Use of a cage molecule for the detection of androstenone in the gas phase, method and kit for the detection of androstenone in the gas phase, in a porcine sample

Abstract

The present invention relates to the use of a cage molecule for the detection of androstenone in the gas phase, said cage molecule being selected from cucurbiturils having a size greater than 17.5 Å with an internal diameter greater than 6.9 Å.

Description

[0001] METHOD FOR DETECTING ANDROSTENONE

[0002] The present invention relates to a method for detecting boar odor, more particularly by using specific cage molecules to detect the presence of molecules responsible for boar odor.

[0003] Boar taint, or sexual odor, is a strong, unpleasant smell released during the cooking of meat from certain adult pigs. This boar taint results from the accumulation of androsterone and skatole, and to a lesser extent indole molecules, in the fatty tissues. To prevent the accumulation of these molecules in adult pigs destined for slaughter, they are castrated at a young age before the sexual odor permeates their meat.

[0004] However, to comply with a recent European directive prohibiting the castration of male pigs without anesthesia for ethical reasons, slaughterhouses in the European Union that choose to raise intact males will have to implement a systematic sorting process for uncastrated male pig carcasses in the coming years. This process will be designed to detect carcasses containing specific molecules (skatole, androsterone, indole) that produce a boar taint when cooked, rendering them unfit for direct consumption. Currently, the preferred method (“human nose”) involves heating the fat on the carcass (using a soldering iron or blowtorch) and submitting it (online or offline) to a specialist operator who can determine the carcass's acceptability by smell. This is the industry standard.This operation is relatively difficult to reproduce (disparity of operators, fatigue...), subjective, non-quantitative and presents a high degree of hardship leading to the rotation of the operator every 30 minutes.

[0005] There are also colorimetric methods that offer rapid analysis time. However, they require fat sampling and an extraction step that necessitates a laboratory and dedicated personnel. Furthermore, this step takes 40 minutes and poses a risk in terms of carcass identification.

[0006] Other detection methods exist for analyzing these products, but they require heavy and expensive equipment, and these measurements cannot be performed in the field. In the context of detecting the molecules responsible for boar taint, a few reports exist demonstrating the feasibility of this approach. Liu et al. (Liu, X., H. Schmidt, and D. Morlein, Feasibility of boar taint classification using a portable Raman device. Meat Sci, 2016. 116: p. 133-9) used portable Raman probes to qualitatively detect the presence of the target molecules. The Raman signal obtained without plasmonic probes (SERS) is weak, making this direct approach too time-consuming for the applications targeted in this project. Sorensen et al (Sorensen, KM, et al., Simultaneous quantification of the boar-taint compounds skatole and androstenone by surface-enhanced Raman scattering (SERS) and multivariate data analysis. Anal Bioanal Chem, 2015. 407(25): p.7787-95) proposed a liquid-phase SERS detection approach, enabling the detection of considerably lower concentrations (ppm). However, the liquid-phase detection applied in this study requires an extraction step, which is incompatible with online detection and the desired productivity.

[0007] The present invention aims to provide a simple, rapid and reproducible method for detecting the molecules responsible for boar odor, and in particular androstenone.

[0008] The present invention also aims to provide a method for detecting in the gas phase the molecules responsible for boar odor, and in particular androstenone.

[0009] The present invention also aims to provide a more reliable method for quantifying the molecules responsible for boar odor, and in particular androstenone.

[0010] The present invention also aims to provide a rapid detection method, usable on the slaughter line, and which can be carried out by a single operator.

[0011] Thus, the present invention relates to the use of a specific cage molecule for the detection of at least one molecule responsible for boar odor, and in particular for the detection of androstenone.

[0012] The present invention relates in particular to the use of a cage molecule for the detection of androstenone in the gas phase, wherein the cage molecule is selected from cucurbituriles larger than 17.5 Å with an internal diameter greater than 6.9 Å.

[0013] The present invention is therefore based on the use of a cage molecule chosen from cucurbituriles of size greater than 17.5 Å with an internal diameter greater than 6.9 Å.

[0014] Preferably, said cage molecule is chosen from cucurbituriles of size ranging from 17.5 Å to 21 Å and / or with an upper internal diameter ranging from 6.9 Å to 11 Å.

