Analysis of Food Toxins and Toxicants

By Richard J. Lewis, Sr.

Analysis of Food Toxins and Toxicants consists of five sections, providing up-to-date descriptions of the analytical approaches used to detect a range of food toxins. Part I reviews the recent developments in analytical technology including sample pre-treatment and food additives. Part II covers the novel analysis of microbial and plant toxins including plant pyrrolizidine alkaloids. Part III focuses on marine toxins in fish and shellfish. Part IV discusses biogenic amines and common food toxicants, such as pesticides and heavy metals. Part V summarizes quality assurance and the recent developments in regulatory limits for toxins, toxicants and allergens, including discussions on laboratory accreditation and reference materials.

• Language: English
• Publisher: Wiley
• Release date: Jul 3, 2017
• ISBN: 9781118992708

Book preview

List of Contributors

Amparo Alfonso

Department of Pharmacology

Veterinary Faculty

Universidad de Santiago de Compostela

Lugo

Spain

Gregor Anderluh

Department of Molecular Biology and Nanobiotechnology

National Institute of Chemistry

Ljubljana

Slovenia

Natalia Arroyo-Manzanares

Department of Analytical Chemistry

Faculty of Sciences

University of Granada

Granada

Spain

Till Beuerle

Technische Universität Braunschweig

Institute of Pharmaceutical Biology

Lower Saxony

Germany

Ana M. Botana

Department of Analytical Chemistry

Sciences Faculty

Universidad de Santiago de Compostela

Lugo

Spain

Luis M. Botana

Department of Pharmacology

Veterinary Faculty

Universidad de Santiago de Compostela

Lugo

Spain

Peter A. Brown

Norwich

Norfolk

United Kingdom

Stephen Burrell

Marine Institute

Rinville

Oranmore

County Galway

Ireland

and

School of Chemical and Pharmaceutical Sciences

Dublin Institute of Technology

Dublin

Ireland

Gaelle Catanante

Biocapteurs-Analyses-Environnement

Universite de Perpignan Via Domitia

Perpignan

France

Pui-kwan Chan

Analytical and Advisory Services Division

Government Laboratory

Hong Kong

Joanne Sheot Harn Chan

Food Safety Laboratory

Applied Sciences Group

Health Sciences Authority

Singapore

Samuel Tsz-chun Cheung

Analytical and Advisory Services Division

Government Laboratory

Hong Kong

Winnie Wing-yan Chum

Analytical and Advisory Services Division

Government Laboratory

Hong Kong

Colin Crews

Fera Science Ltd

York

United Kingdom

Jonathan R. Deeds

Office of Regulatory Science

FDA Center for Food Safety and Applied Nutrition

College Park, MD

USA

Martin Danaher

Food Safety Department

Teagasc Food Research Centre

Ashtown

Dublin

Ireland

Michael Ellisor

Chemical Sciences Division

National Institute of Standards and Technology

Charleston, SC

USA

María Luisa Fernández-de Córdova

Department of Physical and Analytical Chemistry

Faculty of Experimental Sciences

University of Jaén

Campus Las Lagunillas

Jaén

Spain

Pasquale Ferranti

Department of Agriculture

University of Naples Federico II

Parco Gussone

Naples

Italy

and

Institute of Food Science and Technology

National Council of Research

Avellino

Italy

Ambrose Furey

Mass Spectrometry Group (MSG)

Department of Physical Sciences

Cork Institute of Technology (CIT)

Cork

Ireland

Monica Gallo

Department of Molecular Medicine and Medical Biotechnology

University of Naples Federico II

Naples

Italy

Laura Gámiz-Gracia

Department of Analytical Chemistry

Faculty of Sciences

University of Granada

Granada

Spain

Ana M. García-Campaña

Department of Analytical Chemistry

Faculty of Sciences

University of Granada

Granada

Spain

Arjen Gerssen

RIKILT Institute of Food Safety

Wageningen University and Research Centre

Wageningen

The Netherlands

Caroline T. Griffin

Mass Spectrometry Group (MSG)

Department of Physical Sciences

Cork Institute of Technology (CIT)

Cork

Ireland

M. Hazel Gowland

Allergy Action

St. Albans

Hertfordshire

United Kingdom

Wai-yan Ha

Analytical and Advisory Services Division

Government Laboratory

Hong Kong

Akhtar Hayat

Interdisciplinary Research Centre in Biomedical Materials (IRCBM)

