Method for obtaining a fluidized food matrix sample and using the resulting sample for the detection of bacteria

Abstract

The invention relates to a method for obtaining a sample (S3) of a food matrix, this method consisting of mixing a sample (S1) of said food matrix with a solution (S2) comprising a secondary alcohol ethoxylate surfactant compound, said method also comprising a step of heating said sample (S3) obtained, or of preheating before mixing the solution (S2) containing said surfactant compound and the sample (S1) of said food matrix, to a temperature suitable to avoid any degradation of target biological species (E) in the sample. Figure to be published with the abbreviation: Figure 1

Description

Title of the invention: Method for obtaining a fluidized food matrix sample and using the resulting sample for bacterial detection. Technical field of the invention

[0001] The present invention relates to a method for obtaining a fluidized food matrix sample and to the use of the sample obtained for the detection of bacteria. State of the art

[0002] Detecting the presence of pathogenic bacteria in complex and fatty food matrices, such as milk, meat, fish, and chocolate, is a major challenge in the food industry. Significant losses in food production have recently been observed following the detection of pathogenic bacteria.

[0003] In the agri-food sector, microbiological analyses remain essential for ensuring food safety. These analyses are reliable because they are based on proven testing methods.

[0004] Now, two types of methods exist on the market: reference (standardized) methods, based mainly on bacterial culture, or methods available in the form of commercial kits (culture or culture + molecular method or culture + immunological test).

[0005] Some known methods for detecting pathogenic bacteria generally focus on steps of bacterial enrichment by culture, bacterial lysis, followed by biomolecular detection of pathogen DNA, notably by qPCR and LAMP (isothermal amplification). These solutions often involve on-site sampling, followed by outsourced analysis to service laboratories or in-house analysis in an on-site laboratory, making the process lengthy. Furthermore, these known methods often do not allow for the use of a sufficiently large volume to ensure reliable analysis without prior sample enrichment. However, this enrichment phase can last several hours to detect major pathogenic bacteria such as Salmonella, Listeria, and E. coli. In general, sufficiently sensitive and rapid analytical methods are therefore currently impossible to deploy in the field.

[0006] The object of the invention is to provide a method for obtaining a fluidized food matrix sample, ultimately enabling reliable, rapid and robust analysis said sample, made possible in particular by a large volume commitment, without prior enrichment of the sample. Description of the invention

[0007] This goal is achieved by a process for obtaining a sample of food matrix, this process consisting of mixing a sample of said food matrix with a solution comprising a surfactant compound of the type Secondary Alcohol Ethoxylate, said process also comprising a step of heating said sample obtained, or of preheating before mixing the solution containing said surfactant compound and the sample of said food matrix, to a temperature suitable to avoid any degradation of target biological species in the sample.

[0008] According to one particular feature, the solution containing said surfactant compound is added to the food matrix sample so as not to exceed 25% concentration in the sample obtained.

[0009] According to another feature, the food matrix sample and the solution containing the surfactant compound are heated separately to a temperature between 35°C and 50°C.

[0010] The invention also relates to the use of the fluidized food matrix sample obtained by the process described above, this use consisting of implementing a filtration step of said sample obtained through a filter in order to isolate said target biological species.

[0011] According to one particular feature, a lysis step of said isolated biological species is implemented in order to release the DNA molecules.

[0012] According to another feature, an elution step of the released DNA molecules using an elution buffer is implemented after said lysis state.

[0013] According to another feature, the elution buffer is an amplification reagent. Brief description of the figures

[0014] Other features and advantages will become apparent in the detailed description that follows, given in relation to the accompanying drawings in which: - Fig. 1 illustrates the different stages of the process of the invention; - Figure 2 shows an example of a microfluidic component that can be used for the implementation of the process of the invention; - Fig. 3 shows a diagram illustrating the tracking of bacteria in a milk-type food matrix during its preparation and analysis; - Figures 4A to 4C show diagrams of results obtained for a minced steak type food matrix.

[0015] Detailed description of at least one embodiment

[0016] The invention relates to the preparation of a food matrix sample, in order to be able to analyze this sample and detect the presence of pathogenic bacteria.

[0017] The food matrix is, for example, composed of dairy products (milk, cheese) or other products such as meat, fish, or chocolate. The food matrix is ​​advantageously in liquid or liquefied form (i.e., a solid ground / disintegrated in a liquid – for example, meat or fish ground in a culture medium at a ratio of 1 / 10 or 1 / 4) or slightly pasty. It is advantageously fatty and protein-rich.

