ES2273531A1 - Set of polynucleotides as foodstuffs biological tracers comprises exogenic additives for recovery of foodstuff extracellular genetic material - Google Patents

Abstract

The set of polynucleotides as foodstuffs biological tracers has exogenic addition to the foodstuff, for recovery of the extracellular genetic material of the marked foodstuff. The process authenticates and guarantees the quality of the foodstuff.

Description

Polynucleotides as product biotracers food.

Field of the Invention

The invention relates, in general, to the use of polynucleotides as biotracers to trace and Verify food products. In particular, the invention is relates to a method to mark a food product and verify its authenticity later by detecting an exogenous biotracer polynucleotide previously added to said food product during its manufacturing process.

Background of the invention

Food traceability is defined as the ability to track a food from its origin to the consumer, giving rise to a reliable identification of their ingredients, sanitary control, and food monitoring throughout the production chain. Traceability is, by therefore, a fundamental tool at the service of quality food

The production and marketing of materials premiums for the food industry as well as production and commercialization of food products of lower quality than  those that are specified or that do not meet the quality standards required, for example, the characteristics of a Denomination of origin, supposes food fraud and constitutes a serious problem that both have to face administrations such as manufacturers and distributors of materials Raw and food products. On the one hand, the consumer health and quality marching associated with a certain denomination of origin and, on the other hand, are the losses caused to the food industry and denominations of origin by fraudulent marketing of products (copies or imitations) of worse quality.

The identification of animal species and plant, as well as the detection and quantification of transgenic, they are the two main areas that have been defined as fundamental for the food sector today.

The need to comply with regulations and fight fraud, as well as the growing demand from the consumer towards food that inspires confidence, points towards  a growing interest in technologies that allow the food traceability

DNA analysis has been shown as a very effective tool to determine food traceability, although with certain limitations. So far, the analyzes of DNA commonly used in food is based on the characterization of nuclear molecular markers (mainly microsatellites) or in the analysis of conserved regions of the Mitochondrial DNA in the starting material and its subsequent search in the processed product. This method of analysis is very useful in the Raw material identification and fraud detection of composition but presents many drawbacks in traceability. The fundamental is the high cost of genetically typing the starting material and processed food and the number of leaks that the system can have if a large number of samples. On the other hand, endogenous DNA analysis is not very useful for the identification of appellations of origin since in these cases, more than the raw material, the characteristic is usually the elaboration process.

There is, therefore, the need to develop an alternative, reliable and economical method that allows to determine food traceability and make it more effective in detecting fraud.

Compendium of the invention

The invention relates, in general, to the use of polynucleotides as exogenous biotracers that can be add at any stage of product development dietary, thereby allowing the food traceability of such food products at all times. Saying polynucleotide, hereinafter referred to as "polynucleotide tag ", can be customized and associated with a specific characteristic, for example, quality, denomination of origin, quality certification, producer, ingredients, sanitary controls, monitoring of the productive chain, date of production, expiration date, etc., so that the detection of said polynucleotide label on a food product determined, which has been previously marked at some stage of your elaboration process, is indicative of the authenticity and characteristics of said food product. The authenticity of a food product can be checked by detection of the existence of said polynucleotide tag.

A characteristic that the label polynucleotide to add to the food product lies in which is an exogenous polynucleotide, that is, a polynucleotide that is added to the food product, and that although It has the same chemical nature as the endogenous, it is not part of the biological material (cells, nucleic acids, etc.) that they could eventually be present in the food product at trace and therefore is not found naturally in the food product to trace. Therefore, said polynucleotide label must have characteristics of size (length) and composition (sequence) that guarantee its stability against the degradation and allow its subsequent detection accurately and reliable, by any appropriate technique, regardless of possible existence of other nucleic acids in the product own food of said food product.

