ISO 22000: Traceability in Food Supply Chains /Molecular Based Traceability Techniques - IV

ISO 22000 & Food Traceability

Part IV

Molecular Based Traceability Techniques 

Food traceability is an emerging topic that is becoming increasingly relevant especially in terms of international trade. For the export and import of food, the development of traceability systems has been identified as a priority, especially in connection with food safety. Therefore, the implementation of food traceability mechanisms, including analytical methodologies for verification, is particularly relevant for developing countries who wish to increase extending their share in international food trade. International organizations, such as the IAEA, can play a role towards providing equal access to such technologies in the future as well as assisting developing countries to build the necessary capacities to use them.

A deeper understanding of how meteorological and geochemical signatures are transferred into food systems would allow the generation of isotopic and multi-element maps for foods from different geographical locations, which could be incorporated into traceability systems. Comparative databases constructed from these data can then be used as benchmarks in ongoing scientific developments of the future.

Advanced Technologies Used in Food Traceability Systems

Producing safe and high quality food is a prerequisite of every food manufacturer to ensure consumer health and successful domestic and international trade which is critical to the sustainable development of national agricultural resources. Systems to trace food or feed products through specified stages of production, processing and distribution play a key role in assuring food safety. Such traceability systems are typically based on a continuous “paper-trail” and effective labeling. However, analytical techniques that enable the provenance of food to be determined which can provide an independent means of verifying “paper” traceability systems. It also helps to prove product authenticity, to combat fraudulent practices and to control adulteration, which are important issues for economic, religious or cultural reasons.

Food traceability primarily focuses on food safety and quality, which also have positive impacts on food security - the quantity and overall availability of food. Applying food traceability techniques can reduce food losses by minimizing recalls of food consignments if the production region can be determined scientifically.

Proof of provenance has become an important topic in the context of food safety, food quality and consumer protection in accordance with national legislation and international standards (ISO 22000, HACCP, etc) and guidelines. Provenance means to identify and ensure the origin of a commodity and thereby the region where it was produced. Earlier incidents, such as the outbreak of food poisoning from Salmonella in contaminated peppers from Mexico, which occurred in the USA in 2008, have demonstrated the need for effective traceability systems and the deficiencies in current paper-based systems. The failure to trace the contaminated batch of peppers to their origin resulted in a wide-scale, costly and lengthy recall procedure involving many producers in Mexico and retail outlets in the USA.

Therefore, an independent and universally applicable analytical strategy to verify the declared country of origin of food can be an invaluable tool to enable regulatory authorities to trace contaminated foods back to their source. Isotopic and elemental fingerprinting provides a robust analytical tool to determine the origin of food. These techniques, when used in conjunction with food safety surveillance programmes, provide independent verification of food traceability systems, thereby helping to protect human health and facilitate international trade worldwide. The availability of verified traceability systems can also facilitate the targeted withdrawal and/or recall of contaminated products from the market if necessary. Such action can reduce the enormous economic impact of a ‘blanket withdrawal/recall’ and the subsequent damage to industry and consumer confidence. Furthermore, public awareness of the existence of tools and protocols to determine product origin, in a food safety context, can act as a deterrent to traders who knowingly re-route contaminated products to mislead importers, thus preventing the occurrence of incidents related to “third country” dumping of unsafe commodities.

The capability to certify food origin or authenticity is of significant economic importance to many stakeholders in developing countries. For example, some food products can be marketed using labels, e.g. Geographic Indication (GI) or organic produce, that are based on standards of identity or composition related to a very specific production area or production practices. This adds value to such products in terms of marketability and increased export value. Basmati rice from India and Pakistan, for example, is defined by its cultivar and also by its area of production. Genomic techniques can easily confirm the cultivar of Basmati rice, while isotopic and elemental fingerprinting is essential to determine its geographical origin. Isotopic parameters have also recently been added to the Protected Denomination of Origin (PDO) technical specification of Grana Padano Cheese (Italy) and other food commodities are undergoing similar characterizations.