[0018] By way of non-limiting evidence, classic pathogenic bacteria to be detected include, for example, Salmonella, Listeria, E. coli, Cronobacter, Campylobacter, Bacillus cereus, or yeasts and molds. Other known bacteria, yeasts, and molds could be cited.

[0019] The principle of the invention consists of preparing a sample of the food matrix by adding a fluidizing agent to the sampled food matrix. This fluidizing agent must have certain characteristics: - It must be added in an appropriate quantity to fluidize the food matrix, so that the resulting mixture can pass through the filter of a microfluidic component intended for sample preparation; - It must be added in an appropriate quantity to avoid any degradation of the target biological species, i.e. the potentially present target bacteria; in particular, it may be present in a concentration of between 5% and 25% of the final mixture. - It must allow for the fluidity of the food matrix, without having to deploy excessively large and restrictive resources, thus allowing for easy deployment in the field; - It must be added in an appropriate quantity to obtain a final mixture of reasonable volume, in order to be engaged in a microfluidic component; - It must not be an inhibitor (persistent even after washing) for the molecular amplification reaction implemented subsequently for detection;

[0020] As indicated above, to allow at least partial preparation of the sample and its analysis, a microfluidic component such as that shown in [Fig.2] is advantageously used.

[0021] This microfluidic component 1 comprises a housing including a lower wall 10, a side wall 11, and an upper wall 12. All the walls of the housing will be made of one or more materials. These materials will, for example, be able to withstand heating within a temperature range of 20°C to 100°C. Preferably, some of the walls of the housing will be made of a material transparent. Preferably, the material used will be a plastic, for example of the PMMA (Poly(Methyl Methacrylate)) or COC (Cyclic Olefin Copolymer) type.

[0022] Component 1 includes a chamber 13 formed in the housing. This chamber represents the location in which the purification / concentration and, optionally, the detection of the target biological species can be carried out (when amplification is performed in situ). The chamber 13 is closed at the bottom by the lower wall of the housing.

[0023] The microfluidic component includes a first channel 14 formed in the housing and arranged for injecting fluids into the chamber 13 or for expelling fluids from the chamber. The first channel 14 has a first end having an opening formed, for example, through the upper wall 12 of the housing, and a second end opening into said chamber 13. The first end of the first channel 14 is, for example, arranged vertically and its second end opens, for example, horizontally into the chamber 13. The first end of the first channel is, for example, flared to accommodate the tip of a pipette or is adapted to the type of device used to inject the fluid into the device. For example, it may be an opening with a Luer-type fitting for connecting a syringe or adapted for connecting a fluidic circuit.

[0024] The microfluidic component includes a second channel 15 formed in the housing. This second channel 15 also has a first end that communicates with the outside, forming an opening made, for example, through the upper wall of the housing, and a second end that communicates with the space formed by the chamber 13. Fluids can also be injected into or discharged from this second channel 15. Its first end is, for example, arranged vertically and its second end horizontally. The chamber 13 is located between the first channel 14 and the second channel 15. Similarly, the first end of this second channel is, for example, flared to accommodate the tip of a pipette or will be adapted to the type of device used to inject the fluid into the device.For example, it could be an opening with a "luer" type fitting for connecting a syringe or adapted for connecting a fluidic circuit.

[0025] At the top, the chamber 13 can be closed by an advantageously flexible and stretchable membrane 18, preferably transparent. The upper wall 12 of the device housing thus has an opening which is hermetically covered by said membrane 18. Said membrane is thus anchored in the housing by any suitable fastening solution, for example by adhesive. This membrane 18 will, for example, be composed of a film, for example a self-adhesive PET film, of thickness, dimensions and constitution adapted to deform elastically, relative to its anchor points, particularly to the bottom of chamber 13.

[0026] The term "transparent" means that the material used is at least partially transparent to visible light, fluorescence, or luminescence, so as to allow at least 80% of this light to pass through. It should therefore be understood that it will be sufficiently transparent to see the interior of chamber 13, at least the second space located above the filter 16 mentioned below.

