Method for authenticating and detecting adulteration of oils of marine origin

Abstract

The present invention relates to a method for authenticating and detecting adulteration of an oil of marine origin, characterised in that the method comprises: obtaining molecular data including: - determining the fatty acid and / or unsaponifiable compound composition of said oil; obtaining isotopic data including: - determining the ratio of the stable isotopes of carbon (13C / 12C) of fatty acids and / or of unsaponifiables, - obtaining a profile of said oil on the basis of 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 identical to one of the reference profiles and therefore indicating the origin, if, at the end of the comparison, the profile corresponds to a recorded profile and, otherwise, concluding that the oil is a mixture or of different origin than a pure fish oil or a pure microorganism oil, and estimating the proportions of this mixture.

Description

[0001]DESCRIPTIONTITLE: METHOD FOR AUTHENTICATING AND DETECTING ADULTERATION OF OILS OF MARINE ORIGIN The present invention belongs to the technical field of oils of marine origin, in particular microalgae oils, intended for nutraceuticals, pharmacy, human or animal food and cosmetics. 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 possible adulteration of this oil, i.e. for detecting a possible mixture of several oils of different natures. The invention also relates to a method for creating a library of profiles of marine oils of different natures for implementing the authentication method,as well as the resulting library. STATE OF THE ART 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,becomes the dominant source of omega 3 in the multi-billion dollar dietary supplement market. 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 sell for more than fish oils. Such a market attracts fraud, which mainly involves 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. 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 the 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. These analyses remain limited in reliably identifying frauds concerning refined products. PURPOSE OF THE INVENTION The purpose of the present invention is to propose a reliable solution for authenticating the nature of an oil of marine origin, in particular a heterotrophic microalgae oil, and for detecting a possible adulteration of this oil, i.e. for detecting a possible mixture of several oils of different natures,in particular a mixture of microalgal oil and fish oil. OBJECTS OF THE INVENTION 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 microalgal oil. According to a second aspect, the invention relates to a method for creating a library, or database, of reference profiles of oils of marine origin. According to a third aspect, the invention relates to a library of reference profiles of oils of marine origin. Thus, the 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 ditunsaponifiable compounds of said oil,obtaining isotopic data including:- determining the ratio of stable carbon isotopes (13C / 12C) of fatty acids and / or unsaponifiables of said oil,- 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, concluding that said oil is a mixture or of another origin than a pure fish oil or a pure microorganism oil, and an estimation of the proportions of this mixture. In particular, the reference profiles of oils of determined marine origin, i.e. whose nature is known, include the profiles of a pure fish oil, a pure photosynthetic microalgae oil,of a pure heterotrophic microalgal 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. Advantageously, the reference profiles are available in the form of a library, or database, obtained according to a method as described below. 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 microalgal oil, supposed 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 for determining the molecular and isotopic composition of the lipids in these oils. The invention presented here is a molecular and isotopic diagnostic tool for certifying the purity of an oil of marine origin, in particular a microalgal oil. 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. According to one embodiment, obtaining isotopic data further comprises determining the ratio of stable hydrogen isotopes (, 2 H / 1H) total and / or hydrogen of fatty acids and / or unsaponifiables. Advantageously, the determination of the molecular composition of 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. Advantageously, the determination of the molecular composition of unsaponifiable compounds includes the quantification and calculation of the relative abundance of sterols, in particular cholesterol, as well as squalene or squalane. 