COMSATS Institute of Information Technology

Lahore

Pakistan

and

Biocapteurs-Analyses-Environnement

Universite de Perpignan Via Domitia

Perpignan

France

Vesna Hodnik

Department of Biology

Biotechnical Faculty

University of Ljubljana

Ljubljana

Slovenia

Xiaofeng Hu

Oil Crops Research Institute of the Chinese

Academy of Agricultural Sciences

Wuhan

China

and

Key Laboratory of Detection for Mycotoxins

Laboratory of Risk Assessment for Oilseeds Products

Ministry of Agriculture

Wuhan

China

José F. Huertas-Pérez

Department of Analytical Chemistry

Faculty of Sciences

University of Granada

Granada

Spain

Marco Inserra

Institute for Molecular Bioscience

The University of Queensland

Queensland

Australia

and

School of Pharmacy

The University of Queensland

Queensland

Australia

Takeshi Ito

Department of Mechanical Engineering

Kansai University

Osaka

Japan

Alun Jones

Institute for Molecular Bioscience

The University of Queensland

Queensland

Australia

Akbar S. Khan

Chemical Biological Directorate

Defense Threat Reduction Agency

Fort Belvoir, Virginia

USA

Mirjam D. Klijnstra

RIKILT Institute of Food Safety

Wageningen University and Research Centre

Wageningen

The Netherlands

Yelena Lavrukhina

Institute for Molecular Bioscience

The University of Queensland

Queensland

Australia

Tin-yau Law

Analytical and Advisory Services Division

Government Laboratory

Hong Kong

Richard J. Lewis

Institute for Molecular Bioscience

The University of Queensland

Queensland

Australia

Angela Li

Food Safety Laboratory

Applied Sciences Group

Health Sciences Authority

Singapore

Peiwu Li

Oil Crops Research Institute of the Chinese

Academy of Agricultural Sciences

Wuhan

China

and

Key Laboratory of Biology and Genetic

Improvement of Oil Crops

Key Laboratory of Detection for Mycotoxins

Laboratory of Risk Assessment for Oilseeds Products

Ministry of Agriculture

Wuhan

China

Chee Wei Lim

Food Safety Laboratory

Applied Sciences Group

Health Sciences Authority

Singapore

Eulogio J. Llorent-Martínez

Department of Physical and Analytical Chemistry

Faculty of Experimental Sciences

University of Jaén

Campus Las Lagunillas

Jaén

Spain

Stephen E. Long

Chemical Sciences Division

National Institute of Standards and Technology

Charleston, SC

USA

M. Carmen Louzao

Department of Pharmacology

Veterinary Faculty

Universidad de Santiago de Compostela

Lugo

Spain

Sameeh A. Mansour

Environmental Toxicology Research Unit (ETRU)

Pesticide Chemistry Department

National Research Centre

Giza, Cairo

Egypt

Angel Maquieira

Instituto interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico

Departamento de Química

Universitat Politècnica de València

Valencia

Spain

Jean-Louis Marty

Biocapteurs-Analyses-Environnement

Université de Perpignan Via Domitia

Perpignan

France

Sara C. McGrath

Office of Regulatory Science

FDA Center for Food Safety and Applied Nutrition

College Park, MD

USA

Karen Murphy

Chemical Sciences Division

National Institute of Standards and Technology

Gaithersburg, MD

USA

Muhammad Azhar Hayat Nawaz

Interdisciplinary Research Centre in Biomedical Materials (IRCBM)

COMSATS Institute of Information Technology

Lahore

Pakistan

Frances Nilsen

Chemical Sciences Division

National Institute of Standards and Technology

Charleston, SC

USA

Chiara Nitride

Department of Agriculture

University of Naples Federico II

Parco Gussone

Naples

Italy

Pilar Ortega-Barrales

Department of Physical and Analytical Chemistry

Faculty of Experimental Sciences

University of Jaén

Campus Las Lagunillas

Jaén

Spain

Suiyan Ouyang

Oil Crops Research Institute of the Chinese

Academy of Agricultural Sciences

Wuhan

China

and

Key Laboratory of Biology and Genetic

Improvement of Oil Crops

Key Laboratory of Detection for Mycotoxins

Laboratory of Risk Assessment for Oilseeds Products

Ministry of Agriculture

Wuhan

China

Sajid Rauf

Interdisciplinary Research Centre in Biomedical Materials (IRCBM)