[0027] The microfluidic component 1 comprises a filter 16 arranged in said chamber 13, this filter 16 being arranged to separate said chamber 13 into two spaces. The two spaces are, for example, superimposed and designated as such lower space 130 located below the filter and upper space 131 located above the filter and below the membrane 18. This filter 16 is preferably made in whole or in part in the form of a flexible and thin film, held in the space formed by the chamber so as to allow passage from one space to the other only through the pores of the filter 16. The film advantageously has elastic deformability allowing it to stretch when a support force is applied in a substantially vertical direction, this elastic deformability having a level sufficient to reach the lower wall of the chamber 13. The filter 16 has an average pore diameter of between 0.2 pm and 50 pm, for example between 0.2 µm and 1 µm for the separation of microorganisms or from 0.2 to 2 µm for retaining bacteria.

[0028] The pore diameter is, of course, adapted to ensure separation between different biological species present in the sample. The filter 16 will, for example, be composed of a film of a thickness, dimensions, and composition adapted to deform to the bottom of the chamber 13 relative to its anchoring points. According to a particular embodiment, the filter may also be made of a transparent material, for example, with the same transparency characteristics as the membrane.

[0029] One of the active treatments that can be applied to the sample may consist of lysis of the biological species present in the sample. In the case of lysis, the microfluidic component may advantageously include a rough bearing surface 17 arranged on the bottom of the chamber 13. This rough bearing surface 17 extends over a major part of the bottom of the chamber. It has an average surface roughness parameter between 0.0 µm and 10 µm, preferably between 0.2 µm and 3 µm. This rough bearing surface 17 is intended to allow mechanical lysis of the biological species present in the biological sample placed in the device. Preferably, the mechanical lysis is carried out by grinding said biological species by abrasion on said rough bearing surface. The grinding operation is implemented by a frictional movement of the biological species against the rough support surface, using a suitable grinding element. This element will be, for example, a spatula or a rod, made of plastic or metal. This element is applied from outside the chamber 13 and its end is pressed against the outer surface of the membrane 18 so as to stretch the membrane 18 and the filter towards the bottom of the chamber and thus rub the biological species present in a sample against the rough support surface 17.

[0030] The microfluidic component can also be adapted to allow active thermal treatment of the sample. In this case, the housing can advantageously incorporate heating means for the internal space of the chamber, consisting, for example, of at least one heating element. The heating element is, for example, fixed under the lower wall of the housing. A power supply will, for example, be provided to power the heating element. The power supply will, for example, include one or more batteries, providing sufficient energy to heat the chamber to a temperature within the range defined above, i.e., from 20°C to 100°C. Of course, other heating means could be used, including, for example, conductive ink deposited by printing or screen printing under the lower wall of the housing.

[0031] Thus, to summarize, the microfluidic component may advantageously comprise the following "multilayer" structure: - A rough lower support surface 17, - A lower space 130 of chamber 13, located above the rough bearing surface 17, - A filter 16, advantageously flexible and stretchable, located above the lower space 130, - An upper space 131 of the chamber 13 located above the filter 16, - A membrane 18, advantageously flexible and stretchable, located above the upper space 131, hermetically closing the chamber and accessible from outside the device.

[0032] The lower wall of the component and the membrane can be made of transparent materials, in particular to implement detection by biomolecular amplification, directly in the component.

[0033] By way of non-limitation, the microfluidic component may have the following dimensional characteristics: - A first channel 14 consisting of an inlet channel of 1mm in diameter x 3mm in height, then a rectangular section channel of lmmxl50pm of 3mm long; - A chamber 13 consisting of a lower space 130 for concentration / lysis which has a diameter of 8mm x 50pm in height and an upper space for elution with a diameter of 8mm x 300pm in height; - Filter 16 with porosity adapted to the target to be retained (virus, yeast, mold...), for example with a porosity of 0.2 to 2 pm to retain bacteria; - A second channel 15 consisting of a rectangular channel of 1mm x 150pm and 3mm long, then a channel of 1mm diameter x 3mm height;

[0034] It should be noted that the microfluidic component has the particular advantage of optimizing the quantity of target biological species obtained and concentrating them to the maximum in the lower space 130 of the chamber 13 of component 1.

[0035] In the process of the invention, a food matrix is ​​initially taken, this food matrix being able to be as defined above, i.e. in liquid or slightly pasty form. In the case of a meat-type food matrix, the sample will be liquefied / dissolved / disintegrated in a liquid, and is thus in liquid form.