In addition to their application to enhance food safety, these techniques can be applied to address religious or cultural issues. For example, whilst safe for human consumption, the animal or botanical source of a food may render it unfit for some consumers. For instance, gelatin derived from porcine sources and ethanol derived from wines or spirits are not compliant with Halal guidelines, nor are they in accordance with definitions given by Codex Alimentarius (1997).

Nuclear and Nuclear-related Techniques for Food Traceability

Nuclear techniques are uniquely tailored for the determination of food provenance. These methodologies, such as those listed in Table 1, may be used for traceability and authentication of food and have the potential to be applied in many developing countries, thereby enhancing their capacities to improve food safety and quality. Where appropriate, these techniques can also be complemented by conventional, non-nuclear approaches.

The following nuclear techniques can be used to measure the isotopic (e.g. hydrogen-3/ hydrogen-2/ hydrogen-1, carbon-13/ carbon-12, nitrogen-15/ nitrogen14, oxygen-18/ oxygen-16, sulphur-34/ sulphur-32, strontium-87/ strontium-86, lead-208/ lead-207/ lead-206) and elemental (e.g. macro, micro, and trace) composition of food:

Glossary of Terms for Analytical Methods Used for Food Traceability and Their Fingerprinting Characteristics

Method	Abbreviation	Fingerprint
Neutron activation	NAA	Isotopic and elemental
Multicollector inductively coupled plasma mass spectrometry	MC-ICP-MS	Isotopic and elemental
Nuclear magnetic resonance	NMR	Isotopic
Thermal ionization mass spectrometry	TIMS	Isotopic
Isotope ratio mass spectrometry	IRMS	Isotopic
Cavity-ring-down spectroscopy	CRDS	Isotopic
Atomic absorption spectrometry	AAS	Elemental
Atomic emission spectrometry	AES	Elemental

In order to determine an isotopic fingerprint of a specific product, an extensive quality assured database of authentic food samples is required. Appropriate modeling of the isotopic and elemental data, using multivariate statistics, is a prerequisite for identifying isotopic and elemental fingerprints. Only a few databases covering wide regional distribution exist so far. A considerable amount of additional data needs to be collected to provide universally applicable information that could make data comparable from diverse regions worldwide.

The application of the cavity-ring-down spectroscopy (CRDS) technology to distinguish between C3 (sesame, soybean) and C4 (corn) plants through isotopic techniques is an example. Photosynthetic carbon isotope fractionation is related to carbon dioxide uptake and enzymatic processes. The C3 plants, named due to the number of carbons in an intermediate molecule in the relevant biochemical pathway, discriminate more heavily against carbon-13 than the C4 plants and therefore have more negative δ13C values (calculated from the ratio of carbon-13 to carbon-12 in the plant). Plotting the data from δ13C measurements therefore shows a dramatic difference between the oils from C3 plants (sesame, soybean) and that from corn, which is a C4 type plant. From the plot, it can be observed that even the two oils from C3 plants are clearly distinct. Accordingly, the adulteration of respective plant oils with materials of lesser quality and value can be determined.

Supplementary and Combined Analytical Techniques Supporting Food Traceability

In addition to nuclear and nuclear-related approaches, various non-nuclear techniques can provide complimentary data to complete or confirm results obtained by isotopic and elemental fingerprinting. For instance, seafood is a highly perishable food item which is increasingly traded globally. Particular conditions and difficulties have to be taken into account compared to other food products and different analytical techniques are applicable for characterizing seafood. More than 500 species are traded in the European market alone. A large number of processed fish products have lost their morphological characteristics and for instance fraud, where low grade fillets are substituted for high-value fillets, can only be discovered by means of a combination of reliable analytical methods to determine the species and geographical origin.

