ratios of fatty acids and unsaponifiables greater than -20%o, particularly:

[0250] The carbon isotope ratio of DHA must be greater than -15.5%o

[0251] The carbon isotope ratio of EPA must be greater than -14.5%o

[0252] The carbon isotope ratio of DPA3 must be greater than -15%o

[0253] The carbon isotope ratio of DPA6 must be greater than -16.5%o

[0254] The carbon isotope ratio of Cholesterol must be greater than -10%o

[0255] The carbon isotopic ratio of Squalene must be greater than -8%o

[0256] - A microorganism oil having been fermented from a carbon substrate (glucose, dextrose, etc.) from a plant with the C3 Calvin cycle (wheat, beet, etc.) must have carbon isotopic ratios of fatty acids and unsaponifiables lower than -20%o, particularly:

[0257] The carbon isotope ratio of DHA must be less than -27.5%o

[0258] The carbon isotope ratio of EPA must be less than -28%o

[0259] The carbon isotope ratio of DPA3 must be less than -29.5%o

[0260] The carbon isotope ratio of DPA6 must be less than -28%o

[0261] The carbon isotope ratio of Cholesterol must be less than -25%o

[0262] The carbon isotopic ratio of Squalene must be less than -22%o

[0263] - A pure microorganism oil must present:

[0264] - A difference in carbon isotope ratio analyzed by GC-c-IRMS between the DHA, EPA, DPA3 or DP6 less than 3%

[0265] - A difference in carbon isotope ratio analyzed by GC-c-IRMS between the cholesterol and EPA or DHA less than 5%©

[0266] - A pure microorganism oil must have a total isotopic ratio of hydrogen analyzed by EA-IRMS greater than -210%o

[0267] - A pure fish oil must have a total hydrogen isotope ratio analyzed by EA-IRMS less than -220%©

[0268] - A pure microorganism oil must have an isotopic ratio of EPA hydrogen analyzed by GC-p-IRMS greater than -205%©

[0269] - A pure fish oil must have a hydrogen isotope ratio of EPA analyzed by GC-p-IRMS less than -210%©

[0270] - A pure microorganism oil must have a difference in ratio hydrogen isotope ratio between DHA and EPA less than -20%©

[0271] - A so-called microorganism oil exhibiting isotopic ratio values Fatty acid carbon outside the limits expressed above has a high probability of being adulterated with fish oil or having undergone extensive refining processes.

[0272] - A fish oil must have carbon isotopic ratios of acids fat and unsaponifiables between -20%© and -35%©, particularly:

[0273] The carbon isotope ratio of DHA must be between -28.5%© and -23.5%©

[0274] The carbon isotope ratio of EPA must be between -29.5%© and -24.5%©

[0275] The carbon isotope ratio of DPA3 must be between -31.5%© and -23.5%©

[0276] The carbon isotope ratio of DPA6 must be between -27.5%© and -26.5%©

[0277] 1.4.2 Determination of an isotopic adulteration score

[0278] Based on the threshold values ​​determined in 1.4.1, a score is calculated for each oil ([Fig.l]).

[0279] On a scale of 0 (pure microalgae oil) to 14 (very likely adulteration):

[0280] • An oil of 100% microalgae origin must have a score of less than 2

[0281] • An oil of 100% fish origin must have a score between 5 and 7

[0282] • An oil said to be of 100% microalgae origin with a score between 2 and 4 contains possibly fish oil

[0283] • An oil said to be of 100% microalgae origin with a score greater than 10 contains most likely fish oil.

[0284] 1.5 Processing of molecular data

[0285] Multivariate statistical analyses:

[0286] From the data acquired for each reference oil, principal component analyses (PCA) are calculated separately between fatty acids and sterols.

[0287] The ACPs are constructed from the relative contributions of each fatty acid or unsaponifiable compound to their respective total concentrations.

[0288] The contributions of each variable (i.e. the abundance of each fatty acid or sterol) are studied at the output of the ACP models to determine the most discriminating variables (contribution or value of cosine2 on the first two principal components) and then reduce the number of variables used, because the number of samples must be greater than the number of explanatory variables (abundance of fatty acids or sterols).