[0036] The preparation process of the invention initially consists of adding to the extracted food matrix a fluidizing agent formed from a secondary alcohol ethoxylate type surfactant compound. This type of compound is better known by the trade name Tergitol (registered trademark) and can take various formulations. It is described in particular in patent application US2017 / 037339A1. Generally, it has the following formula:

[0037] R-0 (CH2CH2O)nH in which R is a branch substituted or unsubstituted by an alkyl group of type Cn i5, n corresponds to the degree of ethoxylation between 3 and 20.

[0038] Without limitation, the compound chosen for the implementation of the process of the invention is selected from Tergitol 15-S-9 and Tergitol TMN6. Other compounds from the same family could however be considered.

[0039] With reference to [Fig. 1], the process of the invention follows these steps:

[0040] El: A sample of the food material SI to be analyzed is taken. Preparation is then prepared A solution S2, for example at 20%, of the surfactant compound to be added to the sample is also required. The resulting sample S3 must contain a maximum concentration of 25% of the surfactant compound, advantageously around 10%. Indeed, the volume of surfactant compound added must be controlled to avoid any degradation of the target biological species (pathogenic bacteria) present in the food matrix sample.

[0041] E2: The food matrix sample SI and the surfactant solution S2 are heated (T°C). The heating is carried out, for example, at a temperature between between 35°C and 50°C, advantageously around 45°C. The two solutions can be heated separately (as in [Fig. 1]) or heated after mixing. The heating temperature is chosen to promote fluidization of the food matrix while avoiding the degradation of the target biological species. When the two solutions are heated separately, one could be heated to a higher temperature than the other, provided that the conditions for preserving the biological species are met after mixing.

[0042] E3: The selected surfactant compound is added to the food sample to create a usable sample S3 for use in a microfluidic component 1 as described above. The resulting sample S3 must contain a maximum concentration of 25% of the surfactant compound, advantageously about 10%. As indicated above, the volume of surfactant compound added, the temperature, and the duration of the treatment must be controlled to prevent any degradation of the target biological species E (pathogenic bacteria) present in the food matrix.

[0043] From step E4 described below, the sample S3 obtained after mixing is advantageously treated using the microfluidic component 1 described above.

[0044] E4: The prepared sample S3 is injected through the first channel 14 of the microfluidic component. The fluid passes through the filter 16 and is discharged through the second channel 15, while the target biological species E remain in the lower space 130 of the chamber.

[0045] E5: A washing of the target biological species E retained in the lower space 130 is carried out with a washing buffer LL Then drying is advantageously carried out by injection of air (AIR) through the microfluidic circuit of component 1, via the first channel 14. A pump P can be placed downstream to accelerate the drying.

[0046] E6: The biological species present in the lower space of the chamber are lysed by grinding them against the rough lower surface 17, to release the DNA molecules M. For example, a rod T or spatula is used to exert pressure against the membrane 18.

[0047] E7: DNA molecules are eluted through filter 16 of the component, for example using an elution buffer, for example the amplification reagent R required for the subsequent amplification reaction.

[0048] E8: An AMP amplification reaction, for example of the PCR or LAMP type, is carried out to identify the presence of pathogenic bacteria in the food matrix.

[0049] As an alternative embodiment, it would also be possible, after step E4 described above, to wash the target biological species E retained in the lower space 130 of chamber 13, followed by the injection of a culture medium. Then, The microfluidic component is then closed by blocking these two channels. Bacterial growth is promoted by incubation (for example, by heating at 37°C for 2 hours).

[0050] Once the incubation period is over, the biological species E present in the lower space 130 are washed and then dried. Steps E7 and E8 described above are then carried out in the same way.

[0051] More simply, according to another embodiment, after step E4, a washing is carried out, then an elution of the biological species E retained in the lower space 130 of the chamber 13 is carried out. An analysis is then carried out, according to different methods (analysis in culture, microscopic visualization, antibiogram, immunology...).

[0052] Without limitation, for a food matrix such as milk, the process can be implemented under the following conditions: - Preparation of solutions:

[0053] The surfactant solution is based on Tergitol 15S9, at 20%.

[0054] The washing pad used is, for example: Tris HCl pH8 10mM SALMON DNA 5 mg / mL BSA 0.5% - Heating is carried out separately for the Tergitol solution and the milk, at a temperature of 45°C without stirring for 5 min; - A pre-filter is used, upstream of filter 16 present on the microfluidic component; - A rinse of biological species can be carried out with ImL Ringer's solution; - The wash is carried out with 3mL of the wash buffer; - Drying is carried out with an empty syringe, by injecting 5mL of air onto the component while a pump connected to the second channel 15 creates a suction; - Mechanical lysis is performed manually; - The elution is carried out using the elution buffer from the detection kit used;

[0055] Fig. 3 shows a diagram tracking the presence of bacteria in a milk-type food matrix during its preparation and the different analysis stages.

