METHOD FOR DETECTING SCATOL IN MALE PORK ADIPOSE TISSUE

Abstract

The invention relates to a method for detecting the presence of skatole in adipose tissue from a male pig, comprising at least the steps of: a) preparing an organic extract from a sample of the adipose tissue; b) subjecting the organic extract prepared in step a) to an electrochemiluminescence reaction; etc.; measuring the intensity of the luminescence during step b) and, if the measured luminescence intensity exceeds a predetermined threshold value, inferring the presence of skatole in the adipose tissue sample; and characterized in that step a) comprises the substeps of: i) collecting the adipose tissue sample using a sampling swab; ii) dissolving the adipose tissue sample in a medium comprising an aprotic organic solvent; iii) adding an anhydrous background salt to the aprotic organic solvent, this addition being able to be carried out before or after substep ii).Applications: identification, on slaughter lines, of entire male pigs whose meat carries boar odor; research tool, in particular to study the factors influencing the production of skatole in the adipose tissues of entire male pigs with a view to developing methods to prevent or reduce boar odor.

Description

Description Title of the invention: METHOD FOR DETECTING SCATOL IN A MALE PIG'S ADIPOSE TISSUE Technical field

[0001] The invention relates to the field of agri-food and, in particular, to the field of the production and distribution of pork.

[0002] — More specifically, the invention relates to a method for detecting very rapidly the presence of skatole, or 3-methylindole (3-MIH), in adipose tissue of male pig and, if present, to determine its content, with a very high sensitivity and very high specificity with respect to the other above-mentioned compounds likely to be present in this adipose tissue.

[0003] Scatol being, with androstenone, responsible for a strong and unpleasant odor, so-called boar odor, which is released during the cooking of male pig meat whole, the invention is, firstly, capable of being implemented for sorting in slaughterhouses the carcasses of entire male pigs in order to isolate those whose meat carries boar odor and directs them to a processing circuit adapted.

[0004] It is also likely to be implemented as a research tool, in particular to study the factors influencing the production of skatole in tissues fat from entire male pigs with a view to developing methods for prevent or reduce boar taint, for example by genetic selection or by modification of breeding conditions (feeding, stabling conditions, com- position of animal groups in terms of age, sex, etc.). State of the prior art

[0005] — Boar taint results mainly from an accumulation of skatole and androstenone and, to a lesser extent, indole in adipose tissues — or fatty tissues — from entire, i.e., uncastrated, male pigs.

[0006] — This smell, which is released during the cooking of pork, is generally considered nauseating by consumers.

[0007] — Historically, to prevent boar taint, male piglets were castrated before they reach sexual maturity.

[0008] — However, for about fifteen years, ethical reasons, animal welfare but also economic reasons are leading more and more breeders to no longer castrate male piglets, especially since only 5% to 10% of adults develop the smell of boar.