[0015] By "size" we mean here the maximum distance in the inner cage, in the direction of the principal axis of symmetry of the molecule.

[0016] The term "internal diameter" here refers to the maximum distance within the inner cage, perpendicular to the molecule's principal axis of symmetry. In both cases, the size and internal diameter can be estimated by X-ray diffraction.

[0017] Cucurbituriles are molecules well known to those skilled in the art. They are macrocyclic molecules composed of glycolurile units [=C4H2N4O2=]. In these molecules, the oxygen atoms are oriented towards the center of the molecule, thus forming a partially closed cavity.

[0018] Preferably, the cage molecule is cucurbit[8]uril, with the following formula (I):

[0019]

[0020] Molecule responsible for boar odor

[0021] As mentioned above, the invention relates to the detection of the molecule(s) responsible for boar odor. The term "molecule responsible for boar odor" refers to a molecule that generates boar odor.

[0022] Examples of molecules responsible for boar odor include indole, skatole, and androstenone.

[0023] According to one embodiment, the molecule responsible for the boar odor corresponds to the following formula (II):

[0024]

[0025] Preferably, the molecule responsible for boar odor is androstenone, or (5S,8R,9S,10S,13R,14S)-10,13-dimethyl-1,2,4,5,6,7,8,9,11,12,14,15-dodeca-hydrocyclopenta[a]phenanthren-3-one of the following formula (III):

[0026]

[0027] Detection method

[0028] The present invention also relates to a method for detecting androstenone in a pig sample, in the gaseous phase, said method comprising the following steps:

[0029] a) to bring together a pig sample with androstenone, a solution of a cage molecule and a solution of plasmonic nano-objects (SERS substrate), to obtain a solution containing the cage molecule, the plasmonic nano-objects and the androstenone,

[0030] b) measure the Raman spectrum of the solution obtained at the end of step a); c) compare the Raman spectrum obtained in step b) with the Raman spectrum obtained with the plasmonic nano-objects and the androstenone-free cage molecule, and

[0031] d) deduce whether the pork sample contains androstenone.

[0032] According to a particular embodiment, the detection method according to the invention comprises the following steps:

[0033] a) preparation of a solution of a cage molecule in a mixture comprising two different solvents S1 and S2, solvent S1 being a solvent in which androstenone is insoluble at room temperature and solvent S2 being a solvent in which androstenone is soluble at room temperature, said cage molecule being selected from cucurbituriles of size greater than 17.5 A°;

[0034] followed by the introduction of androstenone vapor into the cage molecule solution, then mixing with a solution of plasmonic nano-objects, to obtain a solution containing the cage molecule, the plasmonic nano-objects and the androstenone,

[0035] b) measure the Raman spectrum of the solution obtained at the end of step a); c) compare the Raman spectrum obtained in step b) with the Raman spectrum obtained with the plasmonic nano-objects and the androstenone-free cage molecule, and

[0036] d) deduce whether the pork sample contains androstenone.

[0037] By "gas-phase detection method" we mean a method for detecting the molecules responsible for boar odor, in particular obtained by direct volatilization of the target molecules from a pig sample without prior liquid-phase extraction of said pig sample.

[0038] According to the invention, said pork sample may be the whole carcass or a part directly found on the whole carcass or a part of meat, carcass or adipose (fatty) tissue taken from said whole carcass.

[0039] In one embodiment, the detection process is carried out on a sample of adipose (fatty) tissue taken from an entire carcass.

[0040] In a preferred embodiment, the pork sample is the whole carcass or a part directly found on the whole carcass.

[0041] Preferably, the pig sample is the adipose (fatty) tissue directly found on the whole carcass. According to one embodiment, the aforementioned step a) consists of preparing a solution of a cage molecule, in a mixture of two different solvents S1 and S2.

[0042] Preferably, solvent S1 is a solvent in which androstenone is insoluble at room temperature.

[0043] Preferably, solvent S2 is a solvent in which androstenone is soluble at room temperature.