A method like the one developed by this invention allows to mark a food product and subsequently verify its authenticity in a simple, reliable and economical way, by detecting a tag polynucleotide (biotracer) previously added to said food product during its process of elaboration Also, said method serves not only to detect, avoid or minimize food fraud but also could be adapted for use in certification processes of foods.

Therefore, one aspect of this invention is relates to a method to analyze the authenticity of a Food product labeled with a polynucleotide label.

Another aspect of this invention relates to a method of marking a food product with a polynucleotide label in order to be able to verify later, if desired, its authenticity The food product marked with said polynucleotide tag constitutes an additional aspect of this invention.

Another additional aspect of this invention is relates to a method to mark a food product and verify its authenticity later by detecting a  biotrazador polynucleotide previously added to said product food during its elaboration process.

Brief description of the figures

Figure 1 is a photograph of the agarose gel in which the detection by PCR and subsequent staining are shown with ethidium bromide of the amplification bands of the label polynucleotides added to wine (lane 1) and loin (lane 2) [see Example 1]. Street 3 corresponds to the control negative amplification [water] and lane 4 with the marker of molecular weight of 1 kilobase (Roche).

Detailed description of the invention

In a first aspect, the invention relates to with a method to analyze the authenticity of a product food labeled with a tag polynucleotide comprising recover the extracellular genetic material present in said labeled food product and analyze said genetic material extracellular recovered to check if it contains such polynucleotide tag.

The food product that can be analyzed according to the method of the invention can be practically any food product, containing or not containing biological material  (for example, nucleic acids). The term "product food "as used here includes both materials premiums for the food industry, for example, cereals, legumes, vegetables, fruits, etc., as food products handled or unhandled, for example, battered products, precooked, frozen, etc. Said food product may be a solid product, for example, meat, fish, poultry, pasta, etc.; liquid, for example, milk, drinkable milk derivatives, both alcoholic beverages (wines, beers, etc.) and not alcoholic (juices, soft drinks, etc.); or semi-solid, for example yogurts, baby pots, purees, etc.

As used in this description, the term "polynucleotide tag" refers to a polynucleotide that allows to determine the food traceability of a food product, so it is useful to identify the origin of the food product, its composition, denomination of origin, quality certification, sanitary controls to which undergone and monitored its productive chain, etc., factors that influence the quality of the food product. The employment of said polynucleotide label helps to ensure the quality of a food product and, in addition, to avoid or minimize the risk of  consumer fraud. Therefore, product manufacturers food may select their label polynucleotides from personalized form, that is, so that they can identify their food products made by other manufacturers. For it would be enough to select some label polynucleotides and associate them with the business origin of such products food. Also, polynucleotides can be selected label indicating the designation of origin of a product nutritional, or guarantees of a certain quality standard, or indicative of the different qualities of the products food they produce, or they can be selected label polynucleotides to identify different batches of production and facilitate the identification of expiration dates of such lots; even polynucleotides can be selected specific label to be incorporated in certification standards of Quality of food products.

The polynucleotide tag can be consisting of a DNA sequence or a sequence of RNA, preferably by a DNA sequence, with a composition and size that allows its subsequent detection, reliably and reproducible, by appropriate techniques.

Said polynucleotide tag may come from an organism, for example, an animal, a plant, a bacterium, a yeast, a fungus, a virus, etc., or it can be of origin synthetic. In a particular embodiment, said polynucleotide label comes from the non-coding DNA of an organism used in food, for example, yeasts, corn, etc.

The tag polynucleotide size is not a limiting factor for the implementation of the method provided by this invention, so it may vary within a wide range; however, in general, they are used label polynucleotides with an appropriate size to avoid or minimize its possible degradation. In a particular embodiment, said tag polynucleotide comprises between 50 and 600 nucleotides, preferably between 80 and 150 nucleotides.

The polynucleotide tag can be obtained by any conventional method, for example, by synthesis chemistry using automatic synthesizers, or by techniques based on enzymatic reactions, for example, PCR, choosing as a template a sequence of an organism, such as a organism used in food, for example, yeasts, corn, etc. Also, the tag polynucleotide can be detected and identified by any appropriate technique that allows detection and identification of a nucleic acid fragment, optionally attached to a marker, as will be mentioned more ahead.