Lahore

Pakistan

and

Department of Physics

COMSATS Institute of Information Technology

Lahore

Pakistan

Rizwan Raza

Department of Physics

COMSATS Institute of Information Technology

Lahore

Pakistan

Xianfeng Ren

Oil Crops Research Institute of the Chinese

Academy of Agricultural Sciences

Wuhan

China

and

Key Laboratory of Biology and Genetic

Improvement of Oil Crops

Ministry of Agriculture

Wuhan

China

Alan Richards

Public Analyst and Head of Scientific Services Ltd

Wolverhampton

West Midlands

United Kingdom

Antonio Ruiz-Medina

Department of Physical and Analytical Chemistry

Faculty of Experimental Sciences

University of Jaén

Campus Las Lagunillas

Jaén

Spain

S. Santiago-Felipe

Instituto interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico

Departamento de Química

Universitat Politècnica de València

Valencia

Spain

Aisling Sheehan

Mass Spectrometry Group (MSG)

Department of Physical Sciences

Cork Institute of Technology (CIT)

Cork

Ireland

Luis A. Tortajada-Genaro

Instituto interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico

Departamento de Química

Universitat Politècnica de València

Valencia

Spain

Andrew D. Turner

Centre for Environment Fisheries and Aquaculture Science

Weymouth

Dorset

United Kingdom

Carmen Vale

Department of Pharmacology

Veterinary Faculty

Universidad de Santiago de Compostela

Lugo

Spain

Irina Vetter

Institute for Molecular Bioscience

The University of Queensland

Queensland

Australia

and

School of Pharmacy

The University of Queensland

Queensland

Australia

Valerija Vezočnik

Department of Biology

Biotechnical Faculty

University of Ljubljana

Ljubljana

Slovenia

Mercedes R. Vieytes

Department of Physiology

Veterinary Faculty

Universidad de Santiago de Compostela

Lugo

Spain

Natalia Vilariño

Department of Pharmacology

Veterinary Faculty

Universidad de Santiago de Compostela

Lugo

Spain

Michael J. Walker

Consulting Analytical Scientist

Government Chemist Programme

Laboratory of the Government Chemist

Teddington

Middlesex

United Kingdom

Laura Wood

Chemical Sciences Division

National Institute of Standards and Technology

Gaithersburg, MD

USA

Wang-wah Wong

Hong Kong Accreditation Service

Quality Services Division

Innovation and Technology Commission

Wanchai

Hong Kong

Huali Xie

Oil Crops Research Institute of the Chinese

Academy of Agricultural Sciences

Wuhan

China

and

Key Laboratory of Biology and Genetic

Improvement of Oil Crops

Ministry of Agriculture

Wuhan

China

Lee Yu

Chemical Sciences Division

National Institute of Standards and Technology

Gaithersburg, MD

USA

Qi Zhang

Oil Crops Research Institute of the Chinese

Academy of Agricultural Sciences

Wuhan

China

and

Key Laboratory of Biology and Genetic

Improvement of Oil Crops

Ministry of Agriculture

Wuhan

China

Zhaowei Zhang

Oil Crops Research Institute of the Chinese

Academy of Agricultural Sciences

Wuhan

China

and

Key Laboratory of Detection of Mycotoxins

Ministry of Agriculture

Wuhan

China

Foreword

I regard it an honour and privilege to have been asked to provide a preface for this significant monograph which surveys the recent and current developments in monitoring food toxins and toxicants which compromise human food safety. In the past, many incidents have caused profound impacts on people's health and on businesses in the food manufacture and distribution sectors. The awareness and knowledge of natural and synthetic toxins and toxicants in food has improved significantly in the last two decades via extensive worldwide collaborative research work and monitoring programmes.

The provision of reliable data from the use of advanced and versatile technologies is now a prerequisite for the investigation of the causes of food poisoning incidents, the prevention of such events and for the production of wholesome and safe food. Attention is drawn herein to the importance of quality assurance activities, as for any analytical measurements with potential as forensic evidential use, and to the need for relevant reference materials. The value of strategic risk assessments and data modelling are shown to be keys to the setting up of appropriate limits for toxin concentrations in foods and in food components. Hence this comprehensive review of such work, much of which has been undertaken with worldwide collaborations, to produce data using validated analytical methodologies is considered to be most timely.