The most appropriate technique is related to the specific condition of the seafood product, e.g. processed vs. unprocessed, or whether or not it is a closely related or a different species. Separation and characterization of specific proteins through isoelectric focusing (IEF) of sarcoplasmic proteins (water soluble proteins) is the method of choice for the identification of fish species. DNA based methods using polymerase chain reaction (PCR) for nucleic acid amplification is the key technology for species identification, as there is no limitation when different processing treatments of fish and seafood are used.

The most important techniques for the determination of geographical origin are the methods using the variability of stable isotopes (hydrogen-2/ hydrogen-1, nitrogen-15/ nitrogen-14, carbon-13/ carbon-12, oxygen-18/ oxygen-16) in different biological tissues. It is well known that freshwater ecosystems are generally carbon-13 and nitrogen-15 depleted in comparison to marine ecosystems. Consequently, the analysis of these stable isotopes ratios can be used for distinguishing between freshwater and marine fish. Furthermore, different feedings often lead to significant fingerprints in terms of stable isotope ratios in the body so that stable isotope analysis can be also used to distinguish between different environments. Nuclear magnetic resonance, coupled with isotopic ratio mass spectrometry (NMR/IRMS) and site-specific natural isotope fractionation by nuclear resonance (SNIF-NMR), are the two main methods to determine the ratios of stable isotopes.

Isotopic Traceability Techniques for Rapid Response to Emerging Food Safety Risks

The food supply is vulnerable to a range of food hazards (microbiological, chemical, physical) that may arise at any stage of the food supply chain. In addition to well publicized food safety incidents such as aflatoxins in maize, dioxins in pork, melamine in dairy products, and Salmonella in peanuts, new hazards and risks are continually emerging. These may be related to unintentional contamination with, e.g. agrochemicals or bacteria, or intentional contamination (adulteration for economic fraud or with the intent to harm consumers). Other issues may also pose threats to food safety which are not yet well understood or characterized, for example the effects of climate change on food production, or emerging technologies such as the use of nanoparticles in food.

Isotopic measurement techniques can provide an effective means for the identification and tracking of food products, allowing a rapid first response to counter any threat by efficiently tracing and removing affected products from the market. Stable isotope and radio-labeling techniques can also provide a second-tier analytical portfolio to help to detect, identify and characterize the hazard. For example, radio-labeling offers a uniquely sensitive traceability method to investigate the fate of nanoparticles in foods. Nanoparticles are increasingly applied on a broad range of applications and may also play a vital role as food additives. However, the respective risk assessment and evaluation are just in their infancy. The development and application of these techniques would address vulnerabilities in the food supply chain due to emerging threats and help establish effective preventative systems and incident response strategies.

Specific future tasks would be to develop isotopic traceability methodologies and systems to facilitate the rapid tracking of contaminated products and their removal from the market. The development of related stable and radio-isotope techniques that can be applied to detect and characterize emerging food safety hazards and assess and control the risks associated with those hazards is also envisioned.

DNA Barcoding Methods

In general, DNA-based methods use specific DNA sequences as markers, and can be divided as:

1.      Hybridization-based markers

2.    Polymerase Chain Reaction (PCR)-based markers

In hybridization based methods, species-specific DNA profiles are discovered by hybridizing DNA digested by restriction enzymes and comparing it with labeled probes (DNA fragments of known origin or sequence). PCR-based methods involve the amplification of target loci by using specific or arbitrary primers, and a DNA polymerase enzyme. Fragments are then separated electrophoretically and banding patterns are detected by different staining methods such as autoradiography.

