food fraud the analytical tools workshop - gov.uk

Food and Environment Research Agency

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Food Fraud – the Analytical Tools Workshop –

27-28th

February 2014, Sand Hutton, York

Summary of the Defra food authenticity conference

Authors: Hez Hird & James Donarski


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Contents

Page number

Introduction 3

Summary 4

Key points arising from the Conference and workshop 5

Annex 1 The Conference and Workshop: summary of abstracts 10


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Introduction

This report is a summary of the Defra food authenticity conference held at the Food and Environment Research Agency (Fera) on the 27th and 28th of February 20141 . It was the first food authenticity event of this kind organised by Defra. The conference contributed to Government activity to raise awareness of the cutting edge science funded by Defra’s Food Authenticity Programme on analytical methods to ensure that the UK food industry and consumer confidence is not undermined by food fraud. The conference brought together international speakers and specialists with a broad range of expertise and perspectives in the arena of food authenticity.

The conference comprised two sessions, the first, on the 27th of February, covered the challenges associated with predicting and detecting food fraud from a national and international perspective, the national and EU policy implications and the response by Defra and the Food Standards Agency on activity to support consumer protection, food law enforcement and ensure a level playing field for industry. The role of the public analyst laboratories was described followed by a series of technical presentations describing the analytical approaches and technologies that can be and have been successfully deployed to detect and prevent food fraud. The first day was concluded by examining the future challenges and a question and answer session from expert panellists in the food authenticity arena.

The second session, held on the 28th of February, was a specialist technical workshop addressing topical country of origin issues. The session explored the policy landscape on food labelling and the technical approaches which are available to verify country of origin from an EU perspective. The scientific challenges faced by Defra on food fraud were outlined and some of the solutions that have been developed in response.

The conference was attended by over 150 delegates from a total of 23 different countries. The conference was part of a week-long event dedicated to the detection of adulteration in food. This event on food authenticity marked the launch of the €12M Food Integrity project. Funded by the European Union's Seventh Framework Programme, comprising 38 international partners from industry, academia and government institutes, Food Integrity will address many of the post-horsemeat issues at a European level. As well as carrying out research on new methods and systems for authenticating food and ingredients, the project will work with stakeholders to better understand consumer behaviour, develop horizon scanning tools and systems for data sharing. An international network of expertise on food authenticity will be established. It will identify gaps and commission €3M worth of research requirements within the project to address emerging issues2.

1 http://fera.co.uk/events/foodIntegrity2014/

2 https://secure.fera.defra.gov.uk/foodintegrity/index.cfm

http://fera.co.uk/events/foodIntegrity2014/

https://secure.fera.defra.gov.uk/foodintegrity/index.cfm


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Summary

The conference and workshop presented a policy and science perspective on the breadth of issues faced in the area of food fraud. It covered the challenges these create in terms of detection, and the scientific approaches, the ‘analytical tool box’, that is currently available to detect food mis-description and verification in terms of chemical, biochemical and DNA markers. It considered the Government’s response to the horsemeat incident, including intelligence gathering, consumer insight and technical innovation around enabling methodologies to detect food fraud. It also explored legislative developments in food labelling including geographic origin and the challenges around putting the tools in place to verify these claims. Over 150 delegates from 23 countries participated in the two day event.

The drivers of food fraud were discussed at length during the course of the meeting. The main driver being for direct economic gain, by either extending a product or ingredient or through substitution. Those committing food fraud actively seek to avoid detection and therefore the development and implementation of routine screening tools can lead to deterrence and ultimately prevention. In the UK, the department of Food, Environment and Rural Affairs (Defra) and the Food Standards Agency (FSA) are ensuring that suitable detection methods are developed, validated and implemented e.g. for use in targeted surveillance to safeguard the UK consumer and support food law enforcement. Following the horsemeat incident, several independent reviews have been completed and the UK Government is currently reviewing these and using the recommendations to augment their procedures and control measures.

The main analytical methods for detection of food fraud rely on either knowledge of specific markers of authenticity / adulteration or use databases that describe authentic products. For example, DNA based methods can detect the presence of genomic sequences to reveal the presence of adulterants, substitution with other components or provenance (e.g. genetically modified organisms, undeclared species). Proteomic and immunological based methods can be applied to situations analogous to those used by DNA based technologies except these are used to detect specific peptides/proteins associated with adulteration and substitution. These techniques are powerful and the DNA methods are routinely used by large numbers of laboratories. The immunological based methods can be used to generate, in large numbers, rapid diagnostic tests that can be used by non-expert staff. Proteomic based methods are currently not as widely used as DNA based methods but have the ability to be applied where DNA technology cannot (e.g. where processing factors have removed / destroyed all DNA) and it is expected that these will be widely adopted in the future.

Metabolomic based methods use non-targeted analysis to generate large amounts of data from groups of authentic samples and statistical tools (chemometrics) to generate mathematical models that describe authentic products. Samples can be compared to these models and non-authentic / adulterated samples which do not fit the model of authentic samples, can be identified. These methods are very powerful in their ability to discriminate


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authentic and non-authentic/adulterated products as they report information from a wide variety of metabolites in a sample and require no a priori knowledge of the adulteration type. Furthermore, it is possible, using advanced statistical tools, to generate specifications for products that can then be transferred to other methods, although it should be noted that once a specification is generated, it can be exploited by a counterfeiter.