The editors are to be congratulated for their selection of relevant and interesting topics. The various section authors have produced a readable, in-depth survey of the current position in the analysis of food toxins and toxicants and also have drawn attention to some important residual problems in certain areas concerning reference materials.

The volume is divided into five main sections: I (Chapters 1–4). Recent developments in analytical technology including sample pre-treatment and food additives; II (Chapters 5–10). Microbial and plant toxins, including plant pyrrolizidine alkaloids; III (Chapters 11–15). Marine toxins in fish and shellfish; IV (Chapters 16–19). Biogenic amines and common food toxicants, such as pesticides and heavy metals; V (Chapters 20–24). Quality assurance and recent developments in regulatory limits for toxins, toxicants and allergens, which includes discussions on laboratory accreditation and reference materials.

Due to the excellent editorial control all the chapters are easy to follow, coherent in layout and are comprehensively referenced, which most helpfully indicate the papers' contents by giving their titles in full.

This up-to-date set of accounts of analytical approaches available and the problems to be encountered in the detection and estimation for a variety of food toxins will be useful to analytical chemists working in academic, manufacturing, distribution and regulatory food control laboratories.

D. Thorburn Burns, DSc, FRSE, MRIA

Professor Emeritus of Analytical Chemistry

The Queen's University of Belfast, and Visiting Research Professor

Institute for Global Food Security

The Queen's University of Belfast

Belfast, UK

Preface

Food toxins and toxicants are widely discussed global issues, and given the threats they pose to human health, they represent one of the most important aspects of analytical chemistry. Over the past two decades, we have experienced various crises due to foodborne toxins causing profound impacts on human health and the food industry. Importantly, in countries where well-developed food management systems with reliable methods of measurement are not established, such outbreaks can lead to potentially life-threatening exposures and resource waste. Our knowledge and awareness of natural toxins and toxicants in foods have improved substantially through extensive research and worldwide networking programs in the field. Thanks to the continuous commitment and cooperation of various organizations, validated analytical methodologies can now detect sub-clinical levels of many food toxins and toxicants using advanced and versatile technologies. A comprehensive monitoring of food toxins and toxicants is a critical prerequisite to substantiate the causes of food poisonings and help prevent similar food catastrophes from taking place. The implementations of quality assurance to food toxin analysis, including the production of reference materials, strategic risk assessment and data modelling for toxin thresholds, are required to validate and strengthen the measurement applications. This book provides an up-to-date and comprehensive overview of the analytical approaches used to detect a range of food toxins. Contributions from more than 70 eminent food toxin scientists across the globe illustrate their expertise and experience to readers. We hope that it can provide useful guidance and instruction to analytical chemists and food scientists, both in industry and academia. In each chapter, the authors aim to provide a concise discussion on the latest methodology currently applied to measure a wide variety of food toxins and toxicants, including a detailed and illustrated overview of different separation and detection approaches used. Finally, we would like to express our sincere thanks to all of our renowned authors who contributed their invaluable time and their expertise to this book.

Yiu-chung Wong

Richard J. Lewis

Section I

Recent Analytical Technology for Food Pathogens and Toxins

1

Omic Analysis of Protein and Peptide Toxins in Food

Pasquale Ferranti,¹,² Chiara Nitride,¹ and Monica Gallo³

¹Department of Agriculture, University of Naples Federico II, Parco Gussone, Naples, Italy

²Institute of Food Science and Technology, National Council of Research, Avellino, Italy

³Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy

1.1 Introduction

The human body is continuously exposed to many substances potentially harmful to health, and therefore defined toxins. Toxins can be of exogenous or endogenous origin. Endogenous toxins are mainly represented by free oxygen radicals that are formed during the normal metabolic processes (cellular respiration, food digestion, excretion) and are waste products of metabolism itself. The level of endogenous toxins may increase in certain situations, that is, prolonged stress, very intense physical activity, large and high caloric meals. On the other side, exogenous toxins enter the body through ingestion, respiration, inhalation or skin adsorption. They may be chemical compound additives contained in many foods, waste products of drugs and heavy metals. These toxins may accumulate insidiously in the body, causing damage at various levels.