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 ( 13 C / 12 C) DHA, EPA, DPA n-3, and DPA n-6. 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 ( 13 C / 12 C) cholesterol, 24-methylenecholesterol, stigmasterol, squalene and squalane. According to one embodiment, the determination of the ratio of stable hydrogen isotopes ( 2 H / 1 H) total is obtained by an elemental analyzer coupled to an isotope ratio mass spectrometer. According to one embodiment, the determination of the ratio of stable hydrogen isotopes of fatty acids includes the determination, by gas chromatography coupled to a pyrolysis oven and an isotope ratio mass spectrometer, of the ratio of stable hydrogen isotopes (2 H / 1H) of DHA, EPA, n-3 DPA, and n-6 ​​DPA. 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 isotopic ratio m 2ass 1e spectrometer, of the ratio of stable hydrogen isotopes ( H / H) of cholesterol and squalene. Advantageously, obtaining isotopic data includes: - determining the difference in the carbon isotope ratio between DHA, EPA, n-3 DPA, or n-6 DPA, and / or - determining the difference in the carbon isotope ratio between cholesterol, squalene, EPA or DHA. and / or- determining the difference in hydrogen isotope ratio between DHA and EPA, DPA3, or DPA6, and / or- determining the difference in hydrogen isotope ratio between cholesterol, squalene, EPA, or DHA.According to one 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. According to another characteristic of the invention, obtaining a profile of said oil includes the allocation of an adulteration score of the oil between 0 and 14 from the isotopic data obtained.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: 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 so-called unsaponifiable compounds, Obtaining a reference profile, for each oil, or each mixture, depending on the data.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. Advantageously, obtaining isotopic data further comprises determining the ratio of stable hydrogen isotopes (. 2 H / 1H) total and / or hydrogen of fatty acids and / or unsaponifiables. 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. 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. According to one embodiment, the determination of the ratio of stable carbon isotopes of the 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, DPA n-3, and DPA n-6.According to one embodiment, the determination of the ratio of stable carbon isotopes of unsaponifiables includes the determination, by gas chromatography coupled with a combustion oven and an isotope ratio mass spectrometer, of the carbon isotope ratio of cholesterol, 24-methylenecholesterol, stigmasterol of squalene and squalane. According to one embodiment, the determination of the ratio of stable isotopes of total hydrogen is obtained by an elemental analyzer coupled with an isotope ratio mass spectrometer. 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.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. Advantageously, obtaining isotopic data includes:- determining the difference in carbon isotope ratio between DHA, EPA, n-3 DPA, or n-6 DPA, and / or- determining the difference in carbon isotope ratio between cholesterol, squalene, EPA, or DHA, and / or- determining the difference in hydrogen isotope ratio between DHA and EPA, n-3 DPA, or n-6 DPA, and / or- determining the difference in hydrogen isotope ratio between cholesterol, squalene, EPA, or DHA.Advantageously, obtaining a profile of said oil includes a principal component analysis of the molecular data obtained. 