PCR-based methods are extremely sensitive, often faster than other technologies and are widely used in agriculture and zootechny (Doulaty Baneh et al., 2007; Grassi, Labra, & Minuto, 2006; Labra et al., 2004; Mane, Tanwar, Girish, & Dixit, 2006; Teletchea, Maudet, & Hänni, 2005). Discontinuous molecular markers such as RAPDs, AFLPs, as well as their variants (i.e. ISSR, SSAP, SAMPL) have been successfully used in the characterization of different kinds of raw material (Chuang, Lur, Hwu, & Chang, 2011; Fajardo et al., 2010;Mafra et al., 2008; Nijman et al., 2003). In recent years, the PCR-denaturing gradient gel electrophoresis (PCR-DGGE) has been largely used in the field of food traceability and safety in order to characterize bacteria and yeasts in fermented products (Dalmacio, Angeles, Larcia, Balolong, & Estacio, 2011; Muyzer, De Waal, & Uitterlinden, 1993; Peres, Barlet, Loiseau, & Montet, 2007; Zheng et al., 2012). By using this technique, microorganism composition is defined on the basis of the migration pattern of PCR-fragments belonging to specific genomic regions such as 16S and 26S rDNA (El Sheikha et al., 2009). PCR-DGGE was also used to monitor bacterial contamination in food products such as fermented drinks (Hosseini, Hippe, Denner, Kollegger, & Haslberger, 2012) and define the origin of raw material starting from the characteristics of its yeast or bacterial communities as in the case of fruit (El Sheikha, Bouvet, & Montet, 2011; El Sheikha, Durand, Sarter, Okullo, & Montet, 2012; El Sheikha, Métayer, & Montet, 2011) and fish (Le Nguyen, Ha, Dijoux, Loiseauet, & Montet, 2008; Montet, Le Nguyen, & El Sheikha, 2008).

The selection of the most suitable molecular approach depends on different aspects, including the amount of genetic variation of the analyzed species, the time needed for the analysis, the cost/effectiveness ratio and the expertise of laboratories. Furthermore, genomic techniques require high-quality DNA to work successfully because their effectiveness can be negatively influenced by altered or fragmented DNA (Hellberg & Morrisey, 2011; Meusnier et al., 2008; Pafundo, Agrimonti, Maestri, & Marmiroli, 2007).

Regarding sequencing-based systems, Single Nucleotide Polymorphisms (SNPs) and Simple Sequence Repeats (SSRs), are largely used nowadays because of their high level of polymorphism and high reproducibility (Kumar et al., 2009). These approaches are used both in the identification of plant cultivars (Labra et al., 2003; Pasqualone, Lotti, & Blanco, 1999) and animal breeds (Nijman et al., 2003) and to prevent fraudulent commercial activities (Chuang et al., 2011). However, being highly species-specific, these approaches require access to the correct DNA sequence of the organisms (e.g. strains/varieties or ecotypes) and their application is often limited to a single taxon, or to closely related taxa.

Lack of both standardization and universality is the most relevant problem of DNA-based approaches. In 2003, a new identification system, DNA barcoding was developed by researchers at the University of Guelph (Canada). This approach is based on the analysis of the variability within a standard region of the genome called “DNA barcode” (Hebert, Ratnasingham, & deWaard, 2003). This approach proved useful in solving taxonomic problems in several theoretical and practical applications (Hollingsworth, Graham, & Little, 2011; Rasmussen, Morrissey, & Hebert, 2009; Valentini, Pompanon, & Taberlet, 2009). In a strict sense, DNA barcoding is not completely innovative, because molecular identification approaches were already in use. However, it has the advantage of combining three important innovations: molecularization of identification processes (i.e. the investigation of DNA variability to discriminate among taxa), standardization of the procedure (from sample collection to the analysis ofmolecular outputs), and computerization (i.e. the not redundant transposition of the data using informatics) (Casiraghi, Labra, Ferri, Galimberti, & De Mattia, 2010).

The name DNA barcoding figuratively refers to the way an infrared scanner univocally identifies a product by using the black stripes of the Universal Product Code (UPC). An ideal DNA barcode requires two fundamental characteristics: high taxonomic coverage, and high resolution (Hebert et al., 2003). High taxonomic coverage (also called ‘universality’) refers to the correct amplification of the genomic region chosen as DNA barcode in the widest panel of taxa. On the other hand, a high resolution ensures the identification of different taxa, based on interspecific differences in DNA barcode sequences. As a general principle, DNA barcode regions should have a high interspecific, and low intraspecific variability.