Because the food product that comprises the polynucleotide tag may or may not contain a biological material, for example, DNA or RNA, the composition (sequence) of the polynucleotide tag should be chosen so that the risk of providing results is avoided or minimized incorrect or inaccurate, that is, that does not correspond to the reality. Therefore, the polynucleotide label to add to the product food must be an exogenous polynucleotide, that is, a polynucleotide that is not part of the biological material (cells, nucleic acids, etc.) that may be present in the food product to be traced and therefore not found naturally in the food product to be traced. Also, the tag polynucleotide sequence must be chosen in such a way that structures are not created that hinder or diminish the performance or reliability of the analytical technique used to check the existence of the label polynucleotide in the product food tested. In a particular embodiment, the generation and Subsequent detection of the polynucleotide tag is performed by an enzymatic amplification reaction, for example, by the polymerase chain reaction (PCR), so the main criteria when selecting the polynucleotide sequence label consists of obtaining some ends from which initiator oligonucleotides can be designed that meet all efficacy criteria, such as absence of structures secondary, similar Tm, that do not present sections of a single nucleotide repeated more than three times, etc. [PCR protocols, A guide methods and applications, Ed. Innis, Gelfand, Sninsky and White] so as to specifically amplify the polynucleotide tag in question.

The polynucleotide tag may contain, also, if desired, any modification, in any of its nucleotides, which allow or facilitate their detection and / or binding to molecules of the food product or its subsequent recovery and / or concentration for the purposes of checking and / or detection. Said modification may be, for example, a marker, such as a member of a specific binding pair, a fluorophore, etc. Examples Illustrative of specific binding pairs include the pairs formed by, for example, avidin (or streptavidin) / biotin, avidin / phycoerythrin, etc., as well as systems based on antigen / antibody type interactions, or fragments of same that contain recognition sites. In a particular embodiment, the tag polynucleotide is attached to biotin

The use of a fluorophore allows to detect the label polynucleotide by fluorimetric methods. Therefore in A particular embodiment, the tag polynucleotide comprises a modified polynucleotide containing a fluorescent compound, such as digoxigenin, calcofluor, fluorescein, rhodamine, etc.

The introduction of the marker in the polynucleotide can be performed directly, for example, by reaction with formation of a chemical bond between the polynucleotide and the marker, or by simple coupling, or through intermediate ligands.

The implementation of the method to analyze the authenticity of a food product previously marked with a polynucleotide tag comprises material recovery extracellular gene present in said labeled food product and its analysis to check if it contains said polynucleotide label. The extracellular genetic material to recover will include any nucleic acid that is present in the product food marked but outside cellular structures possibly present in said labeled food product, among which I will find the added label polynucleotide exotically.

The recovery of genetic material extracellular present in the labeled food product can be performed by any conventional method. In one embodiment in particular, the extracellular nucleic acid fraction can recover using conventional purification techniques by affinity. This way of collecting the polynucleotide tag is particularly interesting because said tag polynucleotide has been added externally and therefore there would be no need for break any cell structure. Alternatively, the fraction of nucleic acid can be recovered by extraction / solubilization with an appropriate and subsequent solvent purification / concentration by selective precipitation, filtration or by affinity.

The analysis of extracellular genetic material recovered to check if it contains said tag polynucleotide It can be done by any conventional method that allows detect and / or identify a nucleic acid sequence. By way illustrative, not limiting, said analysis can be performed by using a nucleotide probe labeled with a group detectable marker, or by an enzymatic reaction specific, for example, amplification or restriction, or through a method based on the detection or identification of marker optionally present on the tag polynucleotide, or either by infrared spectroscopy, etc. In one embodiment in particular, the presence of said tag polynucleotide is analyzed by using a labeled oligonucleotide probe with a detectable marker group. In another particular embodiment, the presence of tag polynucleotide is analyzed by a specific enzymatic reaction, such as, for example, PCR, both classical PCR as any of its variants, for example, nested-PCR, ELISA PCR, real-time PCR, etc.