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: - Molecular and isotopic profiling of oils, preferably of marine origin, by the combined analysis of: fatty acids, unsaponifiables, in particular sterols, the overall composition in stable isotopes of hydrogen (δ. 2 H-IRMS bulk), of the stable carbon isotope composition of fatty acids (δ 13 C-CSIA-AG), of the stable carbon isotope composition of sterols (δ 13 C-CSIA-STEROL), of the stable hydrogen isotope composition of fatty acids (δ 2H-CSIA-AG),- The creation of a database of these molecular and isotopic profiles on different reference oils or aquaculture feeds (fish, microalgae, mixtures, genetically modified terrestrial plants),- The selection of molecular and isotopic indices allowing to distinguish the different sources of oils by multivariate statistical approaches such as PCA or other,- The distinction between different carbon substrates used to cultivate certain strains of microalgae by δ 13 C-CSIA-AG / STEROL,- The identification of refined or blended oils on the basis of the various criteria chosen above,- The assignment of a purity score to an oil relative to its origin.DETAILED DESCRIPTION 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 :[FIG.1] illustrates a score scale obtained after isotopic analyses of different oils, [FIG.2] illustrates the score plot of the PCA of fatty acid profiles of marine oils from different sources, [FIG.3] illustrates the abundance of cholesterol in marine oils from different origins, [FIG.4] illustrates the isotopic signatures 13 C of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in marine oils of different origins, [Fig.5] illustrates the isotopic signatures 13 C of cholesterol, squalene, DHA and EAP in marine oils of different origins, [FIG.6] illustrates the isotopic signatures 2 H global and 13C of DHA in marine oils of different origins, [FIG.7] illustrates the hydrogen isotopic ratio of EPA and DHA in marine oils of different origins.EXAMPLE 1: Preparation of a database of oils of determined marine origin A database was prepared on crude or refined oils, microalgal culture extracts, aquaculture feeds and oil mixtures made manually:• 30 fish oils• 34 Thraustochytrid oils• 5 photoautotrophic microalgal oils• 4 fish-based aquaculture feeds• 16 microalgal and fish oil mixtures.1.1 Extractions of fatty acids and unsaponifiable compounds Fatty acids and unsaponifiable compounds are extracted, for each oil or feed, by methods known to those skilled in the art.Extraction of fatty acids. :In this example,The fatty acids are extracted according to the following method: - Heat the crude oils at 50°C for 10 to 15 minutes to homogenize them, - Weigh 40 µl of heated crude oil (between 20 and 40 mg) in a borosilicate glass tube and dissolve in a chloroform-methanol mixture in the proportions 2:1 (by volume), dilute to obtain a concentration of approximately 100 mg / l, - Take 2 ml of this solution, transfer them to a glass tube and add 2 µg of tricosanoic acid (C23:0) as an internal standard, - For each analysis batch, prepare an analytical blank that does not contain 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,- Evaporate under vacuum the 2 ml of diluted oil,- Add 1 ml of a mixture of potassium hydroxide-methanol at 0.5 mol / l,- Drive the air out of each tube with nitrogen gas,- Heat the tubes to 80°C for 30 min to saponify the bound fatty acids (ester and triacylglycerols),- Allow to cool,- Add 1600 µL of a sulfuric acid-methanol mixture (3.4%, v / v),- Expel the air from each tube with nitrogen gas,- Heat the tubes to 100°C for 10 min to break the residual ester bonds,protonate the carboxylate ions and transmethylate the released fatty acids to fatty acid methyl esters (FAME),- Allow to cool,- Add 800 µL of hexane and 1.5 ml of hexane-saturated distilled water,- Mix,- Separate the organic (upper) and aqueous (lower) phases by centrifugation at 1000 rpm for 1 min,- Collect and discard the lower aqueous phase using with a Pasteur pipette,- Add 1.5 ml of distilled water to the organic phase,- Mix,- Centrifuge,- Take the lower aqueous phase and discard it,- Repeat the washing of the organic phase a second time with 1.5 ml of distilled water,- Mix,- Centrifuge,- Place the tube in the freezer for at least 3 hours so that the aqueous phase is completely frozen,- Transfer only the unfrozen organic phase (FAME) into a chromatography bottle,- Expel the air from the bottle with nitrogen gas and store the bottle at -20°C before analysis. Extraction of unsaponifiable compounds: In this example, the unsaponifiable compounds are extracted using the following method:- Heat the crude oils at 50°C for 10 to 15 minutes to homogenize them,- Weigh 40 µl of heated crude oil (between 20 and 40 mg) in a borosilicate glass tube and dissolve in a chloroform-methanol mixture in the proportions 2:1 (by volume), dilute to obtain a concentration of approximately 100 mg / l,- Take 2 ml of this solution, transfer them to a glass tube and add 2 µg of 5β-cholan-24-ol as internal standard,- For each analysis batch,prepare an analytical blank that does not contain 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,- Evaporate