[0009] A certain number of processes are known which make it possible to identify, on the chains slaughter, the carcasses of male pigs whose meat carries the odor of boar. In particular, international application PCT WO-A-2021 / 009438, hereinafter reference [1], has been described a method for detecting and measuring skatole present in a sample of pig adipose tissue with very high sensitivity - since this method has a detection limit of the order of 20 nmol / L of sample - and very high specificity with respect to other indole compounds likely to also be present in this adipose tissue. This process, which also has the advantage of being able to be implemented in a slaughterhouse, consists of preparing an organic extract from the adipose tissue sample and subjecting this extract to an electrochemiluminescence (ECL) reaction. The preparation of the organic extract of the adipose tissue sample, which is proposed in reference [1], comprises a succession of steps, namely: a separation of the fat present in the adipose tissue sample from the non-fatty elements also present in this sample, a dehydration of the fat present in the adipose tissue sample, a dissolution of the dehydrated fat in an aprotic organic solvent, a heating of the organic solution resulting from this dissolution to allow the skatole to be extracted, a degreasing of the organic solution and, finally, an addition of an anhydrous stock salt to this organic solution. It turns out that, given the slaughter rate to which abattoirs are subject, the time between the slaughter of pigs and the dispatch of carcasses to cutting plants and processing lines is very short. However, the identification of carcasses (which can number several hundred per day) whose meat carries boar taint must be carried out during this very short period of time. As a result, the time needed to detect the presence of skatole in these carcasses and, if present, to measure it must be optimized. Statement of the invention The invention relates to an improvement of the method described in reference [1], which makes it possible to reduce as much as possible the time necessary to detect the presence of skatole in adipose tissue of a male pig and, where appropriate, to measure this skatole by simplifying the preparation of the organic extracts intended to be subjected to the electrochemiluminescence reaction, without the performance of this method, in terms of sensitivity and specificity, being affected. The invention therefore relates to a method for detecting the presence of skatole in adipose tissue of a male pig, which comprises at least the steps of: (a) preparing an organic extract from a sample of adipose tissue; b) subjecting the organic extract prepared in step a) to a reaction electrochemiluminescence: and c) measuring the luminescence intensity during step b) and, if the measured luminescence intensity exceeds a predetermined threshold value, deducing the presence of skatole in the adipose tissue sample; and which is characterized in that step a) comprises the sub-steps of: i) collecting the adipose tissue sample using a sampling swab; ii) dissolving the adipose tissue sample in a medium comprising an aprotic organic solvent; iii) adding an anhydrous base salt to the aprotic organic solvent, this addition being able to be carried out before or after sub-step ii); whereby the organic extract is obtained. Thus, according to the invention, the adipose tissue sample is collected using a sampling swab and then dissolved or dissolved (the two terms being considered synonymous) in a medium comprising an aprotic organic solvent and, optionally, an anhydrous background salt. If the anhydrous background salt is not already present in the medium at the time of dissolution of the sample, then it can be added after this dissolution. The expression "sampling swab" means any swab of the type conventionally used for taking biological or microbiological samples, such as a laboratory cotton bud and, more generally, any plastic, wooden, aluminium or other stick, fitted at one end with a tip made of an absorbent material, this material being able to be natural (cotton or wadding for example) or artificial (viscose or polyester foam for example). In accordance with the invention, the sampling is preferably carried out by rubbing the adipose tissue with the swab for a time sufficient to saturate the swab with adipose tissue. This time will obviously depend on the size of the tip of the swab used for the sampling but, generally, 20 seconds of continuous rubbing will be sufficient to obtain saturation of this tip. Then, once the adipose tissue sample is collected using the swab, it is put into solution so that it can be analyzed by the ECL reaction. In the foregoing and following, the term "aprotic", when applied to an organic solvent, is taken in its usual meaning, namely that it denotes an organic solvent whose molecule is free of acidic hydrogen atoms, that is to say linked to a heteroatom such as a nitrogen, oxygen or sulfur atom. In accordance with the invention, the aprotic organic solvent used in sub-step ii) is advantageously a polar aprotic solvent, i.e. one having a non-zero dipole moment, such as