Detection of the polynucleotide tag is can be carried out by any conventional method, by example, by using a nucleotide probe capable of hybridize with the tag polynucleotide, said probe comprising a detectable marker, and detect said marker by methods conventional, for example, by direct display in a gel. Alternatively, the detection of said tag polynucleotide It can be performed by gel electrophoresis and staining, or by capillary electrophoresis and detection of a fluorophore, or either using micro or nanodevices that detect signals electrical (potential changes, etc.), or by detection or identification of the marker optionally present in The polynucleotide tag.

In accordance with the provisions of the method previously described, if the analyzed food product contains the polynucleotide tag, because it has been previously added in a stage of its productive process, said polynucleotide Tag will be detected and / or identified. However, if the analyzed product does not contain said label polynucleotide, it is not can detect, so you can distinguish some products food produced by a particular producer of others food products produced by other producers or from fraudulent food products.

A method to analyze the authenticity of a food product labeled with a polynucleotide labeled as the provided by this invention allows to authenticate a certain food product and guarantee the consumer its quality, business origin, designation of origin, composition, sanitary controls, monitoring of the productive process, etc., and contributes to avoid or minimize the risk of fraud, imitation or unfair competition. Additionally, said method could be Used in product quality certification processes food.

The use of a polynucleotide label as food additive, in particular, as a molecular marker for determine the food traceability of a food product, It has numerous advantages, among which are the following:

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the DNA It is a molecule of great mechanical, chemical and thermal stability, for what can be used to biotrace virtually every product food, both processed and unprocessed;

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its registration as a food additive (as a marker / tracer) no It should generate problems since it is a substance that is ingested usually in the diet and that added exogenously behaves as an organic acid that does not alter the organoleptic properties of food;

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how sources of tag polynucleotides can be molecules coming from of organisms used in food;

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the enormous diversity associated with DNA allows unlimited creation of labels, so these could be customized (producers, products, expiration dates, quality standards, etc.); Y

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he Verification / authentication analysis can be performed using specific techniques that guarantee a high sensitivity of the detection process, which allows the polynucleotide to be added label in trace quantities.

The implementation of the method to analyze the authenticity of a food product developed by this invention requires that said product has been previously marked food with a polynucleotide label.

Therefore, in another aspect, the invention is relates to a procedure to mark (trace) a product food with a polynucleotide label in order to be able to subsequently verify, if desired, its authenticity, that comprises adding a label polynucleotide to said product food, in which when the food product comprises a biological material (eg, a nucleic acid), said tag polynucleotide is an exogenous polynucleotide, that is, no is found naturally in the food product a mark.

The characteristics of the polynucleotide tag correspond to those previously defined in relation to the method to analyze the authenticity of a food product developed by this invention.

The addition of the tag polynucleotide to the food product is made by conventional methods depending on the type of food product to be traced. To do this, in In general, a solution of the label polynucleotide is prepared in a suitable solvent, in which the label polynucleotide is soluble, and said solution is contacted with the product food by any appropriate method, for example, by simple mixing and homogenization after the addition of said solution to the food product at any stage of the process productive, preferably, in the early stages, or by spray on food products, or by immersion of the food products in said solution containing the polynucleotide tag, etc. In another particular embodiment, the polynucleotide label is added in powder form, optionally mixed with an excipient.

The tag polynucleotide is added to the product food in a concentration that allows its subsequent detection by the chosen technique, so it is necessary to know the  level of sensitivity or theoretical detection of the technique of detection chosen to make the corresponding adjustments that the final concentration of polynucleotide label in the food product exceeds the theoretical detection limit of The technique used in the detection.