Stable isotope methods use naturally occurring isotopes of elements to determine the geographical origin of a sample. Natural processes alter the distribution of the stable isotopes (e.g. biological processes in plants alter carbon isotope ratios, precipitation and altitude can affect hydrogen isotope ratios and geology influences strontium isotope ratios). Geographical origin (based on probability) can be confirmed through the measurement of these ratios. These methods are potentially extremely powerful in determining the likely geographical origin although large databases of authentic reference samples are required to extrapolate data and the methods do have some limitations. Published research shows that many methods rely on very small datasets which does not span sufficient seasons of years of production. Therefore the resolution of the separation for these techniques falls as data are added to the database from more sampling points and/or geographical locations. Trace element analysis can be also be confounded by a change in agricultural practice. Databases therefore need to take account of knowledge of provenance and mode of the production of the samples they contain and need to be curated. Data are generated and referenced to international reference samples therefore potentially databases are transferable between laboratories (assuming equivalent sample preparation) and fears on the transferability of databases / methods have proved unfounded. Existing databases are held by several different organisations (public and private) and are used for commercial purposes, which can limit their transferability due to exploitation of intellectual property issues. A recurring theme of the conference was the request for database sharing by organisations and the need for complementary approaches for determining origin to support food law enforcement recognising the limitations of isotope ratio analysis, and the need for certified reference materials. The Food Integrity project will specifically address the need for database sharing and aims to look at solutions that address the desires of the scientific community balanced by those of commercial organisations.

Key messages and outcomes arising from the conference and workshop.

The main discussion points and outcomes arising from the event are summarised below. The key messages can be broadly grouped into those that relate to food fraud, comments relating to the horsemeat incident, food law, intelligence and detection strategies.


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Food fraud

One of the key points that was discussed by several researchers, and in particular by Dr Spink, was that the ultimate aim of the food authenticity is not the detection of adulteration and substitution of foods, but the deterrence and prevention of food fraud. This is facilitated by the use of robust detection methods. Those that commit food fraud actively seek to remain undetected, therefore when methods are available for use by industry and enforcers, awareness of an issue is raised which may in turn discourage food fraud.

Dr Spink also outlined a new concept, building on HACCP (Hazard Analysis and Critical Control Point) to deliver TACCP (Threat Assessment Critical Control Point) and VACCP (Vulnerability Analysis Critical Control Point). TACCP concentrates on the analysis of threats to business (therefore relates to food defence) and VACCP on vulnerabilities (therefore relates to food fraud). The mechanisms for utilising TACCP and VACCP are based on those of the recognised industry standard of HACCP and therefore adaptation for using these approaches is easy for businesses. In particular, VACCP is key to understanding which parts of the food production chain are vulnerable to fraudulent behaviour and therefore resource to prevent fraudulent behaviour can be used in a more cost effective manner and can be directed where it is most effective.

Potential solutions for food fraud may be found by looking at exploiting approaches used by other industries (e.g. automotive and pharmaceutical sectors),. It was noted that fraud often occurs after the last testing point. An innovative solution used in other industries is to use the consumer to participate in verification procedures (which can be disguised with an incentive) thereby, greatly reducing the capacity for fraud.

Horsemeat Incident

Several presentations highlighted the impact of the horsemeat incident on consumer confidence in the integrity of the food chain and the science and research completed in response to the incident to support consumer protection and food law enforcement. The horsemeat incident had caused short and long term economic impacts and following it, consumer buying patterns were initially significantly altered. Although some return to normality has been observed, the effects can still be measured in consumer food purchasing preferences. The nature of the incident, i.e. that of beef substitution with horsemeat, was not the primary concern of the typical consumer, instead their concerns centred on that any fraud of this magnitude across global supply chains could occur.

A significant amount of European and UK testing has been performed since the horsemeat incident by industry and enforcers, the vast majority of which was found to be negative for undeclared horsemeat. However this incident has resulted in a loss in consumer confidence in food systems, food industry practices and evidence from analytical techniques to detect and deter. The policy challenge is to restore this change in consumer confidence. ‘The


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Review of the Integrity an Assurance of Food Supply Networks (‘Elliott Review’) will inform the Government’s response to the horsemeat incident.

Food fraud affects consumers, industry, regulators and by nature it can be very sophisticated and can involve organised crime. The FSA has established an intelligence hub; it is developing a Food Crime Intelligence Unit and also undertaking data mapping to inform data sharing and availability.

Food law

All EU food law is based on the principle that the consumer comes first, and that the key requirement is that the consumer is not mislead. Regulation (EU) No 1169/2011 will come into force in December 2014, which outlines the mandatory information on food labels and which will result in providing the consumer with more information and harmonisation throughout Europe.

The utilisation of HACCP like processes (TACCP and VACCP, Threat Analysis Critical Control Point and Vulnerability Analysis Critical Control Point, respectively) should be considered by food manufacturers / suppliers to determine relevant threats and vulnerabilities in their supply chains. The UK Government perspective is facilitating work with industry to assure the integrity of the food chain. It is the responsibility of industry to put the necessary checks and testing in place to support consumer protection against food fraud..

Several innovative and emerging technologies provide results that taken in isolation will not be accepted by law for prosecution purposes. Currently, these screening methods can be used to supplement prosecution evidence and to warrant deeper examination of traceability information and audit. For example the use of multivariate methods which provide classification through statistical modelling, which causes difficulties as current food law enforcement relies on the avoidance of doubt and therefore these methods must be used in combination of traceability / audit information.

Intelligence

There is a need to strengthen the joining up and sharing of intelligence between government, industry and enforcers to predict food fraud and inform action needed in response. Collaboration across the research community and those involved in developing and using detection methods is also needed to standardise the scientific approaches to tackling food fraud. For example sharing of methods , for validation, standardisation and harmonisation purposes and ultimately the sharing of databases. The issues and challenges around database sharing was raised throughout the meeting. For example, some databases have commercial value (and intellectual property issues) which limits wider accessibility; and where non-harmonised methods have been used, the comparability of databases needs to be addressed. Databases exist in several areas (geographically and in terms of commodities) and it should be investigated if these databases can be: compared; combined; transferred and shared. The EU funded Food Integrity project will examine the


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issues associated with database sharing and seek to find practical solutions to address this issue.