acetonitrile, dimethyl sulfoxide (or DMSO), propylene carbonate or γ-butyrolactone, with preference given to acetonitrile. The anhydrous base salt, which is added to the aprotic organic solvent, can be chosen from a very large number of salts, it being understood that it must be, on the one hand, soluble in the aprotic organic solvent and, on the other hand, chemically and electrochemically inert so as not to disturb the ECL reaction or induce an undesirable reaction with the skatole. As is well known to electrochemists, this salt may in particular be a tetrafluoroborate, a hexafluorophosphate or a tetraalkylammonium perchlorate whose alkyl group comprises from 1 to 6 carbon atoms, such as tetrabutylammonium tetrafluoroborate or tetrabutylammonium hexafluorophosphate (or TBAHFP), this type of salt having, in fact, remarkable stability in an organic medium, preference being given to TBAHFP. Furthermore, the anhydrous bottom salt is added to the aprotic organic solvent in an amount such that its concentration in this solvent is typically between 0.01 mol / L and 1 mol / L, preferably between 0.05 mol / L and 0.5 mol / L, and, even better, equal to 0.1 mol / L. In accordance with the invention, it is preferred that the organic extract prepared in step a) comprises at most 0.1% by volume of water. Indeed, above this threshold, if skatole is present in the organic extract - the male pig being contaminated with skatole - the intensity of the luminescence signal measured by the ECL reaction risks being weakened due to the interaction between water and superoxides, then interfering in the formation of the conjugate base of skatole according to the following reaction: [Math.1] 3-MIH+ O,>3-MI +H0, Also, knowing that a pig adipose tissue contains from 10% to 20% by mass of water, it may be necessary, depending on the mass of the adipose tissue sample taken, to add in addition to the aprotic organic solvent, either before sub-step ii) or after sub-step ii), a dehydrating agent or drying agent (the two terms being considered synonymous) such as a hygroscopic salt insoluble in the aprotic organic solvent such as anhydrous sodium sulfate, anhydrous potassium sulfate or anhydrous magnesium sulfate, or a molecular sieve (for example, of 3 or 4 angstroms) to remove all or part of the water likely to be released during the dissolution of the adipose tissue sample. If so, this dehydrating agent can then be removed from the dissolution medium, for example by decantation, filtration or centrifugation, before proceeding to step b). In accordance with the invention, it is preferred to facilitate the dissolution of the adipose tissue sample by means of an abrasive agent which is added to the aprotic organic solvent before proceeding to sub-step ii). In the foregoing and the following, the expression "abrasive agent" means any material insoluble in the aprotic organic solvent and in a very finely divided form which, when present in the medium comprising the aprotic organic solvent, is capable of removing the fatty tissue from the swab by simple rubbing. Thus, it may in particular be a powder consisting of a salt insoluble in the aprotic organic solvent such as a salt of an alkali or alkaline-earth metal such as sodium sulfate, potassium sulfate, magnesium sulfate or a powder which is not composed of a salt such as a silica powder, a powder of a metal oxide (alumina for example), a pumice stone powder, a diatomaceous earth powder, etc. Among these abrasive agents, preference is given to an abrasive agent which can also act as a dehydrating agent, which is particularly the case of a hygroscopic salt such as anhydrous sodium sulfate, anhydrous potassium sulfate or anhydrous magnesium sulfate. Typically, the abrasive agent is added to the organic solvent at a rate of 0.5 g to 1 g of abrasive agent per 10 mL of solvent. If an abrasive agent is added to the aprotic organic solvent, then the dissolution of the adipose tissue sample is preferably carried out by introducing the swab into a container (typically cylindrical or conical in shape such as a test tube, pillbox or centrifuge tube) in which there is the medium comprising the aprotic organic solvent, the abrasive agent and, optionally, the anhydrous background salt, by immersing the swab in this medium and rubbing it, for example by rotation, against the wall of the container for at least 20 seconds. Stirring the container, for example using a vortex-type laboratory stirrer, can advantageously complement this protocol. In any case, the abrasive agent can then be removed from the dissolution medium, for example by decantation, filtration or centrifugation, before proceeding to step b). As known per se, the ECL reaction is preferably carried out in an electrochemical cell, the term "electrochemical cell" here designating the assembly formed by a container of the cuvette type or the like, in which the organic extract is placed for the ECL reaction, and at least two electrodes, namely a working electrode and a counter-electrode. In accordance with the invention, it is preferred: - that the ECL reaction is initiated by the application of a cathodic potential to a working electrode, which is immersed in the organic extract, so