under vacuum the 2 ml of diluted oil,- Add 3 ml of a 1 mol / l potassium hydroxide-methanol mixture,- Expel the air from each tube with nitrogen gas,- Heat the tubes at 100°C for 10 min to saponify the bound sterols (ester of sterols, steryl glycosides etc.),- Allow to cool,- Add 1600 µl of hexane and 1.5 ml of distilled water,- Mix,- Separate the organic (upper) and aqueous (lower) phases by centrifugation at 1000 rpm for 1 min,- Collect and discard the lower aqueous phase using a Pasteur pipette,- Add 1.5 ml of distilled water to the organic phase,- Mix,- Centrifuge,- Collect the aqueous phase lower and discard it,- Repeat the washing of the organic phase a second time with 1.5 ml of distilled water,- Mix,- Centrifuge,- Place the tube in the freezer for at least 3 hours so that the aqueous phase is completely frozen,- Transfer only the unfrozen organic phase into a vial for chromatography,- Add 100 µl of a mixture of bis(trimethylsilyl)trifluoroacetamide-chlorotrimethylsilane (BSTFA-TMCS, 99:1, v / v) to the tube,- Add 100 µl of pyridine,- Purge the air from each tube with nitrogen gas,- Heat the tubes at 70°C for 30 min to trimethylsilylate free sterols and other unsaponifiable lipids with a hydroxyl group,- Allow to cool,- Add 800 µl of hexane and 1.5 ml of distilled water saturated with hexane,- Mix,- Separate the organic (upper) and aqueous phases (lower) by centrifugation at 1000 rpm for 1 min,- Take and discard the lower aqueous phase using a Pasteur pipette,- Add 1.5 ml of distilled water to the organic phase,- Mix,- Centrifuge,- Take the lower aqueous phase and discard it,- Repeat the washing of the organic phase a second time with 1.5 ml of distilled water,- Mix,- Centrifuge,- Place the tube in the freezer for at least 3 hours so that the aqueous phase is completely frozen,- Transfer only the unfrozen organic phase into a vial for chromatography,- Add 2 µg at 100 ppm of 5α-cholestane as an injection standard.1.2 Obtaining isotopic data1.2.1 Analysis of the stable carbon isotope composition of fatty acid methyl esters by gas chromatography coupled with a combustion oven and an isotope ratio mass spectrometer (GC-c-IRMS)- ThermoFisher Scientific ISOLINK Trace ULTRA GC gas chromatograph.- DB-WAX polar column (30 m × 0.25 mm and a phase thickness of 0.25 μm, Agilent).- 1 µl of the FAME fraction is injected in splitless mode at 250°C.- The helium flow is set to 1,5 ml / min. 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 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 are detected (m / z 44, 45 and 46).- The 13C / 12C isotopic ratios are expressed according to the delta (δ) notation compared to international standards (Vienna Pee Dee Belemnite for δ, 13 C), according to the equation: [Math 1] ^ ^ × 10 ^^^^^^^^^ Rsample 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 value δ 13 C known.- Each sample / fraction is analyzed three times (analytical triplicates). The average value of the three replicates is used as the value of 13 C of the same sample.- The δ13C values ​​are calibrated by an external calibration with the mixture of certified standards in δ 13 C F8-3 from Schimmelmann Research (Indiana University Stable Isotope Reference Materials) 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 δ values 13C of the fatty acids in the sample.- Correction: The methyl group of the methanol grafted to the fatty acids during transmethylation is corrected according to the following equation: [Math 2] ^ − ] δ 13 CFA is isotopic ratio 13 C / 12 C of the free, unmethylated fatty acid, analyzed from a standard in CPG-c-SMRI. δ 13 CFAME is the isotope ratio 13 C / 12 C of fatty acid methyl ester, the form analyzed in CPG-c-SMRI. δ 13 C MeOH is the isotopic ratio 13 C / 12C of the methanol used during transmethylation determined by difference between δ13CFA and δ13CFA values ​​of fatty acid standard. n is the number of carbon atoms of the free fatty acid considered. 1.2.2 Analysis of the stable hydrogen isotope composition of fatty acid methyl esters by gas chromatography coupled with isotope ratio mass spectrometer (GC-p-IRMS)- Gas chromatograph GC ISOLINK Trace ULTRA Thermo FisherScientific DB-WAX polar column (30 m × 0.25 mm and a phase thickness of 0.25 μm, Agilent).- 2 μl of the FAME fraction are injected in splitless mode at 250°C.- The helium flow is fixed at 3 ml / min during the 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 column outlet flow 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 are detected. main (m / z 2, 3 and 4).