Detection strategies

Several key technologies exist in the ‘authenticity toolbox’ which exploit properties of elements, chemicals/metabolites and biological molecules to verify foods in terms of provenance, production, quality and composition. This toolbox has been developed through public funding of the UK Government’s Food Authenticity Programme from both Defra and historically FSA, who continue to support its continued development through enabling transferable methodologies to prevent and detect food fraud. Established methods are validated and transferred to laboratories to perform food surveillance to support food law enforcement. As advancements in key technologies are realised they should be assessed and where applicable exploited and taken up into practice to determine authenticity. Examples using emerging technologies to develop detection methods under the Food Authenticity Programme include: application of next generation sequencing; digital PCR; and proteomics. The strengths and weaknesses of different technologies necessitate the development of several key technologies that are able to detect specific biomarkers. For example, any technology that relies on the detection of specific biomolecules (e.g. DNA or protein based technology) can be circumvented through removal or external addition of those biomarkers. A new technology, which is gaining in popularity, is digital PCR, which allows the absolute quantification of the DNA in a sample and may circumvent some of the issues inherent in other methods of DNA quantification. In particular there are outstanding issues for the measurement of DNA in a sample which need to be addressed, including:

• Lack of standardisation

• Agreement on expression units (w/w or cp/cp)

• Use of mitochondrial vs. nuclear DNA

• DNA quantitation: better definition in terms of relationship to

total meat

– total DNA

– specific meat

– mammalian DNA

• Relationship between DNA copy numbers and actual meat content

• Evaluation and assessment of impact of food processing on DNA

measurement

• Understanding contributing factors

• Investment in technology to help facilitate better traceability and

monitoring.

Technologies that are broadly untargeted (i.e. metabolomics or isotope ratio measurements on bulk components) require the development of exhaustive databases. The creation of databases requires the acquisition and curation


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of authentic samples and this is a significant challenge for database construction. These database samples should take into consideration the natural variation of the sample. A method of storing these samples for future utilisation (sharing) by alternative technologies would be a significant resource. Finally, linked to the ability to acquire authentic samples there is a need to produce specific food based certified reference materials for ring trials and proficiency testing.

Concluding remarks

In summary, some of the key future challenges for securing the food chain from fraud are outlined below:

Method standardisation

• Better authentic reference material provision - for method

development, quality control, UKAS accreditation, proficiency testing.

• Reducing Measurement uncertainty (DNA)- in PCR-based methods

and variability of DNA extraction/amplification to support their use as a

rapid tool for food fraud detection.

Enabling methodologies

• Development of ‘Non targeted’ multi-analyte methods to

simultaneously detect multiple species and varieties (molecular

methods); and multi-analytes (chemical methods)

• Development of portable reliable rapid authenticity tools - for use

in in the field

• A step change is needed on molecular methods – for example

utilisation of next generation sequencing techniques

• Novel quantification methods - development of novel molecular

biology, genomic and proteomic methods and their application to food

to detect emerging food fraud issues.

Collaboration and knowledge transfer

• Method standardisation, validation and database sharing.

Recognition and standardisation of robust analytical methods within

EU. Collaborative programmes on authenticity

• Transferability of methods to support food law enforcement

In the future, an innovative analytical toolbox of detection approaches will be required based on cutting edge technology to police and prevent fraud. Rapid, non-targeted methods to support enforcement will allow rapid


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deployment in a new food fraud scenario. The advent of field based, and non-targeted testing as opposed to laboratory based, analytical tests will facilitate greater screening of commodities, by both enforcers and producers, at all points along the food production chain, particularly where VACCP has identified vulnerable links in that chain. Taken together, the field based rapid screening tests and the analytical toolbox will provide a powerful deterrent to food fraud in the future.


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Annex 1. The Conference programme 27th February 2014

09:30 - 10:00 Registration and refreshments

Introduction Chairs: Sandy Primrose, Food Authenticity Programme Advisor & Lucy Foster, Defra

10:00 - 10:05 Welcome and introduction - Sandy Primrose, Food Authenticity Programme

Advisor

10:05 - 10:20 Food Fraud - an international perspective - John Spink, Michigan State

University

10:20 - 10:35 Food Fraud - a policy perspective - Lindsay Harris, Department for

Environment Food & Rural Affairs, and Will Creswell, Food Standards Agency

10:35 - 10:50 The UK Food Authenticity Programme - Lucy Foster and Sophie Rollinson,

Department for Environment Food & Rural Affairs

10:50 - 11:20 Coffee break - attendees are invited to put up challenges and priorities on 'post-

its' for discussion in the afternoon Q&A session

Using chemical markers Chairs: Paul Brereton, Fera and Gerard Downey, TEAGASC

11:20 – 11:40 An overview of analytical approaches to verify country of origin labelling

(COOL) claims - Simon Kelly, University of East Anglia

11:40 – 12:00 Identifying oil species in oil mixtures - Tassos Koidis, Queen’s University

Belfast

Using biochemical markers Chairs: Paul Brereton, Fera and Gerard Downey, TEAGASC

12:00 - 12:20 Mass spectrometry and the analysis of food composition and authentication - Paul Fraser, Royal Holloway University

12:20 - 12:40 A proteomic approach to the detection of offal and added serum in meat

products - Ellen Billett, Nottingham Trent University

12:40 - 13:00 Detection of unlabelled meat in meat products using proteomics and

genomics - Adrian Charlton, The Food and Environment Research Agency

13:00 - 13:40 Lunch - Posters and networking

Using DNA markers Chairs: Sandy Primrose, Food Authenticity Programme Advisor & Hez Hird,

Fera

13:40 - 14:00 Meat speciation analysis - current and emerging approaches - Malcolm

Burns, LGC

14:00 - 14:20 Genetic tools for geographic traceability and breed identification of food -

Gill Murray-Dickson, Trace Forensics

14:20 - 14:40 A Public Analysts' perspective - Liz Moran, Association of Public Analysts

The Future Chairs: Sandy Primrose, Food Authenticity Programme Advisor & Hez Hird, Fera

14:40 – 15:00 European research initiatives on food authenticity - Paul Brereton, The Food

Environment Research Agency

15:00 - 15:15 Meeting the challenges – future development in the Food Authenticity

Programme - Lucy Foster, Department for Environment Food & Rural Affairs

15:15 - 16:00 Panel Q&A session

16:00 Close and tea


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Food fraud – an international perspective.