as to induce the formation of superoxide ions by reduction of the dioxygen dissolved in this extract, then the formation of the conjugate base of skatole and hydroperoxide radicals; and - that the application of a cathodic potential is followed by the application of an anodic potential so as to oxidize the conjugate base of skatole which, once oxidized, will react with the hydroperoxide radicals to lead to the formation of N - 5(2-acetyl-phenyl)formamide in the excited state which, by de-excitation (or, in other words, by return to its ground state), emits a measurable luminescence. For details on this process, the reader is invited to refer to reference [1] as well as to the article published by T. Okajima and T. Ohsaka in Journal of Electroanalytical Chemistry 2002, 523, 34-39, hereinafter reference [2]. The application to the working electrode of a cathodic potential then of an anodic potential can be carried out according to different electrochemical protocols and, in particular, by: - a potential sweep, in which case a sweep towards the negative potentials is applied to the working electrode, then a sweep towards the positive potentials; - a potential jump, in which case a constant negative potential is applied to the working electrode, for a time sufficient to saturate the surface of this electrode with superoxide ions, then a constant positive potential, also for a time sufficient to saturate the surface of the working electrode with oxidized skatole; or - by a series of pulses of alternating cathodic and anodic potential. However, within the framework of the invention, it is preferred that the application to the working electrode of a cathodic potential then of an anodic potential be carried out by potential jump because this is the electrochemical protocol which allows the ECL reaction to be carried out most quickly. For example, for an electrochemical cell equipped with a working electrode and a counter electrode made of boron-doped diamond as well as a pseudo-reference electrode made of platinum, excellent results have been obtained by applying to the working electrode a constant negative potential less than -1.5 V, for example 1.8 V, for 10 seconds to 60 seconds, then a constant positive potential greater than +0.5 V, for example +0.8 V, for 1 second to 15 seconds. The choice of the container for the electrochemical cell is not critical in itself. However, it is desirable that this container be made of a material resistant to organic solvents and saline environments. Furthermore, this container should be made of an optically transparent material in the range of luminescence emission wavelengths or should have at least one wall made of a material exhibiting such transparency if the detection of the emitted photons is carried out by an optical detector located opposite one of its walls. The choice of electrodes for the electrolytic cell is not critical either. If the ECL reaction is based on the formation of superoxide ions, then the working electrode can be made of any electrode material that allows the formation of such ions, such as carbon (graphite, glassy carbon, doped diamond, e.g., with boron or nitrogen, etc.), a noble metal (gold, platinum, palladium, iridium, etc.), or an alloy of noble metals. The counter electrode may be made of a different electrode material or the same electrode material as that which constitutes the working electrode. In the context of the invention, it is preferred that the working electrode and the counter-electrode be made of doped diamond, in particular with boron, because this material is highly conductive, has very high stability, and a natural resilience to fouling due to the high atomic density of diamond. This resilience to fouling is particularly interesting given that the organic extract may comprise a certain number of compounds derived from pig adipose tissue, including fatty acids, which can quickly foul the surface of the electrodes. In addition, in the event of fouling, this type of electrode can be easily cleaned electrochemically, for example by the process described in US patent 9,121,107 B2, hereinafter reference [3]. Finally, this type of electrode has a large potential window which makes it possible to apply high potentials without electrolyzing the solvent present in the organic extract. If a reference electrode is used, then this may in particular be a saturated calomel electrode (SCE) or a silver chloride electrode (Ag / AgCl), possibly with a double junction, as traditionally used in electrochemistry. However, in the context of the invention, it is preferred to use a pseudo-reference electrode made of a noble metal such as platinum because this type of electrode can also be easily cleaned, for example by flaming. Advantageously, the electrochemical cell (i.e. container and electrodes) is made of low-cost materials (plastic container, carbon paste electrodes, etc.) so as to be disposable, which makes it possible, on the one hand, to overcome the problems of fouling of the electrodes and, on the other hand, to ensure traceability of the samples of adipose tissue analyzed, for example by referencing each electrochemical cell in relation to a pig carcass. As