- The 2H / 1H isotopic ratios are expressed according to the delta (δ) notation compared to international standards (Vienna StandardMean Ocean Water for δ 2 H), according to the equation: [Math 3] × ^ ^ 10 ^^^^^^^^ Rsample is the isotopic ratio 2 H / 1H measured in the sample, here for each methylated fatty acid. R standard is the isotopic ratio 2 H / 1 H measured in an international reference, the VSMOW (Vienna StandardMean Ocean Water). Each sample / fraction is analyzed three times (analytical triplicates). The average value of the three replicates is used as the value 2 H of the same sample. The values ​​of δ 2 H are calibrated by an external calibration with the mixture of certified standards in δ 2 H F8-3 from Schimmelmann Research (Indiana University Stable Isotope Reference Materials) 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 δ values 2H of the fatty acids in the sample. Correction: The methyl group of the methanol grafted to the fatty acids during transmethylation is corrected according to the following equation: [Math 4] ^ − ] ^1δ2H FA is the 2H / 1H isotopic ratio of the free, unmethylated fatty acid δ2H FAME is the 2H / 1H isotopic ratio of fatty acid methyl ester, the form analyzed by GC-p-IRMS. δ2H MeOH is the 2H / 1H isotopic ratio of methanol used during transmethylation determined by the difference between the δ2HFA and δ2HFA values ​​of the fatty acid standard. n is the number of hydrogen atoms in the free fatty acid considered.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-IRMS):- Thermo FisherScientific ISOLINK Trace ULTRA GC gas chromatograph.- RTX-65 column (30 m × 0.25 mm and a phase thickness of 0.25 μm, Restek).1 μl of the unsaponifiable 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 250°C at 60°C / min, then to 280°C at 2°C / min, and maintained at 280°C for 5 min.The column outlet flow 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 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. The isotope ratios. 13 C / 12 C are expressed using delta (δ) notation compared to international standards (Vienna Pee Dee Belemnite for δ 13 C), according to the equation: [Math 5] × 10^ ^ ^^^^^^^^ The sample is the isotopic ratio 13 C / 12 C measured in the sample, here for each trimethylsilylated sterol R standard is the isotopic ratio 13 C / 12 C measured in a reference, here the co-injected CO2(g) having a value δ 13Certified C. Each sample / fraction is analyzed three times (analytical triplicates). The average value of the three replicates is used as the value 13 C of the same sample. The values ​​of δ 13 C are calibrated by an external calibration with the mixture of certified standards in δ 13 C F8-3 from Schimmelmann Research (Indiana University Stable Isotope Reference Materials) 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 δ values 13C of the fatty acids in the sample. The combustion reactor is re-oxidized 1 hour after the three analyses of the same sample (triplicate).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-IRMS):- Gas chromatograph GC ISOLINK Trace ULTRA Thermo FisherScientific.- RTX-65 column (30 m × 0.25 mm and a phase thickness of 0.25 μm, Restek).1 μl of the unsaponifiable fraction is injected in splitless mode at 250°C. The helium flow is set at 1.5 ml / min. The analysis is done in continuous flow. 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.The column outlet flow goes to an ISOLINK reduction reactor (Thermo Fisher) where the molecules are reduced and converted to H2 (g) by pyrolysis at 1400°C in a ceramic tube. The H2 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. Isotopic ratios 2 H / 1 H are expressed using the delta (δ) notation relative to international standards (Vienna StandardMean Ocean Water for δ2H), according to the equation: [Math 6] ^ ^ × 10 ^^^^^^^^ R échantillon is the isotopic ratio 2 H / 1 H measured in the sample, here for each methylated unsaponifiable. Rstandard is the isotopic ratio 2 H / 1H measured in an international reference, the VSMOW (Vienna StandardMean Ocean Water). 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. The values ​​of δ 2 H are calibrated by an external calibration with the mixture of certified standards in δ 2 H F8-3 from Schimmelmann Research (Indiana University Stable Isotope Reference Materials) 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 δ values 2H of the unsaponifiables in the sample. 