Most exogenous toxins derive from contaminated water, beverages and foods. They may contain a wide variety of xenobiotics, either naturally or in consequence of voluntary/involuntary addition. A large class of food toxins is that including those of protein and peptide nature. Well-known examples of toxic proteins are bolesatine and ricin. Bolesatine is a glycoprotein isolated from the mushroom Boletus satanas that causes serious gastroenteritis in humans. This lectin, at very low concentrations, has mitogenic activity on human lymphocytes, while at higher concentrations it inhibits protein synthesis (Ennamany et al. 1998). Ricin, a protein found in the seeds of the plant Ricinus communis, is a potent natural cytotoxin: it may cause cell death by blocking the protein synthesis activity on ribosomes. Because of their toxicity, accurate and sensitive methods for detection of protein toxins are needed. However the large complexity of these molecules (high molecular weight [HMW], presence of subunits, glycosylation, micro-heterogeneity) has made this task very difficult. In the last years, however, the application in food analysis of novel analytical ‘omic’ platforms, mostly based on mass spectrometry technologies, has made possible either qualitative and quantitative proteome or peptidome analysis. McGrath et al. (2011) have developed a sensitive and selective MS-based method to detect and quantify ricin in beverages, such as tap water, milk, apple juice, and orange juice, using isotope dilution mass spectrometry with a linear ion trap operating in product-ion-monitoring mode.

Extensive research in the last years has shown that data generated by the combined omic technologies represent a unique resource for food technology. The focus of this chapter is on foods' peptide and protein toxins and on the most recent development in their methods of analysis. In particular, we shall see that ‘omics’ techniques constitute a potentially comprehensive class of methods for monitoring of food quality, allowing simultaneous qualitative and quantitative toxin measurement in a variety of food categories. The omic approach may provide as a global perspective of knowledge on biological systems, and this also includes foods, their evolution over time, and their impact on human health. Proteomics and metabolomics (along with their derived branches) are already mature - but still evolving - technologies capable of tackling composition and contamination of complex food matrices (Table 1.1). By these approaches, even low amounts of toxins in food samples can be rapidly detected also in the presence of interfering components (Boyer et al. 2011).

Table 1.1 Peptide and protein toxins in foods and their reference analytical methods.

1.2 Methods of Food Toxin Analysis

The impressive increase in food production, processing and packaging amounts in the beginning of the new millennium to meet the food demand for a world population exceeding 9 billion people, has been paralleled by an increase of reported cases of food contamination with toxic substances, resulting in various outbreaks of human poisoning or intoxication.

The issue of food safety has an extremely high relevance for both human health and the food market economy and imposes the urgent need to improve the robustness of the available analytical methods for its assessment. The growing consumer awareness of food safety and quality, the increased demand for legal regulation and adequate labelling, together with the evolution of the deceptive strategies, are fuelling the development of up-to-date procedures of food control that have to be developed, standardized and validated.

Briefly, a food product can be contaminated if one of the food ingredients has been produced with contaminated or diseased organisms or when foodstuffs are incorrectly processed or packed. The presence of potentially harmful ingredients or contaminants has to be assessed by the detection of the target molecule(s) and by monitoring the biomolecular composition of the food.

Over the years, an arsenal of analytical methods, mainly based on morphological/anatomical analysis, organoleptic markers (odour, colour, texture) or chemical testing, have been developed to check for food contaminants. In general, there are three basic detection strategies used for verifying a toxin contamination: i) demonstration of the presence of the toxin itself or of a surrogate marker; ii) indirect demonstration by verifying biological properties of the substance, e.g. agglutination or enzyme-linked immunosorbent assays (ELISA) positivity; iii) demonstration of an altered analytic profile compared to the uncontaminated food. Among these, the strategy of direct characterization of a toxin or of an appropriate surrogate marker is considered as the most reliable. For the above reasons, in the recent years new approaches have been developed to improve food characterization. Determination of a stable isotope ratio, especially on trace elements, provides a stable isotope signature useful to establish a close link between products and their environment.