[0289] By simple visual analysis of the fatty acid and sterol profiles of the oil database, other molecular indices are chosen based on the ability to discriminate different oil sources. More specifically:

[0290] • A pure microorganism oil must have a composition of unsaponifiables with less than 60% cholesterol.

[0291] • A pure fish oil must have an unsaponifiable composition with:

[0292] - more than 90% cholesterol

[0293] - less than 10% 24-methylenecholesterol

[0294] - less than 5% of other sterols.

[0295] • An unrefined microorganism oil must have an acid composition fat with a DPA n-6 / DP n-3 ratio greater than 15.

[0296] • An unrefined microorganism oil must have an acid composition fat with DHA / EPA ratio less than 5.

[0297] • An unrefined fish oil must have a fatty acid composition with DHA / EPA ratio less than 5.

[0298] Purity calculations:

[0299] A PCA model is calculated based on the chosen molecular criteria and indices. This model makes it possible to identify the source of an oil (microalgae or fish), or whether it is a mixture of microalgae and fish oil, and whether the oil is crude or refined.

[0300] The purity of a so-called microalgae oil can thus be characterized according to the equation: X = sx £1.5:0 + b X £17:0 Pcx £18:0 + dx £19:0 + e -F- f + i X £20: in —9 + JX £22: in-11 + & + / X £22: in — 9 + m X ~ + nx + ox £&otestexoi 4- px?

[0301] where:

[0302] - X is the so-called 'mixing' coordinate of an oil along axis 1 (PCI) of the model ACP

[0303] - Alphabetical letters are proportionality factors

[0304] - Cx:yn-z corresponds to the relative abundance of this fatty acid compared to the total of fatty acids of the oil determined by GC-FID

[0305] - 'sum(omega3) / sum(omega6)' corresponds to the ratio of the sum of the relative abundances of unsaturated fatty acids called 'n-3' over that of 'n-6'

[0306] - 'DHA / DPA n-6' corresponds to the ratio of the relative abundances of the acid docosahexaenoic acid (C22:6n-3) over that of docosapentaenoic acid (C22:5n-6)

[0307] - 'DHA / DPA n-6' corresponds to the ratio of the relative abundances of the acid docosahexaenoic acid (C22:6n-3) over that of eicosapentaenoic acid (C22:5n-3)

[0308] - 'Cholesterol' is the relative abundance of cholesterol compared to the sum of unsaponifiables and phytosterols analyzed by GC-FID.

[0309] Threshold values ​​of X make it possible to estimate a percentage of purity of a microalgae oil and to identify mixtures with fish oils or an advanced refining process.

[0310] Table 1 represents the threshold values ​​and calculation of microorganism / fish purity

[0311] [Tables 1] Nature Mixture Threshold value _____Pure oil Mixed and / or refined oil Pure oil 100% Microalgae > 90% Microalgae 70-90% Microalgae X<-1.6599 -1.6599 <X< -1.1005 -1.1005 <X<0 30-50% 0<X< 1.1283 70-90% Poisson 0.1283<X< 2.095 > 90% Fish 100% Fish 2095 < K < 2.577 X> 3.2625

[0312] Furthermore, the origin (species, fishing area) of a fish oil can be determined according to the following equation: F = a' XX 07:0 4 c" + f' x Cl&ln-U 4 V x Œh ln-1 + x 4 fx £2C:ln — 9 4 / X £22: In — 11 TF x £22:1«-7 4fx£22; 1«— 9 4 x — 3^ — 6)] 4 n' x (D / / 4 / DP6) 4- x Oo / esterol 4 p* x

[0313] Y is the so-called 'poisson' coordinate of an oil along axis 1 (PCI) of the PCA

[0314] Table 2 represents the threshold values ​​and calculation of Poisson origin:

[0315] [Tables2] Fish Geographical area Threshold value Small pelagic (anchovy, sardine, sardine Island, etc.) Pacific Oceans and Ancient Ati ¥<-0.3637 Small pelagic Black Sea 0.8 <Y<0.9 Thon Océan Indien ¥> 2.5665

[0316] EXAMPLE 2: Determining the origin of an oil

[0317] Since the origin of an oil is to be determined, test samples are prepared according to the methods of Example LL. Isotopic and molecular data analyses are carried out according to the methods of Examples 1.2 to 1.5.