[0044] According to one embodiment, the aforementioned step a) consists of preparing a solution of a cage molecule, in a mixture of two different solvents S1 and S2, solvent S1 being a solvent in which androstenone is insoluble at room temperature and solvent S2 being a solvent in which androstenone is soluble at room temperature, said cage molecule being selected from cucurbituriles of size greater than 17.5 Å, in particular with an internal diameter greater than 6.9 Å, preferably said cage molecule being the cucurbit[8]urile.

[0045] In particular, solvent S1 is such that the solubility limit of androstenone in S1 at room temperature is less than 10 -3 mol.L -1 .

[0046] In particular, solvent S2 is such that the solubility limit of androstenone in S2 at room temperature is greater than 10 -3 mol.L -1 .

[0047] According to one embodiment, solvent S1 is chosen from among the protic polar solvents, preferably solvent S1 is water.

[0048] According to one embodiment, solvent S2 is chosen from the Ci-C4 alcohols, preferably from methanol and ethanol, preferably solvent S2 is methanol.

[0049] Other solvents that can be used include tetrahydrofuran (THF) or acetone.

[0050] Preferably, the mixture of the two solvents S1 and S2 is a mixture of water and methanol or ethanol, preferably a mixture of water and methanol.

[0051] Preferably, the mixture of the two solvents S1 and S2 is a mixture of water and methanol, with a water content varying from 50% to 80% by mass relative to the mass of the mixture of the two solvents.

[0052] Advantageously, the two solvents S1 and S2 are present in the solution of step a) in a volume ratio of S1 to S2 ranging from 1:5 to 5:1, in particular from 1:2 to 2:1, preferably from 1.5:2 to 2:1.5, and preferably in a volume ratio of S1 to S2 of 1:1. The SERS substrate allows, in particular, for enhanced surface Raman scattering. The SERS substrate can be made from plasmonic materials, for example, with noble metals such as gold, silver, or copper.

[0053] According to one embodiment, plasmonic nano-objects are noble metal plasmonic nano-objects, preferably chosen from noble metal nano-stars, preferably gold nano-stars.

[0054] The SERS substrate solution is preferably prepared by dissolving chloroauric acid HAuCl, silver nitrate AgNCh and ascorbic acid CeHsOe in water.

[0055] According to another embodiment, the gas-phase detection method further comprises a heating step, preferably before or concurrently with step a), consisting of heating the pig sample by means of a thermal device which may be powered, for example, by electricity, a fuel, or an optical source.

[0056] According to one embodiment, the heating step consists of heating a pig sample to a temperature of 30°C to 300°C, in particular 50°C to 300°C, preferably 200°C to 300°C, better 200°C to 250°C, to obtain androstenone vapor.

[0057] In one particular embodiment, the detection method according to the invention further comprises a pre-concentration step, preferably after or concurrently with the aforementioned heating step, of the androstenone vapor. This step consists of maintaining the aforementioned sample heating step for 0 to 20 minutes, preferably for 5 to 15 minutes, preferably for 8 to 12 minutes, and in particular for 10 minutes. Preferably, this step consists of maintaining the aforementioned sample heating step for 0 to 5 minutes, preferably between 0 and 1 minute, more preferably between 0 and 30 seconds, and even more preferably between 1 and 10 seconds.

[0058] According to one embodiment of step (a), the androstenone vapor is brought into contact with said cage molecule solution, and then the mixture is brought into contact with a solution of plasmonic nano-objects, to obtain a solution containing the cage molecule, plasmonic nano-objects and androstenone.

[0059] In one embodiment, the contacting of the androstenone vapor with the cage molecule solution is carried out, for example, using a syringe, a capillary system, or a microfluidic channel system. According to a particular embodiment, the contacting of the androstenone vapor with the cage molecule solution is carried out by bubbling using a capillary system, preferably for a duration of 0 to 1 min, in particular by injecting at least 30 mL of the gas phase (at 298K and 1 atm).

[0060] At the end of step a), a step of measuring the Raman spectrum b) of the solution obtained above in step a) is carried out.

[0061] In general, the Raman spectrum comprises bands characteristic of the molecules present in the solution. These bands are said to be the spectral signature of the molecule in question.