The 5′-end portion of mitochondrial cox1 gene was suggested by Hebert et al. (2003) as standard DNA barcode region for metazoans. This region does not assure a complete taxonomic resolution, but it does promise proximity (Hebert & Gregory, 2005). Based on preliminary results on cox1 discriminatory power, specimens have been correctly identified at the species level with a success rate ranging from 98 to 100% in birds (Hebert, Stoeckle, Zemlak, & Francis, 2004), fish (Ward, Zemlak, Innes, Last, & Hebert, 2005), and in several other animal groups (Ferri et al., 2009; Galimberti, Martinoli, Russo, Mucedda, & Casiraghi, 2010; Galimberti et al., 2012; Hajibabaei et al., 2006). Nowadays, this region is considered the universal DNA barcode for metazoans, and is used to better distinguish even closely related taxa (see Uthicke, Byrne, & Conand, 2010; Wong et al., 2011), or to identify organisms from their parts, and also from traces of biological material (Dawnay, Ogden, McEwing, Carvalho, & Thorpe, 2007; Shokralla, Singer, & Hajibabaei, 2010; Vargas et al., 2009). In terrestrial plants, mitochondrial DNA has slower substitution rates than in metazoans, and shows intra-molecular recombination (Mower, Touzet, Gummow, Delph, & Palmer, 2007), therefore limiting its resolution in identification. The research for an analogous of cox1 in terrestrial plants has focused on the plastid genome. Several plastidial genes, such as the most conserved rpoB, rpoC1 and rbcL or a section of matK, which shows a fast evolution rate, have been proposed as barcode regions (Shaw, Lickey, Schilling, & Small, 2007). Intergenic spacers such as trnH-psbA, atpF-atpH and psbK-psbI were also tested, because of their fast evolution rate (Fazekas et al., 2008, 2009). In 2009, the CBoL (Consortium for the Barcode of Life) Plant Working Group (Hollingsworth et al., 2009), suggested the use of 2-locus combination of rbcL and matK as core-barcode regions, because of the straightforward recovery rate of rbcL, and the high resolution of matK. Unfortunately, matK is difficult to amplify by using a single primer pair (Dunning & Savolainen, 2010). On the contrary, despite its limited resolution, rbcL is less problematic in terms of amplification, sequencing and alignment, and provides a useful backbone in the creation of plant DNA barcode datasets (De Mattia et al., 2012). Among other sequences, the trnH-psbA intergenic spacer is straightforward to amplify, and has a high genetic variability among closely related taxa (Bruni et al., 2010; Kress et al., 2010; Shaw et al., 2007). The nuclear ITS region was also indicated as supplementary DNA barcode region (Li et al., 2011). Although there is still debate on the effectiveness of these markers especially when users are dealingwith closely related taxa, DNA barcoding showed consistent results when used to identify unknown specimens based on the comparison with reference sequences (Burgess et al., 2011; De Mattia et al., 2012).

Although themolecular approach at the basis of DNA barcoding is not newto science, the strength of thismethod relies on the availability of an international platform. BOLD (Barcode of life database), coordinated by the International Barcode of Life Project (iBOL), is a repository, which supports the collection of DNA barcodes, with the aimof creating a reference library for all living species (Ratnasingham & Hebert, 2007). BOLD is used to relate a given DNA barcode to both a vouchered specimen and other DNA barcode sequences belonging to the same or different taxa. This platform consists of several components, among which the Identification Engine tool (BOLD-IDS) is one of the most useful. BOLD-IDS provide a species identification tool that accepts DNA barcode sequences and returns a taxonomic assignment to the species levelwhenever possible. This engine assumes correct species identification for genetic distances up to 99%. Any researcher can use BOLD-IDS, and, if a reference record belonging to an unknown specimen is available in the database, the system provides identification at the species rank, or a list of the taxa related to that specimen. BOLD is a reliable resource both for research purposes and for practical applications, such as the traceability of food commodities.