1.3 Obtaining molecular data 1.3.1 Analysis of fatty acids by gas chromatography coupled with a flame ionization detector (GC-FID) - Varian CP8400 dual-column gas chromatograph and dihydrogen as carrier gas. - DB-WAX polar column (30 m × 0.25 mm and a phase thickness of 0.25 μm, Agilent). - DB-5 non-polar column (30 m × 0.25 mm and a phase thickness of 0.25 μm, Agilent). Injection into each of the two injectors in splitless mode maintained at 220°C. 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. Both column outlets are equipped with a flame ionization detector maintained at 300°C. Identification of fatty acid methyl esters is done by comparing the retention times on thetwo columns with standard mixtures from Merck (Supelco 37 Component FAME mixtures, PUFA No. 1, PUFA No. 3, Bacterial Acid Methyl Ester). Each fatty acid methyl ester is quantified by comparing its integration area with that of the internal standard. The proportions of fatty acids are expressed relative to the total amount of fatty acids per oil. 1.3.2 Analysis of unsaponifiables by gas chromatography coupled with a mass spectrometer - Shimadzu GC-2010 Plus gas chromatograph with helium as the carrier gas. - RTX-65 polar column (30 m × 0.25 mm and a phase thickness of 0.25 μm, Restek). 1 μl is injected in splitless mode with an injector temperature of 270 ° C. 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. The temperature of the transfer line is 280°C. The source of the ionization mass spectrometerelectron beam has a collision energy of 70 eV and a temperature of 250°C. The analysis is carried out in scanning mode (SCAN) from 50 to 1000 amu. The identification of sterol silyl esters is done by comparison with the mass spectra of the injected standards, those found in the literature and by interpretation of the fragmentations. 1.3.3 Analysis of unsaponifiables by gas chromatography coupled with a flame ionization detector (GC-FID) - Shimadzu GC-2010 Plus gas chromatograph with dihydrogen as carrier gas. - RTX-65 polar column (30 m × 0.25 mm and a phase thickness of 0.25 μm, Restek). 1 μl is injected in splitless mode with an injector temperature of 270°C. 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. The column outlet is equipped with a flame ionization detector maintained at 300°C. Ester identificationThe determination of sterol silyl esters is done by comparing retention times with a mixture of individual standards and by identification by gas chromatography coupled with a mass spectrometer (see below). Each sterol silyl ester is quantified by comparing its integration area with that of the internal standard. The proportions of sterols are expressed relative to the total amount of sterol in each oil. 1.4 Processing of isotopic data From the data acquired for each reference oil, certain data are selected because they are discriminating.1.4.1 In particular:- A microorganism oil having been fermented from a carbon substrate (glucose, dextrose, etc.) coming from a plant with the C4 Calvin cycle (corn, cane, etc.) must present carbon isotopic ratios of fatty acids and unsaponifiables greater than -20‰, particularly: The carbon isotopic ratio of DHA must be greater than -15.5‰ TheCarbon isotope ratio of EPA must be greater than -14.5‰ Carbon isotope ratio of DPA3 must be greater than -15‰ Carbon isotope ratio of DPA6 must be greater than -16.5‰ Carbon isotope ratio of Cholesterol must be greater than -10‰ Carbon isotope ratio of Squalene must be greater than -8‰- A microorganism oil that has been fermented from a carbon substrate (glucose, dextrose, etc.) from a plant with the C3 Calvin cycle (wheat, beet, etc.) must have carbon isotope ratios of fatty acids and unsaponifiables less than -20‰, particularly: Carbon isotope ratio of DHA must be less than -27.5‰ Carbon isotope ratio of EPA must be less than -28‰ Carbon isotope ratio of DPA3 carbon isotope ratio must be less than -29.5‰ DPA6 carbon isotope ratio must be less than -28‰ Cholesterol carbon isotope ratio must be less than-25‰ The carbon isotope ratio of Squalene must be less than -22‰- A pure microorganism oil must have:- A difference in carbon isotope ratio analyzed by GC-c-IRMS between DHA, EPA, DPA3 or DP6 of less than 3‰- A difference in carbon isotope ratio analyzed by GC-c-IRMS between cholesterol and EPA or DHA of less than 5‰- A pure microorganism oil must have a total hydrogen isotope ratio analyzed by EA-IRMS of greater than -210‰- A pure fish oil must have a total hydrogen isotope ratio analyzed by EA-IRMS of less than -220‰- A pure microorganism oil must have a hydrogen isotope ratio of EPA analyzed by GC-p-IRMS of greater than -205‰- A pure fish oil must have a hydrogen isotope ratio of EPA analyzed by GC-p-IRMS less than -210‰- A pure microorganism oil must have a difference in hydrogen isotope ratiobetween DHA and EPA less than -20‰- A so-called microorganism oil with fatty acid carbon isotope ratio values ​​outside the limits expressed above has a high probability of being adulterated with fish oil or of having undergone extensive refining processes.