Dr John Spink

Michigan State University

Abstract

This presentation will provide an international perspective on the countermeasures underway by industry, agencies, non-governmental organizations, and academics from around the world. Food Fraud – and the subcategory of Economically Motivated Adulteration – is attracting world-wide attention by industry and agencies. There is a growing awareness of the scope, scale, and risk of Food Fraud. Beyond the growing costs to companies and economies there is a growing awareness that the “economically motivated” act can lead to significant public health threats. Although there is still room for improvement, there has already been unprecedented collaboration from around the world and across disciplines. It is unique that there has been such an early focus on harmonization of terms and coordination of countermeasures. Interdisciplinary research has expanded beyond the traditional food sciences to include the behavioral sciences, criminology, social anthropology, international public policy, and even the activities related to language translation. The expansion of the research platform has enabled a unified shift from detection to prevention. The focus on prevention – preventing intelligent human adversaries – is enabling a focus on integrated science and technology solutions that proactively reduce the fraud opportunity. The goal is not to just detect more quickly but to prevent the attack in the first place. Expert opinion and insight will be critical to combat the quickly evolving threat. Gathering data and quantifying the public health and economic risk is important, but with vulnerability such as Food Fraud, non-traditional data sets and innovative analysis will be required. The presenter will provide recent insight from the range of recent USA reports with a perspective from interactions from around the world including the EU, the UK, and China. Some of the perspective is from the US Pharmacopeia/ Food Chemicals Codex, International Standards Organization Technical Committee 247 on Fraud Countermeasures and Controls, as well as from the Global Food Safety Initiative’s Food Fraud Think Tank. These topics help frame the conference question to “highlight emerging issues and gaps in analytical technology.” For more information on these topics and to connect to recent academic publications please visit www.FoodFraud.msu.edu.

Massive Open Online Course (MOOC) is freely available from Michigan state University (http://foodfraud.msu.edu/mooc).

Dr Spink was happy to receive comments and to hear from those working in the authenticity field (http://Food Fraud.msu.edu)

http://foodfraud.msu.edu/mooc


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Food fraud – a policy perspective.

Mr Lindsay Harris / Mr Will Cresswell

The Department for Environment & Rural Affairs / Food standards agency

Abstract

The presentation will include:

- Why food authenticity matters - policy context

- Partly about accurate and useful information for consumers

- Partly about consumer trust in food

- Follow up to the horse meat fraud: Elliott Review of the integrity and assurance of food supply networks

- Issues on industry practices for food businesses to follow up

- Issues on regulatory and enforcement systems for Government to follow up

- Follow up at European level

The UK Food Authenticity Programme

Dr Sophie Rollinson

The Department for Environment & Rural Affairs, UK

Abstract

Food authenticity and food fraud are high on the agenda, in particular in the wake of the horsemeat issue in the UK: consumers are increasingly demanding information on and reassurance of the content and origin of their food. Protecting consumers, supporting the integrity of the food chain and preventing fraudulent practices such as the deliberate adulteration of food are important and challenging issues facing regulators, enforcers and the food industry alike.

The law already makes it illegal for food being sold to consumers to be described in a way that is not accurate or is misleading, but without the means of verifying the accuracy of labelling, it is difficult to ensure the law is complied with and consumers protected and that economic fraud is minimised.

With this in mind, the UK Government set up the food authenticity programme about 20 years ago to fund the development and validation of analytical methods to verify food labelling and check for the mis-description of food.


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The Food Authenticity Programme’s science activities have focused on helping the UK develop the analytical tools it needs to help it comply with the law on food description. Over the years the programme has funded the development of a variety of methods, from methods based on staple isotope and trace element analysis to identify the geographic origin of beef, to the use of DNA-based methods to identify the species and variety of meat, fish, fruit and vegetables, and the use of chromatographic and spectroscopic methods to verify vegetable oil species.

However, with the ever-increasing complexity of food composition and matrices and sophistication of food authenticity fraud, innovative science is needed to develop a cutting edge portfolio of methods to tackle the food fraud of the future.

A Public Analysts’ perspective

Ms Liz Moran

Public Analyst, Scientific Services, UK

Abstract

A review of the challenges faced by the food safety enforcement authorities and public analysts during the horse meat incident and the wider analytical challenges facing the ‘food police’ in dealing with food authenticity and fraud.

Overview of the presentation

Ms Moran is the president of the Association of Public Analysts and the presentation gave an overview of the public analysts’ role in food fraud. The background to the public analyst system was provided, how they have evolved and the current duties that they perform and the number of laboratories in the UK and the authorities they serve. The public analysts requires a qualification that covers both a wide range of analytical chemistry techniques for a multitude of sample types and the law associated with those sample types. The range of analyses that can be performed is very large, any food type can be submitted for any type of test. A network exists between the public analysts as food incidents are less likely to be regional and more likely to be national. It is not possible to test all food or to test a single food for everything: sampling is performed by Trading Standards and Environmental Health professionals and is risk based. The public analysts have a wide range of experience and employ a variety of analysts. The analytical challenges of the public analyst were covered. For example the amount of testing required for horsemeat analysis after the horsemeat scandal was revealed. The public analysts utilise a range of techniques, including the DNA profile, stable isotope analysis, mass spectrometry, proteomics and genomics. These techniques often use extremely expensive equipment and have high running costs. Therefore analysis by these techniques is often performed by using links to national reference laboratories or through links with other laboratories. The Public Analysts have access to large amounts of intelligence which can be used to perform trend analysis to inform the FSA and Defra regarding


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upcoming issues on food fraud. Recommendations to assist in food fraud detection were presented including : harmonisation of analytical techniques and standards; adding markers that are difficult to obtain by fraudsters but easy to analyse, the development (and sharing) of DNA and stable isotope databases for animals used in the food chain; and smart food packaging. The presenter gave her thoughts on the future which was collaborative working between Public Analysts and the Food Standards Agency and Defra.