for the optical detector, it can be a photomultiplier coupled to a bialkali, super-bialkali or ultra-bialkali photocathode, an avalanche photodiode, a photomultiplier with a silicon photocathode, a spectrometer and, in particular, a spectrofluorimeter, a CCD sensor detector (from "Charged Coupled Device"), a CMOS sensor detector (from "Complementarity Metal- Oxide-Semiconductor”), etc. According to the invention, the threshold value used in step c) is preferably at least equal to 150% of the average value of the luminescence intensity corresponding to the background noise of the optical detector. As previously indicated, the method of the invention also makes it possible, if skatole is present in the organic extract prepared in step a), to determine its concentration. Thus, if the presence of skatole has been deduced in step c), the method advantageously further comprises a quantification of the skatole present in the organic extract by comparison of the maximum luminescence intensity measured during step c) with a calibration curve, this quantification being carried out during step c) or after step c). The threshold for rejection of pork by the consumer is set at approximately 0.2 ug of skatoon per gram of adipose tissue, which corresponds, assuming complete extraction of the skatoon present in 1 g of adipose tissue in 1 mL of an organic solvent, to a skatoon concentration of 1.53 umol / L. Also, the invention also relates to a method for sorting carcasses of entire male pigs, which is characterized in that it comprises the implementation of a method for detecting skatole as previously defined. Other characteristics and advantages of the invention will emerge from the additional description which follows, which relates to experiments which have enabled the invention to be validated and which is given with reference to the appended figures. It goes without saying that this additional description is given only as an illustration of the subject of the invention and must in no case be interpreted as a limitation of this subject. Brief description of the figures [Fig.1] illustrates the luminescence signals obtained by subjecting, to a potential jump ECL reaction, 3 synthetic solutions, respectively, S1, S2 and S3, all comprising 100 nmol / L of skatole in a medium comprising acetonitrile and 0.1 mol / L of TBAHFP but differing from each other by their volume percentage in water (respectively 0%, 1% and 2%); in this figure, the y-axis corresponds to the number of counts emitted, noted Nc and expressed in arbitrary units (au) while the x-axis corresponds to the duration, noted t and expressed in seconds, of the ECL reaction. [Fig.2] illustrates, in the form of a bar diagram, the maximum intensity of the luminescence signals obtained by subjecting, to an ECL reaction by potential jump, solutions S1, S2 and S3 as well as a fourth synthetic solution, S4, comprising like the previous ones 100 nmol / L of skatole in a medium comprising acetonitrile and 0.1 mol / L of TBAHFP but differing from these in that it comprises 0.1% by volume of water; in this figure, the ordinate axis corresponds to the maximum number of counts emitted, noted Nc and expressed in arbitrary units (au). [Fig.3] illustrates the evolution of the mass, noted m and expressed in mg, of adipose tissue taken using a swab as a function of the duration, noted t, and expressed in seconds, of the sample, that is to say as a function of the contact time between the swab and the adipose tissue. [Fig.4] illustrates the evolution of the maximum intensity of the luminescence signals obtained by subjecting organic extracts to an ECL reaction by potential jump as a function of the sampling duration; in this figure, the ordinate axis corresponds to the number of counts emitted, noted Nc and expressed in arbitrary units (au) while the abscissa axis corresponds to the sampling duration, noted t, and expressed in seconds. [Fig.5] illustrates, in the form of a bar diagram, the maximum intensity of the luminescence signals obtained by subjecting, to an ECL reaction by potential jump, 3 organic extracts, respectively El, E2 and E3, resulting from a dissolution of adipose tissue samples by different modalities; in this figure, the ordinate axis corresponds to the maximum number of counts emitted, noted Nc and expressed in arbitrary units (au). [Fig.6] illustrates the results of a test to compare the ECL assay of skatole in organic extracts prepared in accordance with the invention from 8 samples of adipose tissue from whole male pigs with the HPLC assay of skatole in the adipose tissue of these same pigs; in this figure, the y-axis corresponds to the number of counts emitted, noted Nc and expressed in arbitrary units (au) while the x-axis corresponds to the concentration of skatole found by HPLC, noted [C]upzc and expressed in ug / g. Detailed presentation of specific implementation methods The ECL reactions, the results of which are reported below, were carried out with electrochemical cells such as that referenced 20 in reference [1], this electrochemical cell being described in point IT.1 of reference [1] and illustrated in [Fig.2] of this reference. For total light emission