[0062] In particular, Raman bands can be associated with the cage molecule. Typically, the spectral signature of cucurbit[8]uril is, for example, visible in Figure 1 (solid CB8 curve), with Raman bands at 445 cm⁻¹ -1 838 cm -1 1380 cm -1 , 1427 cm -1 Other, less intense bands are occasionally observed at 915 cm -1 752 cm -1 658 cm -1 , 1232 cm -1 and 1051cm- 1 .

[0063] In one embodiment, when androstenone is present in the solution with the cage molecule and plasmonic nano-objects, a spectral change is observed compared to the spectrum of a substrate prepared in the same way but in the absence of androstenone. Typically, the spectral signature of the cage molecule is attenuated; in other words, the intensity of the Raman bands characteristic of the cage molecule decreases. Specifically, the spectral signature of the cage molecule disappears, and new Raman bands associated with androstenone complexed by the cage molecule appear; in other words, the intensity of the Raman bands characteristic of androstenone complexed by the cage molecule increases. This can be explained by the complexation of the androstenone molecule with the cage molecule.

[0064] The Raman spectrum thus obtained at the end of step c) (for the solution containing the cage molecule, the plasmonic nano-objects and androstenone) is then compared with the Raman spectrum obtained with the plasmonic nano-objects and the cage molecule without androstenone (step d)), in other words with the Raman spectrum obtained with a solution prepared with the plasmonic nano-objects and the cage molecule but without androstenone, in order to then deduce whether the pig sample contains androstenone. According to one embodiment, when there is attenuation, in particular disappearance of the spectral signature of the cage molecule, and appearance of the Raman bands associated with androstenone, the pig sample contains androstenone.

[0065] According to one embodiment of the process of the invention, the cage molecule is chosen from cucurbituriles of size greater than 17.5 Å with an internal diameter greater than 6.9 Å, preferably the cage molecule is the cucurbit[8]urile.

[0066] According to one embodiment, the detection process is carried out directly on the whole carcass by placing the measuring device on or in the adipose (fatty) tissue of the carcass's back. This device comprises at least:

[0067] 1. a heating zone capable of reaching a temperature of 150°C to 250°C, preferably 180°C to 210°C, enabling the generation of vapor of the molecules responsible for boar odor, in particular androsterone; and 2. a detection zone containing the cage molecule solution, the plasmonic nano-object solution and the elements capable of detecting the variation of the Raman spectra produced during the contact of the vapor of the molecules responsible for boar odor and said solutions.

[0068] The present invention also relates to a method for sorting carcasses of whole male pigs, comprising the implementation of a method as defined above.

[0069] The present invention also relates to a device for detecting at least one molecule responsible for boar odor, comprising at least:

[0070] a solution of plasmonic nano-objects; and

[0071] a mixture comprising 2 different solvents S1 and S2, solvent S1 being a solvent in which androsterone is insoluble at room temperature and solvent S2 being a solvent in which androsterone is soluble at room temperature, in which a cage molecule selected from cucurbituriles of size greater than 17.5 A° is solubilized.

[0072] Preferably, the molecule responsible for boar odor is as defined above, and preferably androsterone.

[0073] The present invention also relates to a kit for the detection of androstenone in the gaseous phase, in a pig sample, comprising:

[0074] - a solution of plasmonic nano-objects, - a cage molecule chosen from among cucurbituriles with a size greater than 17.5 Å, in solution in a mixture comprising two different solvents S1 and S2, solvent S1 being a solvent in which androstenone is insoluble at room temperature and solvent S2 being a solvent in which androstenone is soluble at room temperature, and

[0075] - means of heating.

[0076] Preferably, the cage molecule is as defined above, and is in particular cucurbit[8]uril.

[0077] Preferably, the two solvents S1 and S2 are as defined above, and in particular solvent S1 is water and solvent S2 is methanol. Description of figures

[0078] Figure 1 shows the Raman spectrum of pure cucurbit[8]uril in solid phase (solid CB8) and the SERS Raman spectrum of nanostars prepared according to Example 1 complexed with cucurbit[8]uril (SERS CB8). The main Raman bands of both samples are indicated by the vertical dashed lines.

[0079] Figure 2 shows the NMR spectra 1 H (D2O, 0.5mM cucurbit[8]urile solution) of androstenone from Example 3 at T0, T1 and T2.