[0318] On a first batch of test samples, the multivariate statistical analysis in principal components (PCA) presents fatty acid signatures of microalgal oils, fish oils, refined microalgal oils and refined fish oils ([Fig.2]). This approach makes it possible to identify microalgal oils cut with fish oil, whose signature lies between the two signatures microalgae and fish.

[0319] The relative abundance of cholesterol compared to other sterols found in oils can also help identify the origin of an oil ([Fig.3]). Since cholesterol represents more than 90% of the sterols in fish oils and microalgal oils contain less than 60%, a microalgal oil with a cholesterol abundance between these two values ​​can be considered suspect. This is what is highlighted in the two refined oils, which have abnormal cholesterol levels for their source. The mixture of oils highlighted by the fatty acids is also detected because of its abnormally high cholesterol level.

[0320] Analysis of the isotopic composition of carbon in specific fatty acid compounds makes it possible to distinguish different origins of the same molecule within an oil ([Fig.4]).

[0321] The carbon isotopic signature of DHA and eicosapentaenoic acid (EPA) from the same crude microalgal or fish oil is relatively close when the oil is of good quality. On the contrary, in a mixture, this signature differs between the two fatty acids due to the contribution of a different source of fatty acid compared to the base oil. This approach also makes it possible to distinguish oils from microalgae that have been grown on carbon substrates of different origin (C4 corn glucose compared to C3 wheat glucose for example), which is important in order to maximize the traceability of microalgal oils. However, microalgal oils grown on C3 substrate have a 13C signature close to that of fish, and another approach is necessary to distinguish these two sources.

[0322] These 13C signature heterogeneities are also visible between cholesterol, squalene, DHA and EPA ([Fig.5]).

[0323] Unlike the carbon isotopic signature on a specific compound alone, its combination with the overall composition of an oil in stable hydrogen isotopes makes it possible to clearly distinguish the fingerprint of fish oils from that of microalgal oil cultivated on a C3 carbon substrate ([Fig.6]).

[0324] The hydrogen isotopic signature of DHA and EPA from the same crude microalgae or fish oil is relatively close when the oil is of good quality ([Fig.7]). On the contrary, in a mixture, this signature differs between the two fatty acids due to the contribution of a different source of fatty acid compared to the base oil. This heterogeneity of isotopic signature characteristic of fish / microalgae mixtures corroborates that observed for stable carbon isotopes ([Fig.3]).

Claims

1.

2. Claims Method for authenticating and detecting adulteration of an oil of marine origin, characterized in that the method comprises: - the extraction of fatty acids and / or so-called unsaponifiable compounds from said oil, - obtaining molecular data from said oil including: the determination of the composition of fatty acids and / or so-called unsaponifiable compounds of said oil, - obtaining isotopic data of said oil including: the determination of the ratio of stable carbon isotopes (13C / 12C) of fatty acids and / or unsaponifiables, then, - obtaining a profile of said oil based on said data, then, - comparing the profile obtained with reference oil profiles including the reference profiles of a pure fish oil, a pure photosynthetic microalgal oil, a pure heterotrophic microalgal oil fermented from a carbon substrate from a plant with a C3 or C4 Calvin cycle, a terrestrial plant oil genetically modified to produce long-chain omega-3 fatty acids such as DHA, then, - the conclusion that the oil to be identified has a profile substantially equal to one of the reference profiles and therefore the indication of origin, if, at the end of the comparison, the profile corresponds to a registered profile and, if not, the conclusion that the said oil is a mixture or of another origin than a pure fish oil or a pure microorganism oil, and an estimate of the proportions of this mixture. Method according to claim 1, characterized in that obtaining isotopic data further comprises determining the ratio of stable isotopes of total hydrogen (2H / 'H) and / or hydrogen of fatty acids and / or unsaponifiables.