Dr Tassos Koidis

Queens University Belfast, UK

Abstract

Detection of adulteration of non-processed vegetable oil with lesser value seed oils (classic example is hazelnut in virgin olive oil) has been in the centre of scientific attention for many years and several chemical methods were proposed. The recent EC Regulation 1169/2011, however, introduces necessity for different analytical method in a more complicated matrix. From the end of 2014, food businesses required to declare the composition of the refined oil mixture in the food product label. This creates a gap since there is no analytical method currently available to perform such analysis. In the first phase the work focused on 100% oil blends of various oil species of palm oil (and derivatives), sunflower and rapeseed oil before expanding to foodstuffs. Chromatographic methods remain highly relevant although suffer from various limitations which derive from natural compositional variation. Modern multivariate techniques based on machine learning algorithms, however, when applied in FTIR, Raman spectroscopic data have a strong potential in tackling the problem.

Mass spectrometry and the analysis of food composition and authentication

Prof Paul Fraser

Royal Holloway University, UK

Abstract

Hyphenated mass spectrometry approaches can be applied to the analysis of foodstuffs. Both proteomic and metabolomics approaches can provide an alternative to DNA based methods for the detection of food authenticity. Here two examples will be provided of their potential firstly the proteomic detection and characterize the transgenic protein present in Roundup Ready(TM) soya and maize. Genetically modified (GM) soya and maize contain the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4, which confers resistance to the herbicide glyphosate. TheGM soya and maize proteomes were fractionated by gel filtration, anion-exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) prior to MS. This facilitated detection of a tryptic peptide map of CP4 EPSPS by matrix-assisted


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laser desorption/ionization time-of-flight (MALDI-TOF) MS and nanoelectrospray ionization quadrupole time-of-flight (nanoESI-QTOF) MS. Subsequently, sequence information from the CP4 EPSPS tryptic peptides was obtained by nanoESI-QTOF MS/MS. The identification was accomplished in 0.9% GM soya seeds, which is the Current EU threshold for food-labeling requirements. In addition a proteomic-based method has been developed for the detection of meat species within mixed meat preparations. The procedure is robust and simple, comprising the extraction of myofibrillar proteins, enrichment of target proteins using isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Using this approach, it was possible for example to detect 0.5% contaminating chicken in pork meat with high confidence. Quantitative detection of chicken meat was done by using AQUA stable isotope peptides made from the sequence of previously selected species-specific peptide biomarkers. These methods permit the determination of definitive discriminatory sequence, unlike the DNA PCR based methods used.

A proteomic approach to the detection of offal and added serum in meat products

Prof Ellen Billett

Nottingham Trent University, UK

Abstract

From the point of view of labelling of products containing meat as an ingredient, ‘meat’ is defined as skeletal muscle with naturally included or adherent fat and connective tissues (1). The regulations exclude other types of muscle protein (e.g. heart and tongue) and non-muscle products such as offal and blood proteins. The regulations also state that certain carcass parts, e.g. liver, kidney & lung must be explicitly labelled, as the generic term ‘offal’ is not permitted. Such additions to meat products are not illegal; however, the label must accurately reflect the ingredients.

Currently meat content is determined in Public Analyst Laboratories using the Kjeldahl method of nitrogen analysis and the Stubbs and More Calculation. These methods cannot distinguish between nitrogen from ‘meat’ and nitrogen from offal or blood protein. DNA-based methods cannot distinguish between blood and specific organs since DNA is the same in every tissue. Using a proteomic approach, the Nottingham Trent University group has overcome these drawbacks and has developed technologies to analyse the presence of specific offals and added serum in meat and processed meat products. Marker proteins have been identified to (a) distinguish between meat and offal and (b) detect, quantify and establish the species of origin of specific offal in food products. These methods involve analysis of specific proteins by a combination of sensitive immunoassays and peptide mass fingerprinting. We have also produced reagents (species-specific monoclonal antibodies) and developed sensitive immunoassays which can specifically detect and quantify added porcine and bovine sera.


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The presentation will provide an overview of the methods used together with some specific examples of their applications.

(1) The Food Labelling (Amendment), (England) Regulations, 2003) and parallel legislation in Scotland, Wales and Northern Ireland.

Detection of unlabelled meat in meat products using proteomics and genomics

Adrian Charlton, Rosario Romero, Hez Hird

The Food and Environment Research Agency, UK

Abstract

The presence of unlabelled ingredients of animal origin in food products can raise a range of ethical and religious concerns. Consumer choices are often made on the basis of accurate product labelling and for meat products, accurate labelling to legal requirements reinforces consumer confidence that a product only contains meat from the stated animal origin. Shifts in demand within the food supply chain, perhaps driven by increasing food prices, have seen mislabelled meat products emerge in the UK and international markets. This is illustrated most significantly by the presence of horse DNA in food products during 2013 which were labelled as containing beef.

Analytical methods for the detection of horsemeat were the predominant tool for uncovering this fraud. Robust and validated methods for the detection of horse DNA in several food matrices were developed at Fera. These PCR based methods are good indicators of the presence of horse DNA in many food products. However, moving to a robust system for detecting more widely the presence of unlabelled meat in meat products requires the development of new technologies that consider the effect of processing on the molecular composition of food stuffs and how processing might lead to an underestimate of unlabelled meat in meat products using current detection technologies. This presentation will highlight the work that has been undertaken at Fera to move to more holistic and potentially quantitative analytical methods which are able to detect the presence of unlabelled animal products from many animal sources simultaneously. Case studies including the use of proteomics to detect bovine and porcine gelatine in chickens and the potential for the further development of this approach for the detection of horse protein in highly process products such as stock cubes, will be discussed. Similarly, the potential use of the latest generation of genomics technologies for the holistic characterisation of DNA in our food will be presented.