The food product marked with a tag polynucleotide, wherein said tag polynucleotide is an exogenous polynucleotide that allows quality verification, producer, designation of origin, composition, controls health, or monitoring the productive chain of said food product, etc., constitutes an additional aspect of this invention. Said food product labeled with a polynucleotide label can be obtained by a procedure like the described previously mentioned.

In another aspect the invention relates to a method to mark a food product and verify its authenticity later by detecting a polynucleotide label previously added to said product food during its elaboration process. Both the marking of the food product as the verification of its authenticity is performed in the manner previously mentioned in this description.

The following example illustrates the invention and does not It can be considered limiting the scope of it.

Example 1 Identification of a polynucleotide product label food

This test was performed to check the reliability of the method to analyze the authenticity of a product Food labeled with a tag polynucleotide provided by this invention. The food products chosen were wine, as an example of liquid food product, and pork loin marinated, as an example of solid food product. To do this, in First, the selection and production of label polynucleotides, then proceeded to add said polynucleotides label said food products, and, finally, after properly storing food products marked for a certain period of time we proceeded to analyze said labeled food products in order to Check the existence of such label polynucleotides.

1.1 Selection and production of polynucleotides label

For the performance of this assay, a 133 nucleotide genomic sequence of an intron of the filamentous fungus Trichoderma harzianum (SEQ ID NO: 1) was chosen as the tag polynucleotide. The primers generated for the production of said label polynucleotide and its subsequent detection are those identified as INTF (SEQ ID NO: 2) and INTR (SEQ ID NO: 3). Said label polynucleotide was chosen, in view of the food products to be tested, and taking into account that due to its nucleotide composition it was not expected that structures would be created that adversely affected the performance of the PCR (detection method chosen). Since the generation and subsequent detection of the labels is carried out by PCR, the main criterion when selecting the sequence was to obtain ends from which oligonucleotide primers that met all the efficacy criteria (absence) could be designed of secondary structures, similar Tm, that do not have sections of a single nucleotide repeated more than three times, etc.).

For the production of the label polynucleotide in the quantities required, a first PCR is performed with the 1NTF-INTR primers (SEQ ID NO: 2-SEQ ID NO: 3) using T. harzianum genomic DNA as template. A band of 133 nucleotides is obtained that is subcloned into the commercial vector pGEM-T (Promega). Once the donation is validated by sequencing, this construction (pGEM-INT) is used as a template for the production of the tag polynucleotide. For this a GeneAmp 9700 thermal cycler (Applied Biosystems) is used. The reaction is carried out in 96-well plates and in a final volume of 100 µl in which is added: initiators (INTF-INTR) (SEQ ID NO: 2-SEQ ID NO: 3) at a final concentration of 10 mM ; dNTPs (dATP, dGTP, dCTP and dTTP) at a final concentration of 200 mM, plasmid DNA (10 ng) and 2 U of Taq (Biotools), using the following program:

initial denaturation: preheating to 94 ° C, for 2 minutes;

35 cycles of

denaturation at 95 ° C, for 10 seconds;

banding to 55 ° C, for 15 seconds; Y

elongation to 72 ° C, for 15 seconds; Y

final elongation at 72 ° C, for 7 minutes.

After amplification, the resulting products they are purified with the NucleoFast system (Macherey-Nagel) and quantified by gel. He yield per well ranged from 4-10 totals. Subsequently a pool was made with all the wells and quantified by spectrophotometry at 260/280 nm, obtaining 10 ml of a label polynucleotide solution to a concentration of 150 ng / \ mul.

1.2 Addition of the label polynucleotide to the product food

The polynucleotide label solution is added depending on the type of food product to be traced.