- A fish oil must have fatty acid and unsaponifiable carbon isotope ratios between -20‰ and -35‰, particularly: The carbon isotope ratio of DHA must be between -28.5‰ and -23.5‰ The carbon isotope ratio of EPA must be between -29.5‰ and -24.5‰ The carbon isotope ratio of DPA3 must be between -31.5‰ and -23.5‰ The carbon isotope ratio of DPA6 must be between -27.5‰ and -26.5‰ 1.4.2 Determination of an isotopic adulteration score Based on the threshold values ​​determined in 1.4.1, a score is calculated for each oil (Figure 1). On a scale of 0 (pure microalgal oil) to 14(adulteration very likely):• An oil of 100% microalgae origin must have a score of less than 2• An oil of 100% fish origin must have a score between 5 and 7• An oil said to be of 100% microalgae origin with a score between 2 and 4 possibly contains fish oil• An oil said to be of 100% microalgae origin with a score above 10 very likely contains fish oil. 1.5 Molecular data processingMultivariate statistical analyses:From the data acquired for each reference oil, principal component analyses (PCA) are calculated separately between fatty acids and sterols. The PCAs are constructed from the relative contributions of each fatty acid or unsaponifiable compound compared to their respective total concentrations. 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 significant variables.discriminating factors (contribution or cosine² value 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). 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 between different oil sources. More precisely:• A pure microorganism oil must have an unsaponifiable composition with less than 60% cholesterol.• A pure fish oil must have an unsaponifiable composition avec :- more than 90% cholesterol- less than 10% 24-methylenecholesterol- less than 5% other sterols.• An unrefined microorganism oil must have a fatty acid composition with a DPA n-6 / DP n-3 ratio greater than 15.• An unrefined microorganism oil must have a fatty acid composition with a DHA / EPA ratio less than 5.• An unrefined fish oil must have a fatty acid composition with a DHA / EPA ratio less than 5.Purity calculations: A PCA model is calculated based on the chosen molecular criteria and indices. This model allows the source of an oil to be identified (microalgae or fish), or if it is a mixture of microalgae and fish oil, and if the oil is crude or refined. where: -X is the so-called 'mixture' coordinate of an oil along axis 1 (PC1) of the PCA model -The alphabetical letters are proportionality factors- Cx:yn-z corresponds to the relative abundance of this fatty acid compared to the total fatty acids of the oil determined by GC-FID -'sum(omega3) / sum(omega6)' corresponds to the ratio of the sum of the relative abundances of the unsaturated fatty acids called 'n-3' to that of the 'n-6' -'DHA / DPA n-6' corresponds to the ratio of the relative abundances of docosahexaenoic acid (C22:6n-3) to that of docosapentaenoic acid (C22:5n-6) -'DHA / DPA n-6' corresponds to the ratio of the relative abundances of docosahexaenoic acid (C22:6n-3) to that of eicosapentaenoic (C22:5n-3) - 'Cholesterol' corresponds to the relative abundance of cholesterol compared to the sum of unsaponifiables and phytosterols analyzed by GC-FID.X threshold values ​​allow to estimate a percentage of purity of a microalgae oil and to identify mixtures with fish oils or an advanced refining process. Table 1 represents the threshold values ​​and calculation of microorganism / fish purity [TABLE 1] Furthermore, the origin (species, fishing area) of a fish oil can be determined according to the following equation: Y is the so-called 'fish' coordinate of an oil according to axis 1 (PC1) of the PCA Table 2 represents the threshold values ​​and calculation of fish origin: [TABLE 2]EXAMPLE 2: determination of the origin of an oilThe origin of an oil must be determined, test samples are prepared according to the methods of example 1.1. Isotopic and molecular data analyses