In the case of pathogen contamination, most recent genomic and transcriptomic approaches specifically target RNA and/or DNA markers to detect foreign organisms in the final products derived from the contamination of the raw ingredients. DNA-based methods consist of the PCR amplification of DNA fragments arising from foreign organisms (Rodríguez et al. 2012). In this way specific DNA sequences can be identified and/or DNA fingerprints can be obtained. It is obvious that these methods are complicated when contaminants arising from several species, that often are taxonomically related, occur simultaneously. Furthermore, DNA-based analytical methods have a limited efficacy to establish the causes of contamination, for instance the use of noncompliant processed raw materials.

Although the detection of DNA markers benefits from having well-defined target analytes and the combined use of database analysis and experimental specificity minimize false positives, techniques relying on the phenotypic expression of specific protein or metabolite markers are less laborious and, in most cases, more reliable. The presence of the micro-organisms is not direct evidence that protein/peptide toxins actually have been produced. Conversely, for their intrinsic stability, toxins can remain in the food for long time after the microorganism itself is no longer detectable.

Monitoring of contaminant toxins generally relies on immunochemical assays. Commercially available tools are lateral flow devices or dipsticks, normally used for rapid screening, and ELISA, that also provide semi-quantitative determination (Singh et al. 2015). Typical limit of detection (LOD) of the tests based on ELISA kits is in the range of 0.1 to 5 ppm. Major concerns of the immunochemical methods consist of the fact that the targeted epitopes are usually not well characterized and that cross-reactivity with matrix components can result in false-positive determinations. The reliability of the detection strongly depends on specificity and stability of the employed antibodies and can be affected by the changes induced on proteins by thermal or other technological treatments. Furthermore, food processing can modify antigenic sequences by altering the antibody reactivity. Many protein targets may be underestimated or even not detected by the most commonly used sandwich ELISA-based tests.

A wide array of chemical/biochemical techniques such as high-performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), mono-dimensional (1D) or two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), and capillary electrophoresis (Figure 1.1) have also proved to be useful in component identification and adulterant detection in foods (Önal, 2007). Even though they have a relevant impact in contaminant detection and are extensively used for routine analysis, these methods are merely descriptive as they compare a profile or a measured value with that expected for a given genuine product and therefore cannot explain the causes of the altered outcome at the molecular level. In other terms, appearance/disappearance/shift of electrophoretic bands or chromatographic peaks compared to a reference food cannot be considered for sure diagnostic of an instance of food contamination, as the variation of the band/peak could be due to normal food variability (false positive). On the other side, a contaminant might be masked by co-migration/co-elution with a normal food constituent (false negative). In the light of this, conventional electrophoretic and chromatographic techniques alone, routinely used in this kind of analysis, in spite of the tremendous improvements in resolving power and sensitivity due to the technological advances, must be considered inadequate when facing the problem of describing the complex composition of natural or altered foods.

Figure depicting the array of chemical/biochemical analytical platforms used in food toxin analysis that includes high-performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), mono-dimensional (1D) or two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), mass spectrometry (MS) techniques, and enzyme-linked immunosorbent assay (ELISA).

Figure 1.1 The array of chemical/biochemical analytical platforms used in food toxin analysis.

Given the limitations of the classically used methods, confirmatory strategies are also required to provide an unambiguous identification of markers of foreign food components. The proteomic approach can overcome these limitations. Proteomics is a branch of the omics technologies, a family of analytical techniques that rely on well-established analytical platforms, in particular on mass spectrometry (MS) techniques (Gallart-Ayala et al. 2015).

1.3 Analytical Techniques

1.3.1 MS-Based Proteomics

MS plays a fundamental role in the study of food (macro)molecules; this revolution has been triggered by the introduction in the 1990s of soft ionization techniques that allow very sensitive HMW molecules, such as electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI). In protein analysis, using these techniques, it is possible to determine accurate mass of proteins and protein complexes, post-translational modifications (PTMs), correspondence of a protein sequence with that encoded in DNA, and de novo sequencing of peptides (Reinders et al. 2004). MS-based proteomics is based on two main experimental approaches: bottom-up and top-down proteomics. Both methods allow us to recognize the proteins present in a biological sample, following two different strategies: the first approach is based on enzymatic protein digestion followed by MS identification of peptides produced by digestion (peptide mass fingerprint [PMF]), while the second approach provides for the recognition of the protein based solely on the molecular weight and by fragmentation of the undigested protein (Aebersold and Goodlett, 2001).