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Meat speciation analysis - current and emerging approaches

Dr Malcolm Burns

LGC Group, Queen's Rd, Teddington, Middlesex TW11 0LY

Abstract

Current EU legislation concerning authenticity and labelling of foods affects everyone, inclusive of food manufacturers, retailers, traders, enforcers of legislation and consumers. Food authenticity has been highlighted by some of the recent meat speciation issues concerning beef products in 2013. Testing for food authenticity requires access to accurate methods to determine the level of different materials and ingredients present in foodstuffs.

There are a number of pre-existing and validated methods available for testing for food adulteration, all with their associated advantages and disadvantages. For meat speciation, molecular biology approaches that target the DNA molecule are prevalent, and some of the recent meat speciation issues have emphasised the need to both develop and then maintain approaches for the correct identification of food ingredients. This involves engagement and evaluation of new and emerging technologies, as well as investment in R&D coupled with greater harmonisation and agreement on standardised approaches, to help facilitate better traceability and monitoring of food materials. This presentation highlights some of the Defra funded work involved in food authenticity testing using molecular biology methods.

Genetic tools for geographic traceability and breed identification of food

Gill Murray-Dickson

Trace Wildlife Forensics Network, Royal Zoological Society Scotland, UK

Abstract

Developing a robust traceability system to distinguish fish products from different fisheries is highly desirable, as the sustainable exploitation of fish stocks requires management within species at the level of the biological population. Similarly, the ability to authenticate the origin of meat labelled by breed, as well as species, is paramount to protecting both consumers and genuine traditional meat producers in a premium market that is susceptible to mislabelling fraud.

Animals, their parts and derivatives can be identified using molecular genetic markers called Single Nucleotide Polymorphisms (SNPs). For commercially viable species and breeds, vast numbers of SNPs have previously been identified and used to address questions of geographic origin and breed authentication. However, employing a large number of SNPs to address these issues generally requires specialist laboratory experience, is expensive and difficult to execute across a large number of samples.


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Building on existing large scale research, we are deriving reduced panels of informative SNPs to address questions of geographic traceability and breed identification for three species of fish (cod, European hake and common sole) and traditional pig and cattle breeds, respectively. The overall aim is to produce a series of highly efficient, reproducible and cost-effective assays that are well-suited for use by non-specialist laboratories, are cheaper to run and more feasible for processing large sample numbers

An overview of analytical approaches to verify country of origin labelling (COOL) claims

Simon Kelly

Faculty of Science, Science Building, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ

Abstract

In the European Union it is mandatory to include information about country of origin on the packaging of certain foodstuffs and the number of foodstuffs covered by this legislation will increase in late 2014. To ensure the accuracy of country of origin labelling (COOL) it will be necessary to have appropriate test methods in place. A large number of different analytical approaches have been described in the literature and for the purposes of this presentation have been subdivided into three groups; separation techniques, spectroscopic techniques and mass spectrometry techniques. This presentation will give an overview of the methods used to verify COOL claims with an emphasis on arguably the best hypothesis driven methodology stable isotope analysis. The lack of comprehensive and accessible databases for most of the foods covered by COOL legislation will be discussed. Other methodologies such as SNP genotyping, proteomics, metabolomics and possible metagenomics will be covered in other presentations.

European research initiatives on food authenticity

Paul Brereton

The Food and Environment Research Agency, UK

Abstract

The European Commission has for many years funded research into developing methods and systems for detecting food fraud. Methods of analysis were often embedded within legislation as part of “vertical” standards for trade purposes and for ensuring compliance with Common Agricultural Policy. As a result there has been a long history of supporting food authenticity research and many of the key authenticity techniques such as SNIF-NMR and Stable Isotope Ratio Mass Spectrometry have benefitted from Commission support either through its Framework Programmes and/or


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adoption into Regulation e.g. in the development and implementation Council Regulation No 2048/(EU wine databank).

More recently in 2005 the Commission funded TRACE a €20M research programme that made the link between authenticity and traceability and set the global agenda for research into how we can determine the origin of our food. Over recent years there has been a greater emphasis on relying on traceability procedures and systems as a means providing confidence on the integrity of the food we eat. However major food fraud incidents have continued to surface, some with tragic food safety implications e.g. melamine in milk (China), methanol in spirit drinks (Czech Republic). Following a consolidation of EU research at the “What’s for lunch” presentation to members of the European Parliament http://ec.europa.eu/research/agriculture/wfl/index_en.html, it became apparent that further research and coordination activities were needed. This was further emphasised with the horsemeat incident in Europe in 2013. As a result several new European research initiatives are progressing. The largest of these is the FOODINTEGRITY project that aims to address many of the problems encountered by the stakeholders in the horsemeat incident – at a European scale. The €12M project with participation from industry, regulators, researchers and consumers will address some of the major issues raised during “horsegate”. Such as where can I find an independent body of expertise on food authenticity? How can we use existing data to better effect? How can I confirm the integrity of my product more quickly and cheaply? How do consumers react to food fraud incidents? How can I better anticipate food fraud events? What further research is needed? Some of the major European research initiatives on food authenticity will be presented together with examples of methods and systems that have been developed within Europe to assure the integrity of the food we eat.

Meeting the challenges – future development in the Food Authenticity Programme

Dr Lucy Foster

The Department for Environment & Rural Affairs, UK

Abstract

Food fraud covers a broad spectrum of labelling mis-description including misleading claims about food quality, composition, geographic origin and method of production.

Maintaining the integrity of the food chain to ensure consumers are confident about the food they eat, and what it says on the label presents a plethora of technical challenges in terms of developing the analytical tools needed to verify food description. These challenges include, identification of suitable markers to verify authenticity of ingredients method of processing, origin, detection of adulterants and availability of authentic samples for development of robust reference databases. Ensuring that methods are robust enough to

http://ec.europa.eu/research/agriculture/wfl/index_en.html


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provide data interpretation to support food law enforcement is also a critical element in the development of analytical tools.