are carried out according to the methods of examples 1.2 to 1.5.On a first batch of test samples, multivariate principal component analysis (PCA) presents fatty acid signatures of microalgal oils, fish oils, refined microalgal oils, and refined fish oils (Figure 2). This approach allows the identification of microalgal oils cut with fish oil, whose signature lies between the two signatures of microalgae and fish. The relative abundance of cholesterol compared to other sterols found in oils can also help identify the origin of an oil (Figure 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 oil mixture highlighted by fatty acids is also detected due to its abnormally high cholesterol content. Analysis of the carbon isotopic composition of specific fatty acid compounds makes it possible to distinguish different origins of the same molecule within an oil (Figure 4). The carbon isotopic signature of DHA and eicosapentaenoic acid (EPA) in the same crude microalgal or fish oil is relatively similar when the oil is of good quality. Conversely, 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 grown on carbon substrates of different origin (e.g., C4 corn glucose versus C3 wheat glucose), which is important in order to maximize the traceability of microalgal oils.However, microalgae oils grown on C3 substrate have a signature. 13 C close to that of fish, and another approach is needed to distinguish these two sources. These signature heterogeneities 13C are also visible between cholesterol, squalene, DHA and EPA (Figure 5). Unlike the carbon isotopic signature on a specific compound alone, its combination with the overall composition of an oil in stable hydrogen isotopes allows to clearly distinguish the fingerprint of fish oils from that of microalgal oil grown on C3 carbon substrate (Figure 6). The hydrogen isotopic signature of DHA and EPA of the same crude microalgal or fish oil is relatively close when the oil is of good quality (Figure 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 / microalgal mixtures corroborates that observed for stable carbon isotopes (Figure 3).

Claims

CLAIMS1) Method for authenticating and detecting adulteration of an oil of marine origin, characterized in that the method comprises:- obtaining molecular data of said oil including: determining the composition of fatty acids and / or so-called unsaponifiable compounds of said oil,- obtaining isotopic data of said oil including: determining the ratio of stable carbon isotopes ( 13 C / 12C) 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 having been fermented from a carbon substrate originating from a plant with a C3 or C4 Calvin cycle, a terrestrial plant oil having been genetically modified to produce long-chain omega 3 fatty acids such as DHA, then,- 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 registered profile and, if not, concluding that 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.2) Method according to claim 1, characterized in that obtaining isotopic data further comprises determining the ratio of stable isotopes of hydrogen (, 2 H / 1H) total 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 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 (. 13 C / 12 C) cholesterol, 24-methylenecholesterol, stigmasterol, squalene and squalane.7) Method according to one of the preceding claims, characterized in that the determination of the ratio of stable hydrogen isotopes ( 2 H / 1 H) total 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 isotopes of hydrogen ( 2 H / 1 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 ( 2 H / 1 H) 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 isotopes of hydrogen ( 2 H / 1H) 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 purity or mixture proportion 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 the determination of reference profiles, each profile corresponding to a pre-identified oil, characterized in that the method comprises, for each profile: - obtaining molecular data including: determining the composition of fatty acids and / or so-called unsaponifiable compounds of each oil, - obtaining isotopic data including:. the determination of the ratio of stable carbon isotopes ( 13 C / 12C) fatty acids and / or so-called unsaponifiable compounds of each oil, then, - obtaining a reference profile, for each oil, based on 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) Library of oil profiles obtained by a method as described in one of claims 13 to 15.

Images