[0080] a) spectrum of cucurbit[8]uril alone at T0,

[0081] b) spectrum of cucurbit[8]uril in the presence of androstenone at T1,

[0082] c) spectrum of cucurbit[8]urile in the presence of androstenone at T2.

[0083] Peak 1 and peak 2 represent the signals from the methyl groups of androstenone.

[0084] Chemical shift in ppm is represented on the x-axis, and intensity in arbitrary units is represented on the y-axis.

[0085] Figure 3 shows the Raman spectrum of pure androstenone in solid phase (spectrum obtained in 1 min with excitation by a 633nm laser beam, focused on approx. 1 pm) 2 (The acquisition time for the spectrum was one minute).

[0086] Figure 4 represents the Raman spectra of three SERS substrates prepared from D1, D2 and D3 according to Example 2 from solutions that have been in contact with androstenone in the gas phase, according to the description of Example 2.

[0087] Figure 5 shows the Raman spectra of three SERS substrates prepared from solutions that had been in contact with gaseous phases from three different pieces of pork fat with varying androstenone levels, prepared according to Example 2. EXAMPLES

[0088] Example 1: Preparation of a SERS substrate solution

[0089] The SERS substrate prepared here consists of noble metal nanostars. The nanostar synthesis method is based on the protocol detailed in Shvai He et al. (RSCAdv. 2017.7, 16264), with some modifications.

[0090] The solution prepared according to the protocol below is solution A.

[0091] Reagents:

[0092] - HAuCL at 10 mM

[0093] - AgNChà 10 mm

[0094] - CeHsOe at 100 mM

[0095] Protocol:

[0096] - Mixture 1: Using a precision balance, 2.38 mg of HAuCL are taken and introduced into a 4 mL pillbox, then dissolved in 700 pL of milli-Q water. The pillbox is then covered with aluminum.

[0097] - Mixture 2: Using a precision balance, 0.85 mg of AgNCh are taken and introduced into a 4 mL pillbox, then dissolved in 500 pL of milli-Q water.

[0098] - Mixture 3: Using a precision balance, 1.76 mg of CeHsOe are taken and introduced into a 4 mL pillbox, then dissolved in 100 pL of milli-Q water.

[0099] - In a 15 mL tube, introduce 3 mL of milli-Q water, then add 108 pL of mixture 1 and 6 pL of mixture 2. Shake the solution using a vortex for 10 seconds.

[0100] - Add 18 pL of mixture 3 to the tube. Vortex the solution for 20 seconds. The clear solution will turn blue.

[0101] - Next, centrifuge the resulting solution A at 1500 rpm for 20 minutes. Example 2: Preparation of a functionalized SERS substrate from a solution containing a cage molecule, plasmonic nanoobjects, and androstenone (step b))

[0102] The solution is prepared from solution A of Example 1 and solution B and androstenone C vapors prepared below.

[0103] Solution B of a cage molecule

[0104] Initially, a 1 mM solution B of cucurbit[8]urile in a 1:1 mixture of milli-Q water and methanol is prepared.

[0105] Androstenone C vapor

[0106] A sample of pork fat (approx. 1 cm 3 ) is heated in a flask on a hot plate at 190°C for approximately 10 minutes, in order to create concentrated C vapors of androstenone.

[0107] Introduction of vapor C into solution B

[0108] Vapor obtained above is drawn using a syringe (approximately 90 mL) and then introduced into solution B (1 mL) by bubbling for approximately 10 seconds. This gives solution B'.

[0109] Mixture with the plasmonic nano-object solution A

[0110] Solution A is then added to the vapor-concentrated solution B' C, in a volume ratio of 4:3 of solution A to solution B', resulting in a total volume of 2.3 mL. The mixture is stirred, yielding solution D. A small portion of this solution (20|JL) is deposited onto a glass substrate maintained at a temperature between 20 and 150°C and left for a few seconds until it evaporates completely. This yields a functionalized plasmonic substrate.