1.3.2 Bottom-up and Top-down Proteomics

Bottom-up proteomics allows the identification of protein based on information derived from the mass or the amino acid sequence of the peptides generated following digestion of the protein with an appropriate agent. The approach is based on the assumption that proteins which generate the same peptide map are characterized by the same primary structure and thus coincide; with this approach, it is possible to compare the sequence of a protein with that of a particular gene, to verify the sequence of synthetic proteins, and to detect PTMs. Protocols for the digestion of proteins separated using gel electrophoresis (in-gel digestion) have also been standardized (Dass, 2007). The determination of the molecular weight and amino acid sequence of the peptides produced by the digestion is carried out by MS/MS sequencing using LC-ESI-MS or MALDI-MS (Thiede et al. 2005). Bottom-up proteomics, therefore, allows fast and simple identification of a protein. However, a significant limitation of this approach is the quality of the results, which depends greatly on the purity of the protein treated. For this reasons, MS analysis is preceded and combined with appropriate chromatographic or electrophoretic techniques. Electrophoretic detection can be aided by use of appropriate immunochemical protocols. One example is the search for allergens which can be present only in trace amounts or be unexpected. Exemplary is the case of the discovery of a novel hazelnut allergen, which has been detected and characterized by combined immunological, electrophoretic, and MS/MS de novo sequencing (Nitride et al. 2013).

The top-down experimental approach for proteome analysis consists of the analysis of intact proteins (Figure 1.2). Protein identity is obtained by ESI or MALDI MW measurement and can be confirmed by MS/MS fragmentation of the intact protein. In top-down proteomics, a basic issue is instrumental resolution, and therefore the recent improvements in the technology of mass analysers have been important in its development. Fourier transform-ion cyclotron resonance (FT-ICR) and Orbitrap instruments are providing the higher resolution. Also, hybrid instruments (such as Q-TOF hybrid between quadrupole (Q) and time of flight mass analyser (TOF) that ensure a high resolving power and a fast scanning speed), which are easier to use, are able to provide adequate accuracy and resolution.

Figure depicting the flowchart of proteomic strategies in food toxin analysis, where the top-down experimental approach for proteome analysis consists of the analysis of intact proteins. The bottom-up approach is more profitable. The protein is typically subjected to 1D- or 2D-PAGE separation, the protein band of interest degraded enzymatically, and the resulting peptide mixture characterized by MS/MS.

Figure 1.2 The flowchart of proteomic strategies in food toxin analysis.

With the top-down approach, PTMs can be revealed for only relatively low molecular weight (LMW) proteins (10–20 kDa). Furthermore, when used for proteins contained in a complex sample, or in order to identify proteins present in very low concentration, a preliminary concentration or purification step is generally necessary. In fact, analysis of complex mixtures has two drawbacks: the first concerns the phenomenon of ion suppression due to the different ionization yield of proteins; the second is related to the limited dynamic range of MS, in particular MALDI, which does not allow obtaining valid signals for proteins present at low concentrations (Zhou et al. 2012).

In order to overcome the intrinsic limitations of both approaches, in recent years the intermediate ‘middle-down proteomics’ has been introduced, giving rise to the peptidomic branch of the omic family. It is based on limited peptide bond breakdown in order to obtain peptides with a greater number of amino acids (>20) compared to those produced in the bottom-up proteomics. This step is followed by determination of the amino acid sequence, which can provide information on protein isoforms and on PTMs. Moreover, in contrast to the top-down proteomics, which involves only the analysis of intact proteins, peptides considered in middle-down proteomics are easier to handle, ionize and fragment. These peptides, having a molecular weight characteristic of about 5–10 kDa, can be generated through an enzymatic or chemical digestion of the protein. The last step, as with all other approaches seen so far, is the comparison of experimental data with those of the literature for the identification of peptides/proteins.