For wine (liquid food product) the Label polynucleotide is added directly to the bottle, to a final concentration of 1 pg / ml (6 orders of magnitude above of the theoretical limit of PCR detection for a molecule of 133 nucleotides) For marinated pork loin (food product solid) the polynucleotide tag is added in any of the steps processing thereof at a concentration of 1 ng / ml (9 orders of magnitude above the theoretical PCR detection level for a molecule of that size). In this specific case, the label polynucleotide is added to the initial brine solution in which the piece is maintained for two days, according to the protocol of elaboration followed by the company Embutidos Artesanos Domenecha (Relleu, Alicante).

1.3 Detection of the molecule

Once the polynucleotide label is added to the food products, both products were preserved in the usual storage conditions (ambient temperature in the case of wine, and refrigerated in the case of marinated loin) during 1 month and later samples were taken for the collection of Externally added DNA and its subsequent detection by Enzymatic amplification (PCR).

In the case of wine, it was based on a sample of 10 ml Since the molecule was added externally and there was no need to break any cellular structure, collecting DNA (polynucleotide tag) was performed by purification by affinity to a matrix (diatomaceous earth) avoiding the steps breakage initials.

In the case of marinated loin the process was similar with the exception that the sampling was performed rubbing a swab dipped in water on the surface of the loin since the tag polynucleotide was not embedded. To do this, it macerated the swab in a solution with water and detergent for 2 hours at room temperature. The obtained supernatant was submitted to the same purification process used in the wine.

Once the DNA was recovered, a PCR was performed classical under the same conditions described above with the difference that on this occasion 50 cycles of amplification. PCR analysis was performed by 1% agarose gel electrophoresis and staining with bromide ethidium As shown in Figure 1, in both cases it was obtained a band of the expected size (133 nucleotides), the signal from wine than from the loin.

Therefore, this essay shows that the method to analyze the authenticity of a food product labeled with a polynucleotide tag provided by this invention works correctly.

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Claims (13)

1. A method for analyzing the authenticity of a labeled food product characterized in that said food product is labeled with an exogenously added label polynucleotide and because said method comprises recovering the extracellular genetic material present in said labeled food product and analyzing said extracellular genetic material to check If it contains such label polynucleotide. 2. Method according to claim 1, wherein said tag polynucleotide is selected from a polynucleotide of DNA and an RNA polynucleotide. 3. Method according to claim 1, wherein said tag polynucleotide comprises between 50 and 600 nucleotides 4. Method according to claim 1, wherein said tag polynucleotide is a polynucleotide that comes from an animal, a plant, a bacterium, a yeast, a fungus, or a virus, or is a polynucleotide of synthetic origin. 5. Method according to claim 4, wherein said tag polynucleotide comes from the non-coding DNA of a organism used in food. 6. Method according to claim 1, wherein said food product comprises biological material and said tag polynucleotide is an exogenous polynucleotide that is not present naturally in said food product. 7. Method according to claim 1, wherein said tag polynucleotide further comprises a marker. 8. Method according to claim 7, wherein said marker is a member of a specific binding pair or a fluorophore 9. Method according to claim 1, wherein the recovery of the nucleic acid present in the product labeled food is done by purification / concentration by selective precipitation, filtration or affinity, optionally prior solubilization with a solvent. 10. Method according to claim 1, wherein the analysis of the recovered nucleic acid to check if contains said tag polynucleotide is done by the use of a nucleotide probe labeled with a marker group detectable, either by a specific enzymatic reaction, or either by a method based on the detection or identification of marker optionally present on the tag polynucleotide, or either by infrared spectroscopy. 11. A procedure to mark a product food with a polynucleotide label in order to be able to subsequently verify, if desired, its authenticity, that comprises adding a label polynucleotide to said product food, in which when the food product comprises a nucleic acid, said tag polynucleotide is a polynucleotide exogenous that is not found naturally in the product food to mark. 12. A food product marked with a tag polynucleotide, wherein said tag polynucleotide is an exogenous polynucleotide that allows quality verification, producer, designation of origin, composition, controls health, or monitoring the productive chain of said food product. 13. Use of a polynucleotide as a biotracer Food products.