measurements, electrochemical and ECL measurements were performed using an AutolabTM PGSTAT128N potentiostat / galvanostat (Autolab) or a PDM03-9107-USB photodetector module (ET-Enterprises). To obtain spectral data, ECL measurements were performed using a PalmSens4TM portable potentiostat (PalmSens) and a spectrofluorometer. FluoromaxTM 4P (Horiba Jobin Yvon) which allows the evolution of luminescence to be monitored in real time using integrated software. At the beginning of each measurement session, the electrodes were carefully cleaned by electrochemical activation by immersing them in a solution comprising 0.1 mol / L of TBAHFP in acetonitrile and applying 0.5-second pulses of 2 mA and -2 mA for 200 cycles. During the ECL reactions, the electrochemical cells were placed in absolute darkness to limit background noise from the photodetector or spectrofluorimeter. As previously indicated, the type of ECL reaction which is preferred within the scope of the invention comprises the application of the cathodic potential to the working electrode of an electrochemical cell to introduce the formation of superoxide ions, followed by the application of an anodic potential to this same electrode to introduce the oxidation of the conjugate base of skatole if it is present in the organic extract. As also previously indicated, it is preferred that this be achieved by a potential jump, that is to say by applying to the working electrode a constant negative potential, for a time sufficient to saturate the surface of this electrode with superoxide ions, then by applying to it a constant positive potential, also for a time sufficient to saturate the surface of the working electrode with oxidized skatole. This is therefore the type of protocol that was chosen for all ECL reactions, the results of which are reported below. In this case, these reactions were carried out by applying a constant negative potential of -1.8 V to the working electrode for 40 seconds and then a constant positive potential of +0.8 V for 5 seconds. The measurement of the emitted luminescence was initiated from the beginning of the application of the negative potential to the working electrode. I - Influence of the presence of water in the reaction medium on the measured luminescence signal: In order to assess the influence of the presence of water in the reaction medium on the emitted luminescence signal, 3 synthetic solutions, respectively S1, S2 and S3, differing from each other only by their water content are subjected to an ECL reaction. These solutions are prepared by dissolving synthetic skatole in acetonitrile to give them a skatole concentration of 100 nmol / L, and adding TBAHFP to the resulting solutions to give them a background salt concentration of 0.1 mol / L. Then water is added to only solutions S2 and S3, at a rate of 1% by volume for solution S2 and 2% by volume for solution S3. Solution S1 is therefore free of water. The results are illustrated in [Fig.1] which illustrates the luminescence signals obtained for the 3 solutions. This figure shows that when water is present in the reaction medium, the maximum intensity of the emitted signal is weakened due to the interaction of water with superoxide ions. Thus, solution S3, comprising 2% by volume of water, leads to a luminescence signal whose peak is weakened by approximately 70% compared to that obtained for solution S1, free of water. A fourth synthetic solution S4, also comprising 100 nmol / L of skatole in acetonitrile and 0.1 mol / L of TBAHFP, but to which 0.1% by volume of water is added, is prepared and also subjected to an ECL reaction. As visible in [Fig.2] which illustrates the maximum intensity of the luminescence signals obtained for solutions S1 to S4, that obtained for solution S4 is similar to that obtained for solution S1. This means that, if an optimized luminescence signal is desired, the organic extract subjected to the ECL reaction should not contain more than 0.1% by volume of water. II - Preparation of organic extracts: The tests described below are carried out using adipose tissue from whole male pigs and using laboratory cotton buds as sampling swabs. IL.1 - Influence of the sampling duration: In order to assess the influence of the duration of collection of a fatty tissue sample using a swab on the mass of fatty tissue collected, a series of samples is taken by rubbing a fatty tissue, from the same whole male pig carcass, for 1, 5, 10, 20, 30 or 40 seconds with one of the tips of the cotton swabs. The cotton swabs are weighed before and after the samples are taken, which makes it possible to determine, for each sampling period, the mass of adipose tissue taken. As can be seen in [Fig.3], which illustrates the evolution of the mass of adipose tissue taken as a function of the duration of the samples, there is a correlation between this mass and this duration since the mass of adipose tissue taken increases with the duration of the sample but up to a certain threshold. Indeed, this figure shows that from 20 seconds of sampling, the mass of adipose tissue collected remains constant, equal to approximately 45 mg. This means that the tip of the cotton swab is saturated with adipose tissue and that