[0056] From this diagram, it is understood that: - When milk is pasteurized, there are no more bacteria (Pasteurization); - Addition of the "Salmonella" bacteria to milk (Enrichment); all the Bacteria are therefore present;

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[0064] - The pretreatment of milk consisting of separate heating of compound S2 (Tergitol 20%) and milk to 45°C + mixing of the two, does not result in any loss of Salmonella bacteria, therefore the process thins the milk without damaging the bacteria; - The bacteria are retained on the filter during injection into the component, so the process does not damage the component's filter, which remains intact and captures the majority of the bacteria; - After washing, no bacteria are eluded, so the bacteria are stuck on the filter; The principle of the invention has also been successfully applied to unenriched samples of minced steak with 15% fat (ground in EPT or Ringer's after dilution to 1 / 5). Figures 4A to 4C show output diagrams for a minced steak type food matrix. Two separate samples are therefore used: - First sample: minced steak 15% fat, 4 days before the use-by date, contaminated with salmonella; - Second sample: minced steak 15% fat, J-4 of the use-by date, not contaminated with salmonella; Three separate samples are then prepared by diluting the food matrix: 25g of minced steak in 100g of liquid. Dilution 1 / 5 in buffered peptone water (EPT) or 1 / 5 in Ringer's solution in a mixing bag. - Sample A: 25g minced steak (15% fat), 4 days before the use-by date, not contaminated with salmonella + 100g Ringer's solution - Sample B: 25g minced steak (15% fat), 4 days before the use-by date, contaminated with salmonella + 100g Ringer's solution - Sample C: 25g minced steak (15% fat), 4 days before the use-by date, not contaminated with salmonella + 100g EPT Each sample A, B and C was passed through the grinder / mixer / homogenizer for 1 minute. A sample is taken from each sample A, B and C. The process described above in steps E2 to E8 is then implemented. The diagrams in [Fig.4A], [Fig.4B] and [Fig.4C] are qPCR amplification curves, with measurement of fluorescence (crude, Rn) as a function of the reaction cycle for a positive internal control and Salmonella. In the three diagrams, we can see that the positive internal control is detected in all three trials, indicating the absence of a reaction inhibitor.

[0065] Furthermore, it has been observed that salmonella is only detected in samples of ground beef contaminated with salmonella. The solution of the invention is therefore perfectly reliable and robust.

[0066] The invention thus offers numerous advantages, including: - The ability to process various types of food matrix, without enrichment, thus allowing a significant time saving; - The possibility of engaging a larger sample volume inside the component, and therefore improving the reliability of bacterial detection in the sampled matrix; - A simple, reliable and robust process, easily deployable in the field, in particular because it is fast and uses easily transportable equipment;

Claims

Demands

1. A method for obtaining a sample (S3) of a food matrix, characterized in that it consists of mixing a sample (SI) of said food matrix with a solution (S2) comprising a surfactant compound of the type Secondary Alcohol Ethoxylate, said method also comprising a step of heating said sample (S3) obtained, or of preheating before mixing the solution (S2) containing said surfactant compound and the sample (SI) of said food matrix, to a temperature suitable to avoid any degradation of target biological species (E) in the sample.

2. The method according to claim 1, characterized in that the solution (S2) containing said surfactant compound is added to the food matrix sample (SI) so as not to exceed 25% concentration in the sample (S3) obtained.

3. A method according to claim 1, characterized in that the food matrix sample (SI) and the solution (S2) containing the surfactant compound are heated separately to a temperature between 35°C and 50°C.

4. Use of the fluidized food matrix sample obtained by the process according to any one of claims 1 to 3, characterized in that a filtration step of said sample (S3) obtained is carried out through a filter (16) in order to isolate said target biological species (E).

5. Use according to claim 4, characterized in that a lysis step of said isolated biological species (E) is implemented in order to release the DNA molecules.

6. Use according to claim 5, characterized in that an elution step of the released DNA molecules using an elution buffer is carried out after said lysis state.

7. Use according to claim 6, characterized in that the elution buffer is an amplification reagent (R).

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