[0111] Example 3: Complexation of androstenone in a cage molecule. In NMR, the formation of the insertion complex between a cage molecule and androstenone must be accompanied by shifts in the characteristic peaks of androstenone. To demonstrate the formation of such an insertion complex, a gaseous phase rich in androstenone is prepared by heating a few grains of androstenone in a first flask on a hot plate at 190°C. It is then bubbled through a cucurbit[8]urile solution similar to solution B prepared above. In short, a capillary tube connects the first flask containing the androstenone vapors to the cucurbit[8]urile solution in a second flask. Applying a partial vacuum (10 mbar) to this second flask allows the androstenone-enriched gas from the first flask to be bubbled through the solution in the second flask.

[0112] An NMR spectrum of androstenone in the cucurbit[8]urile solution of the second balloon was acquired three times: at T0 before bubbling, at T1 after 2 minutes of bubbling, and at T2 after 5 minutes of bubbling. These three spectra are shown in Figure 2.

[0113] The signals from the androstenone methyl groups are visible after the first bubbling time (T1), and even more so after T2 (peaks 1 and 2). This spectral evolution is accompanied by relative changes in the cucurbit[8]urile signal: a change in the relative ratios of the two bands observed at approximately 1.8 ppm, and a decrease in the intensity of the band at approximately 0.9 ppm compared to the multiplet observed at 1.0 ppm. These results thus demonstrate that a complex formation has indeed occurred between the cucurbit[8]urile molecule and androstenone.

[0114] Example 4: Measurement and comparison of Raman spectra, detection of androstenone in a pig sample

[0115] Protocol: Raman spectrum acquisition. Raman spectra are measured by illuminating the sample with a laser source (excitation wavelength 633 nm), focused onto the sample using an objective lens. Two high-pass filters (Semrock LP02633RU-25 and LPD02633 RU-25) are used sequentially to completely eliminate the excitation beam. The laser intensity is between 10 pW and 10 mW, focused on an area between 1 pm 2 and 1mm 2 The light reflected and scattered by the sample is collected by the same objective lens and directed to a Raman spectrometer (Jobin Yvon, HR460, detection with a CCD sensor cooled to -80°C). Spectra are collected during acquisition times ranging from 10 to 120 seconds and processed using dedicated software (WinSpec).

[0116] 1. A Raman spectrum of pure androstenone in solid phase is obtained, and shown in Figure 3.

[0117] In the solid phase, it is possible to identify several main Raman bands (indicated by the dotted lines), for example at 247 cm -1 , 402 cm -1 , 434 cm -1 , 517 cm -1 683 cm -1 784 cm -1 , 927 cm -1 1085 cm -1 1118 cm -1 , 1144 cm -1 , 1448 cm- 1 , 1585 cm' 1 , 1712.8 cm' 1 .

[0118] Other bands (indicated by the dotted lines), less intense, can also be observed at 326 cm -1 370 cm -1 490 cm -1 , 553 cm -1 628 cm -1 , 710 cm- 1 , 811 cm- 1 , 858 cm' 1 , 1003 cm' 1 , 1017 cm' 1 , 1243 cm' 1 , 1228.3 crrr 1 and 1351 cm- 1 .

[0119] 2. A Raman spectrum of a plasmonic substrate obtained from a mixture of nano-stars prepared according to example 1 and cucurbit[8]urile prepared according to example 2, with the exception of the bubbling step, as well as a Raman spectrum of pure cucurbit[8]urile in solid phase are obtained, and shown in Figure 1.

[0120] The spectral signature of cucurbit[8]urile is clearly visible on these two spectra, with a shift of approximately 40-50 cm' 1 commonly observed for the same compound in a different chemical environment (SERS spectrum and pure solid molecule).

[0121] 3. Three solutions D1, D2 and D3 are carried out according to example 2 on three different days, and their Raman spectra are carried out according to the protocol described above, and represented in Figure 4.

[0122] For the three solutions D1, D2, and D3, a drastic spectral change is observed compared to a Raman spectrum of nano-complexed with cucurbit[8]uril (for example, the CB8 SERS spectrum in Figure 1), with the disappearance of the spectral signature of cucurbit[8]uril and the appearance of numerous new bands: 1400 ± 5 cm⁻¹ 1 , 1240 ± 5 cm' 1 , 1006 ± 5 cm' 1 and 820 ± 10 cm' 1 , but also 391 cm' 1 , 440 cm' 1 , 603 cm' 1 and other bands of low intensity that appear less systematically. Comparison with the Raman spectrum of androstenone in solid phase (Figure 3) confirms the assignment of these bands, with a slight shift of approximately 150 cm⁻¹ 1This shift, very commonly observed in SERS, can be explained by the influence of the chemical environment (cucurbit[8]urile in SERS spectra, crystal field for the solid molecule). These spectral observations clearly reflect the presence of androstenone in the samples.