it will not allow more than 45 mg of adipose tissue to be collected. Thus, given that a whole male pig adipose tissue naturally comprises at most 20% by mass of water, an organic extract obtained by dissolving 45 mg of adipose tissue in 10 mL of acetonitrile will comprise 0.09 mg of water (provided that the entire adipose tissue is dissolved) and will therefore have a volume percentage of water of 0.09, i.e. lower than the previously mentioned threshold of 0.1%. Under these conditions, no operation aimed at dehydrating the organic extract (heating, addition of a hygroscopic salt or a molecular sieve, etc.) will be necessary, which represents a valuable saving of time. Furthermore, each of the cotton swabs used above is introduced into a test tube containing 10 mL of acetonitrile, 0.387 mg (i.e. 0.1 mol / L) of TBAHFP and 900 mg of sodium sulfate, immersed in this medium and rotated against the wall of the test tube for at least 20 seconds. The test tubes are then subjected to vortex-type agitation for 10 minutes, then left to stand for decantation. The settling supernatants are subjected to an ECL reaction. The evolution of the maximum intensity of the luminescence signals obtained as a function of the sampling duration is illustrated in [Fig.4]. This figure shows that there is a correlation between the sampling duration and the maximum intensity of the measured luminescence signal. Indeed, as previously shown in [Fig.3], the longer the sampling duration, the more intense the emitted signal, this being due to the increase in the mass of the adipose tissue sample collected using cotton swabs. This figure also confirms that from 20 seconds of sampling, the cotton swabs are saturated with adipose tissue, the intensity of the luminescence signal being constant and maximum. IL.2 - Influence of the methods of dissolving the adipose tissue sample: Once the adipose tissue sample has been collected using a swab, it must be put into solution. Different methods of dissolving a sample of adipose tissue taken using a cotton swab are tested. To do this, 3 organic extracts, respectively El, E2 and E3, are prepared: - either by introducing the cotton swab into a test tube containing 10 mL of acetonitrile and 0.387 mg of TBAHFP and simply leaving it to soak in this medium (extract El); - either by introducing the cotton swab into a test tube containing 10 mL of acetonitrile and 0.387 mg of TBAHFP, by immersing it in this medium and rotating it against the wall of the test tube (extract E2); - or again, by introducing the cotton swab into a test tube containing 10 mL of acetonitrile, 0.387 mg of TBAHFP and 900 mg of sodium sulfate (here acting as an abrasive agent), by immersing it in this medium and rotating it against the wall of the test tube (extract E3). The organic extracts thus obtained are subjected to an ECL reaction. The results are illustrated in [Fig.4] which represents the maximum intensity of the luminescence signals obtained for organic extracts E1 to E3. This figure shows that the methods of dissolving adipose tissue samples have an influence on the emitted signal. The maximum intensity of the luminescence signal is obtained for the organic extract E3, which confirms that the addition of an abrasive agent in the aprotic organic solvent allows a better dissolution of the adipose tissue sample, previously collected using a swab. III - Analysis of adipose tissues of male pigs: Organic extracts were prepared using the method of the invention from 8 samples of adipose tissue from whole male pigs with different skatole concentrations, previously determined by HPLC. To do this, a sample of each adipose tissue is taken by rubbing a cotton swab for 20 seconds. Then, each cotton swab is placed in a test tube containing 10 mL of acetonitrile, 0.387 mg (i.e. 0.1 mol / L) of TBAHFP and 900 mg of sodium sulfate, immersed in this medium and rotated against the wall of the test tube for at least 20 seconds. The test tubes are then subjected to vortex-type agitation for 10 minutes, then left to stand for decantation. The extracts thus obtained are then subjected to an ECL reaction by potential jump. [Fig.6] illustrates the intensity of the luminescence signal measured by ECL as a function of the skatole concentration found by HPLC. This figure shows that there is an almost perfect correlation (R2=0.9963) between the intensities of the luminescence signals obtained by the method of the invention and the concentrations of skatole previously determined by HPLC from the same adipose tissues. It also shows that the method of the invention makes it possible to detect skatole concentrations well below the rejection threshold of 0.2 ug of skatole / g of adipose tissue. Furthermore, since the 8 adipose tissue samples were analyzed 5 times each over a 3-day period, the standard deviation is less than 10% for each measurement. As a result, the method of the invention is sensitive, reproducible, reliable and, therefore, perfectly suited to the detection and dosage of skatoon on a slaughter line. entire male pigs. References cited [1] WO-A-2021 / 009438 [2] T. Okajima et T. Ohsaka, Journal of Electroanalytical Chemistry 2002, 523, 34-39 [3] US 9,121,107 B2