3. Method according to claim 1 or 2, characterized in that the determination of the molecular composition of the fatty acids includes the quantification and calculation of the relative abundance of DPA n-6, DPA n-3, DHA and EPA and the calculations of the ratios between DPA n-6 / DPA n-3 and DHA / EPA.

4. Method according to one of the preceding claims, characterized in that the determination of the molecular composition of the unsaponifiables includes the quantification and calculation of the relative abundance of sterols, in particular cholesterol, as well as squalene or squalane.

5. Method according to one of the preceding claims, characterized in that the determination of the ratio of stable carbon isotopes of fatty acids includes the determination, by gas chromatography coupled with a combustion furnace and an isotope ratio mass spectrometer, of the ratio of stable carbon isotopes of DHA, EPA, DPA n-3, and DPA n-6.

6. Method according to one of the preceding claims, characterized in that the determination of the ratio of stable carbon isotopes of the unsaponifiables includes the determination, by gas chromatography coupled with a combustion furnace and an isotope ratio mass spectrometer, of the ratio of carbon isotopes (13 C / 12 C) of cholesterol, 24-methylenecholesterol, stigmasterol of squalene and squalane.

7. Method according to one of the preceding claims, characterized in that the determination of the ratio of stable isotopes of total hydrogen (2H / 'H) is obtained by an elemental analyzer coupled to an isotope ratio mass spectrometer.

8. Method according to one of the preceding claims, characterized in that the determination of the ratio of stable hydrogen isotopes (2H / 'H) of fatty acids includes the determination, by gas chromatography coupled with a pyrolysis oven and an isotope ratio mass spectrometer, of the ratio of stable hydrogen isotopes (2H / 'H) of DHA, EPA, DPAn-6 and DPAn-3.

9. Method according to one of the preceding claims, characterized in that the determination of the ratio of stable hydrogen isotopes of the unsaponifiables includes the determination, by gas chromatography coupled with a pyrolysis oven and an isotope ratio mass spectrometer, of the ratio of stable hydrogen isotopes (2H / 'H) of cholesterol and squalene.

10. Method according to one of the preceding claims, characterized in that obtaining isotopic data includes: - determining the difference in the carbon isotopic ratio between DHA, EPA, DPA n-3, or DPA n-6, and / or - determining the difference in the carbon isotopic ratio between cholesterol, squalene, EPA or DHA, and / or - determining the difference in the hydrogen isotopic ratio between DHA and EPA, DPA3, or DPA6, and / or - determining the difference in the hydrogen isotopic ratio between cholesterol, squalene, EPA or DHA.

11. Method according to one of the preceding claims, characterized in that obtaining a profile of said oil includes a principal component analysis of the molecular data obtained, making it possible to determine a proportion of purity or mixture of said oil.

12. Method according to one of the preceding claims, characterized in that obtaining a profile of said oil includes assigning an adulteration score of the oil between 0 and 14 from the isotopic data obtained.

13. Method for creating a library of oil profiles, the method comprising determining reference profiles, each profile corresponding to a pre-identified oil, characterized in that the method comprises, for each profile: - the extraction of fatty acids and / or so-called unsaponifiable compounds from each oil, - the obtaining of molecular data including: the determination of the composition of fatty acids and / or so-called unsaponifiable compounds of each oil, - the obtaining of isotopic data including: the determination of the ratio of stable carbon isotopes (13C / 12C) of fatty acids and / or so-called unsaponifiable compounds of each oil, then, - the obtaining of a reference profile, for each oil, according to the data.

14. Method according to claim 13, characterized in that the obtaining of molecular data is as defined in one of claims 3 to 4.

15. Method according to claim 13 or 14, characterized in that the obtaining of isotopic data is as defined in one of claims 2 and 6 to 11.

16. A library of oil profiles obtained by a method as described in one of claims 13 to 15.

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