[0123] 4. Three solutions D'1, D'2 and D'3 are prepared according to Example 2 from three different pieces of pork fat obtained from a slaughterhouse. These three pieces are previously classified respectively according to the "human nose" method by odor levels (the said levels ranging from 1 to 5, 1 being the weakest indicating a low presence of androstenone, and 5 the strongest indicating a significant presence of androstenone in the piece), 5 (D'1), 3 (D'2) and 1 (D'3).

[0124] Their Raman spectra are obtained according to the protocol described above, and are shown in Figure 5.

Claims

DEMANDS 1. Use of a cage molecule for the detection of androstenone in the gas phase, wherein the cage molecule is chosen from cucurbituriles of size greater than 17.5 Å with an internal diameter greater than 6.9 Å.

2. Use according to claim 1, wherein the cage molecule is cucurbit[8]uril.

3. A method for detecting androstenone in a pig sample, in the gas phase, said method comprising the following steps: a) to bring together a sample of pig with androstenone, a solution of a cage molecule and a solution of plasmonic nano-objects, to obtain a solution containing the cage molecule, the plasmonic nano-objects and the androstenone; b) measure the Raman spectrum of the solution obtained at the end of step a); c) compare the Raman spectrum obtained in step b) with the Raman spectrum obtained with the plasmonic nano-objects and the androstenone-free cage molecule, and d) deduce whether the pork sample contains androstenone.

4. A method for detecting androstenone according to claim 3, wherein the cage molecule is dissolved in a mixture comprising two different solvents S1 and S2, solvent S1 being such that the solubility limit of androstenone in S1 at room temperature is less than 10 -3 mol.L -1 , S1 being preferably water.

5. A method for detecting androstenone according to claim 4, wherein the solvent S2 is such that the solubility limit of androstenone in S2 at room temperature is greater than 10 -3 mol.L -1, S1 being preferably chosen from C1-C4 alcohols, preferably S1 is ethanol or methanol, preferably methanol.

6. Method for detecting androstenone according to any one of claims 3 to 5, comprising a preliminary step, before step a), of heating a pig sample to a temperature of 180°C to 210°C, preferably 190°C, to obtain androstenone vapor.

7. Method for detecting androstenone according to any one of claims 3 to 6, wherein the cage molecule is dissolved in a mixture comprising two different solvents S1 and S2, the mixture of the two solvents S1 and S2 being a mixture of water and methanol or ethanol, preferably a mixture of water and methanol.

8. Method for detecting androstenone according to any one of claims 3 to 7, wherein the cage molecule is dissolved in a mixture comprising two different solvents S1 and S2, the mixture of the two solvents S1 and S2 being a mixture of water and methanol, with a water content varying from 50% to 80% by mass relative to the mass of the mixture.

9. Method for detecting androstenone according to any one of claims 3 to 8, wherein the pig sample is a sample of meat, carcass or adipose tissue.

10. Method for detecting androstenone according to any one of claims 3 to 9, wherein the plasmonic nano-objects are noble metal plasmonic nano-objects, preferably selected from noble metal nano-stars, preferably gold nano-stars.

11. Method for detecting androstenone according to any one of claims 3 to 10, wherein the cage molecule is cucurbit[8]urile.

12. A method for sorting whole male pig carcasses, comprising implementing a method according to any one of claims 3 to 11.

13. Kit for the detection of androstenone in the gas phase, in a pig sample, comprising: - a solution of plasmonic nano-objects, - a cage molecule chosen from among the cucurbituriles with a size greater than 17.5 Å, in solution in a mixture comprising two different solvents S1 and S2, solvent S1 being a solvent in which androstenone is insoluble at temperature19 ambient temperature and solvent S2 being a solvent in which androstenone is soluble at room temperature, and - means of heating.

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