Claims

Claims

1. Method for detecting the presence of skatole in adipose tissue of a male pig, which includes at least the steps of: a) prepare an organic extract from a sample of the tissue adipose; b) subjecting the organic extract prepared in step a) to a reaction electrochemiluminescence; and c) measure the intensity of the luminescence during step b) and, if the measured luminescence intensity exceeds a predetermined threshold value completed, deduce the presence of skatole in the tissue sample adipose; and which is characterized in that step a) comprises the sub-steps consisting of: 1) collect the adipose tissue sample using a pre-swab lifting; 11) dissolve the adipose tissue sample in a medium comprising an aprotic organic solvent; iii) adding an anhydrous bottom salt to the aprotic organic solvent, this addition that can be made before or after sub-step ii); whereby the organic extract is obtained.

2. A method according to claim |, wherein substep i) comprises rubbing the fatty tissue with the swab for a while sufficient to saturate the swab with fatty tissue.

3. A method according to claim 2, wherein the adipose tissue is rubbed with the swab continuously for at least 20 seconds.

4. A method according to any one of claims 1 to 3, wherein the aprotic organic solvent is acetonitrile, dimethyl sulfoxide, propylene carbonate or γ-butyrolactone, preferably acetonitrile.

5. A method according to any one of claims 1 to 4, wherein the background salt is a tetrafluoroborate, a hexafluorophosphate or a per- tetraalkylammonium chlorate whose alkyl group comprises from 1 to 6 carbon atoms, preferably tetrabutyl hexafluorophosphate- ammonium.

6. A method according to any one of claims 1 to 5, wherein the organic extract prepared in step a) comprises at most 0.1% water volume.

7. A method according to any one of claims 1 to 6, wherein a abrasive agent is added to the aprotic organic solvent before proceed to sub-step ii).

8. A method according to claim 7, wherein the abrasive agent is a powder consisting of a salt insoluble in organic solvent aprotic, a silica powder, a powder of a metal oxide, a pumice powder or diatomaceous earth powder.

9. A method according to claim 8, wherein the abrasive agent is anhydrous sodium sulfate, anhydrous potassium sulfate or anhydrous magnesium sulfate.

10. A method according to any one of claims 7 to 9, wherein the sub-step ii) includes: - the introduction of the swab into a container in which the medium comprising the aprotic organic solvent, the abrasive agent and, possibly, the anhydrous bottom salt; - immersion of the swab in the medium; and - rubbing the swab against the wall of the container for at least minus 20 seconds.

11. The method of claim 10, wherein substep 11) further includes stirring the container.

12. A method according to claim 10 or claim 11, wherein the sub-step ii) further comprises the removal of the abrasive agent from the medium.

13. A method according to any one of claims 1 to 12, wherein, the presence of skatole having been deduced in step c), we also carry out a quantification of skatole present in the organic extract by com- comparison of the maximum luminescence intensity measured during step c) with a calibration curve, the quantification being carried out during step c) or after step c).

14. Method for sorting whole male pig carcasses, characterized in that it includes the implementation of a method according to any of the re- claims | at 13.