Analytical and Managerial Challenges for Food Traceability

Multi-element and isotopic analyses have previously been applied to a range of foodstuffs to develop methods that will permit their geographical origin to be determined with varying degrees of certainty. A vast array of analytical techniques and parameters have been studied to verify the provenance of regional foods, such as aroma, sugar, phenolic and flavour compound profiling, by gas and liquid chromatography and ‘fingerprinting’ or chemical profiling by 1H NMR (using hydrogen-1 as target atom), near Infra-Red and Fluorescence spectroscopy. These techniques can be extremely powerful tools for food origin determination in their own right and NMR profiling is often reported to be used in conjunction with multielement isotopic and trace element analysis.

Food authentication requires a database of genuine samples to which the ‘suspect’ test sample can be compared to establish its authenticity. In order to characterize markers for an authenticity parameter, such as geographical origin, there is a requirement for a large number of independent variables to be measured and statistically ‘screened’ in order to identify key tracers that differentiate the regions or countries of interest. Measuring elemental concentrations and isotopic variation in regional products is arguably the best analytical strategy for accurately verifying geographical origin.

Meat (beef and lamb), dairy products (milk, butter, cheese), beverages (tea, coffee, juice), cereal crops (rice, wheat) and wine have, to date, been the main commodities of interest investigated using the techniques mentioned above. Other commodities such as olive oils have been analyzed for geographical classification using multi-element data together with sensory parameters, combined with multivariate statistics.

Further research activities have also been undertaken to identify the regional provenance of asparagus using strontium isotope ratio measurement by multicollector - inductively coupled plasma - mass spectrometry (MC-ICP-MS). Tracing to origin is also an important issue for protection of the market and trading interests for other commodities such as saffron.

The relative abundance of natural strontium (Sr) isotopes is related to local geological conditions and may therefore provide information on the origin of raw cheese products. Values of Sr isotope abundance ratios in terrestrial vegetation are linked with the Sr isotopic composition of the soil, which is influenced by bedrock, soil/water properties and atmospheric inputs. The typical strontium mass content of mobile strontium in soil and in solution ranges from 0.2 to 20 mg kg-1 (μg Sr leached per g of soil). For different types of geological samples the overall mass contents of Sr range from 1 up to 2000 mg kg-1 and from 0.01 to 7620 mg L-1 for hydrological samples (seawater, rivers, rain) and from 8 to 2500 mg kg-1 for biological samples (wood, roots). Biological processes, whether involved in plant or animal metabolism, do not significantly fractionate strontium isotopes. It has been found that geological properties (e.g. Sr isotope abundance ratios) are reflected directly in the cheeses when the cows are kept under a controlled dietary regime and are not fed with industrially produced feeds or feeds from geographically distant sources.

There is still considerable room for improvement in both sampling and analytical methodology. In particular, there is a need to ensure that:

1. Procedures used by exporting countries are in harmony with those used by the competent authorities in importing countries as provided under applicable legal norms. For example at the European level under the Regulation (EC) 882/2004, on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules;
2. Attributes of food authenticity are clearly identifiable, documented and/or measurable by all involved parties;
3. The limitations inherent in analytical data, in particular concepts such as uncertainty and limits of quantification, are understood by all concerned, and;
4. Mechanisms are developed to assist in the preparation of appropriate commercial specifications as well as making certain that sampling and analytical methods used ‘in house’ (usually rapid methods) are fit for purpose. This involves taking into account not only the analyte but also the food matrix in which it is analyzed.

Also in the field of food authenticity, besides reliable but time consuming analytical methods, there is a need for the development of fast, simple, robust methods of proven efficacy and reliability.