Detecting food fraud requires a range of methods and approaches ‘an analytical tool box’ including stable isotope analysis, DNA methods, proteomics and metabolomics. But food fraud is constantly changing, driven by the economic climate, availability of raw materials and ingredients, regulatory developments and wider impacts of the environment. Cutting edge technology is needed to make available practical, transferable and cost-effective methods for use by enforcers and the food industry alike. These methods also need to overcome challenges around uncertainty and quantitation in measurement in an increasingly complex range of food matrices and demanding processing conditions. Better harmonisation of methods and databases is also needed to tackle food fraud at an EU level and internationally.


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Workshop Programme 28th February 2014

09:00 – 09:30 Registration

1st Session; Chair: Mark Woolfe, Food Authenticity Consultant & Paul Brereton, Fera

09:30 – 09:35 Welcome – Hilary Aldridge, Fera

09:35 – 09:40 Introduction to workshop - Paul Brereton, Fera

9:40 - 10:00 New food labelling requirements – a new opportunity for the

analysts? - Stephen Pugh, Defra

10:00 - 10:20 Techniques for authenticating country of origin labelling - Sandy Primrose, Food Authenticity Programme Advisor

10:20 - 10:40 Requirements for using multi element stable isotope analysis in

food authentication - Andreas Roßmann, Isolab GmbH

10:40 - 11:00 Analytical methods for guaranteeing the authenticity of PDO hard

cheeses - Federica Camin, Research & Innovation Centre -

Fondazione Edmund Mach

11:00 - 11:20 Coffee break

2nd

Session; Chair: Franz Ulberth, JRC-IRMM, Geel

11:20 – 11:40 Tracking food with stable isotopes in practice – Markus Boner,

Agroisolab

11:40 – 12:00 Recent innovations in molecular analysis to trace food using their

microbial ecology (Part 1) – Didie Montet, CIRAD

12:00 – 12:20 Recent innovations in molecular analysis to trace food using their

microbial ecology (Part 2) – Richard Thwaites, Fera

12:20 – 12:50

Next Steps: Round table discussion

Gaps/impediments (data sharing)

Horizon Scanning

Approaches for the future Facilitator: Franz Ulberth , JRC-IRMM, Geel

12:50 – 13:00 Sum-up

13:00 – 14:00 Lunch


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Food Labelling: a UK perspective

Steve Pugh

Head of Food Labelling, Defra, Nobel House, 17 Smith Square, London SW1P 3JR UK

Abstract

The regulation on the Provision of food information to Consumers (1169/2011) is due to come into force at the end of 2014. In the presentation I will highlight the requirements for labelling in the regulations. I will focus on a few of the new provisions brought in through the regulation, such as country of origin labelling and the requirement to labelling different types of vegetable oils in the ingredients list. As the regulations come into force, some of the new provisions will require analytical methods to ensure compliance.

Food composition and particularly food compositional analysis was brought to the forefront of people’s attention during the recent scandal on horse meat. Although food labelling requirements are not of the same scale as deliberate fraud, this scandal has put the spotlight on the accurate description of food and in the presentation I will outline the work being carried out at the moment and some of the requirements for further work.

The information on food labels is the vehicle by which consumers to find out about the products they buy. This information needs to be as accurate as possible and to meet consumer expectations. Analytical methods are required, both by the food retailer and by the enforcement authority, to ensure the descriptions on food labels are as accurate as possible and that the information reflects consumer expectations.

Techniques for authenticating country of origin labelling.

Professor Sandy Primrose

Defra Food Authenticity Programme Advisor, UK.

Abstract

In the European Union it is mandatory to include information about country of origin on the packaging of certain foodstuffs and the number of foodstuffs covered by this legislation will increase in late 2014. To ensure the accuracy of country of origin labelling (COOL) it will be necessary to have appropriate test methods. The experimental method most suited to determining geographic origin is stable isotope analysis with or without trace element analysis. A variant of this method is the use of food isotope maps (isoscapes). These two methods will be covered in detail by other speakers and this presentation will focus on other methods that could complement them. These alternative methods include DNA speciation, SNP genotyping, proteomics, metabolomics and possible metagenomics. None of these methods have the


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broad applicability of stable isotope analysis but examples of where they can be used will be discussed.

Requirements for using multi element stable isotope analysis in food authentication

A. Roßmann

Isolab GmbH, Laboratorium für Stabile Isotope, Woelkestraße 9/I, 85301 Schweitenkirchen, TU München, Center of Food and Life Sciences Weihenstephan, 85350 Freising, Germany

Abstract

Stable isotope analyses of H, C and O have been used for food authenticity control since 1990. Official methods for wine stable isotope analyses were introduced in the EU in 1990, and for products as fruit juices, honeys, maple syrup in the EU and the US between 1990 and 2000. These authenticity investigations aimed to check if cheaper components from other sources had been added to the food, as water and/or sugars to fruit juice or wine. More recently authenticity checks, which refer to the “geographical authenticity” mainly of premium food products, with protected denomination of origin (PDO), were developed. The determination of one or two stable isotope parameters, as applied to detect addition of water or sugars, usually is not sufficient to obtain unambiguous results regarding geographical origin. Evidence of work, including EU project TRACE, strongly supports the use of multi element stable isotope methods, which means combination of stable isotope data of all “bio-elements” (H,C,N,O,S), and sometimes of “geo-elements”, as strontium, in addition.

A prerequisite for application of multi element stable isotope methods to origin assignment, not only for food products, but even in forensic or ecological investigations, is the availability of instrumentation for fully automated analyses of as much stable isotope parameters in a single run as possible, to save time required for the analyses. Systems for simultaneous measurement of CNS or even HCNS isotopic ratios have been developed and their application was demonstrated. In addition, accepted reference materials, or ideally, certified reference materials containing all relevant elements in an elemental composition similar to the samples investigated, should be available. In fact there is still a lack of official reference materials, even if several inter comparison substances have been characterised during TRACE. For routine application of such methods the existence of inter laboratory tested and validated, finally of officially acknowledged methods of analysis, is required. Standard operation procedures for products like defatted dry matter (protein) from meat were elaborated and tested in TRACE. Finally, the most important precondition is the existence of reliable databases and of models for the prediction of stable isotope data of food products from different regions, preferably based on information/data as climate, geography, geology and agricultural practices.


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Analytical methods for guaranteeing the authenticity of PDO hard cheeses

Federica Camin1, Roberto Larcher1, Luisa Pellegrino2

1Fondazione Edmund Mach, via Mach 1, 38010 San Michele all’Adige (TN)

2DeFENS, Università degli Studi di Milano, via Celoria 2, I-20133 Milano

Abstract

European Regulations EC Nos. 510/2006 and 1151/2012 require protection against the mislabelling of foods with Protected Geographical Indications (PGI), Protected Designations of Origin (PDO) and Traditional Specialities Guaranteed (TSG). This is particularly important for PDO hard cheeses such as Parmigiano Reggiano and Grana Padano, which can cost more than the double the generic cheeses imitating them.

The current Standard of Identity for Grana Padano PDO (updated with EU Reg. 584/2011) provides sensorial characteristics, as well as limiting values for humidity and fat content. Besides these specifications, it is stated that the authenticity of marketed cheese is verified on the basis of the free amino acid pattern and the isotopic and mineral profiles. Free amino acids accumulate by up to 28-30% in cheese protein during ripening, following typical and repeatable pathways. The free amino acid pattern is determined using Ion Exchange Chromatography with post-column ninhydrin derivatization. The pattern for authentic Grana Padano PDO has been established and deviation of two or more amino acids from the expected content by more than 3 SD indicates that the cheese is not authentic or unsound. The isotopic ratios of C, H, N and S are measured on defatted cheese using Isotope Ratio Mass Spectrometry, whereas the content of more of 50 elements in cheese is analysed after acid digestion using Inductively Coupled Plasma – Mass Spectrometry. Based on the most significant isotopic and elemental variables, multivariate statistical models were developed able to predict the origin and the type of cheese and in particular able to distinguish Grana Padano PDO from imitations. Finally, the use of raw milk can be assessed by determining alkaline phosphatise activity and the ripening period using capillary electrophoresis of a specific peptide (pyro-γ-3-casein), proved to be stable to proteolysis.

Tracking food with stable isotopes in practice

Marcus Bonner

Agroisolab GmbH, Prof.-Rehm-Str. 6, D-52428 Jülich, Germany

Abstract

The stable isotope method is one of the few scientific applications to check the authenticity of food. Many scientific papers are available dealing with


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stable isotopes to check the origin of food, to verify organic farming or to detect adulteration. Regularly the stable isotopes of the bioelements (COHNS) are in focus as they are delivering various information of the regional water (D/H, 18O/16O), the climate (13C) and the soil (15N/14N, 34S/32S). An additional parameter is always Strontium (87Sr / 86Sr) as a geological parameter which helps to increase the resolution of tracking

On one hand the stable isotopes are a powerful tool but nevertheless it is always necessary to setup a reliable evaluation system. Therefore the use of stable isotopes for tracking the origin is normally depending on several rules. Firstly the database has to be sufficiently developed with a relevant number of samples. The application procedure especially the preparation has to be worked out and validated. Further information of the products are always enhancing the stable isotopic evaluation and are essential for the database. The statistical tool has to be established, as well. Finally the database should always be checked with indepedent blind samples to approve the consistency of the data.

Regarding the mentioned rules, Agroisolab GmbH has developed various extensive databases as the Polish strawberry database, the English pork database, the European egg database, the German Caviar database, the International Teak and Mahogany database or an Olive Oil database in combination with chemical parameters.

The presentation will give insights in the development with their specific problems and the final set up.

Recent innovations in molecular analysis to trace foods using their microbial ecology

Didier Montet1 and Richard Thwaites2

1CIRAD, Zone d'aménagement concerté Baillarguet, 34980 Montferrier-sur-Lez, France

2Fera, Sand Hutton, York, YO41 1LZ, UK

Abstract

EU regulation 178/2002 impose requirements for traceability of food. An innovative method for tracing the geographical origin of a foodstuff is to analyse in a global way the microbial communities of the food samples after their importation. For this purpose, molecular techniques employing 16S rDNA, 26 rDNA and 28 rDNA profiles generated by PCR-DGGE were used to detect the variation in microbial community structures of different food as fish from South Vietnam harvested in different aquaculture farms and during two different seasons. 26 rDNA from yeasts and 28 rDNA from fungi were used to discriminate origin of different fruits from Africa. When the amplicon profiles were analysed by multivariate analysis, distinct microbial communities were


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detected. The band profiles of fish bacteria or fruit yeasts or fungi from different farms were different and specific for each location. These fingerprints could be used as a barcode to discriminate food origins and also discriminate organic food from conventional food. When band profiles within the same location at different seasons were compared, we also observed the same banding pattern for every season and it discriminated farms. Some common bands were found stable throughout the year regardless of the season. These bands can be used as specific markers for these specific locations. This method is proposed as a new traceability tool which provides food products with a unique bar code and makes it possible to trace back the food to their original location. A further development of the technique is to characterise the microbial community of a food in extreme detail using next-generation DNA sequencing (NGS) techniques, which allow us to assign identities to virtually all members of a microbial community. By comparing community composition from NGS data we are able to make highly discriminatory comparisons between foods and cluster them according to total microbial content. An application of this technique to shellfish produced in the UK will be described.

Key words: aquaculture, fruits, traceability, organic, PCR-DGGE, bar-code, rDNA

food fraud the analytical tools workshop - gov.uk

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