Food matrices are extremely complex because they contain a large number of chemical species. This is actually the case of most food toxins, many of which are present in a concentration ranges (parts per million to parts per billion) hard to reveal with the routine analytical techniques, while others interfere with the analysis leading to unsatisfactory results. Furthermore, often food samples are also subject to rapid degradation and need to be stored under conditions of low temperature and in suitable packaging or containers that allow maintaining it unaltered. Also, a food sample can be altered in a more or less marked form by the reactants used or by the various treatments performed. This multiplies incredibly the complexity of a real food. For these reasons, the bottom-up proteomics approach is a valid and simple for single proteins, while it is quite difficult to obtain reliable results from matrices containing a greater number of species, which the usual occurrence in food analysis. This technique, therefore, requires that the food sample is effectively purified by chromatographic or electrophoretic methods; this inevitably causes an increase in the complexity and time required to perform the analysis. The top-down proteomics approach is substantially faster for the study of a complex sample, but the results are not as reliable, based solely on the molecular weight. Therefore, more data is required, such as those from MS/MS fragmentation, which, however, are difficult to obtain from an intact protein. Database screening can be helpful in the identification of the metabolite (i.e. a bacterial or a fungal peptide toxin), but only a limited number of sequences are already recorded at present. Databases are constantly being updated and enriched, and with time they will become more and more complete and reliable.

1.3.3 Data Interpretation and Database Searching

The final step of food proteome analysis of comparison of MS and MS/MS data obtained with those contained in the protein databases. The search can be carried out on a wide number of databases, many of which relate proteomic data with those from genomics. The information that can be found goes well beyond the simple amino acid sequence and includes links to other databases, references, information on the function of the protein identified, possible PTMs and mutations that may be encountered. These databases are handled by independent research groups and are available free of charge via the Internet; they contain complete lists of proteins and nucleotides and are continuously updated.

Databases, together with statistical systems with thematic character, provide a comprehensive and accurate vision of the phenomenon under investigation. Each database is accompanied by various information (methodologies, classifications, definitions) on the subject. Foodomics is based on molecular characterization by metabolomic and proteomic approach, therefore, some databases are www.expasy.ch; GeneBank, EBI, GEO and also BioPEP, PepBank, EROP or APD.

The choice of the most appropriate database essentially depends on the characteristics of the research that is taking place; are typically preferred those containing a higher number of sequences, a small number of errors and repetitions, and a proper amount of bibliographic references (Peri et al. 2004). A non-exhaustive list of relevant sites on foodomics and its applications includes www.foodomics.eu, http://chancefood.eu, www.foodomics.org, www.allergome.com, www.foodchem.it, www.safefoods.nl.

1.4 Food Protein and Peptide Toxins from Micro-organisms

From an analytical point of view, protein and peptide toxins can be arbitrarily divided into two main classes: LMW toxins (peptide toxins, with MW <6–8 kDa) and HMW toxins (protein toxins, MW >6–8 kDa). The structural and quantitative determination of these two classes requires a very different proteomic approach.

HMW toxins are produced by many organisms and may possibly contaminate any kind of vegetal or animal food. In general, after a series of more or less complex purification steps, the intact protein can be analysed by a top-down approach, as described above. This strategy is, however, not applicable for very HMW proteins (>20 kDa) and generally does not allow quantitative measurements.

In these cases, the bottom-up approach is more profitable (Figure 1.2). The protein is typically subjected to 1D- or 2D-PAGE separation, the protein band of interest degraded enzymatically, and the resulting peptide mixture characterized by MS/MS (gel-based proteomics). As an alternative, the protein mixture can be cleaved by action of an appropriate enzymatic or chemical agent and analysed by HPLC-ESI-MS/MS (gel-free or shotgun proteomics). This last option is generally preferred as straightforward and less time and labour intensive. Gel-based approach is however very useful, if not irreplaceable, in the case of very complex samples, where precious information can be achieved by gel separation and specific in-gel detection procedures, for instance, those based on immunochemical assays useful in the location of specific toxic compounds. One example is the search for allergens which can be present only in trace amounts or even unexpected in a given food.

In many cases, for sensitive detection and quantification of harmful protein components (allergens, toxins from pathogens, anti-nutritionals), monitoring of the whole protein is surrogated by analysis of short peptides contained within the protein sequence (proteotypic peptides). These peptides are more easily detected than the parent protein. The targeted monitoring of mass and transitions of selected ‘proteotypic peptides’, overcomes the interference due the presence of a large number of dominant components. In absolute quantification strategy (AQUA), once proteotypic peptides are chosen, it is possible to synthetize peptides which are isotopically labelled to one or more amino acid positions in order to shift molecular mass, without influencing retention time and ionization properties. Protein samples after tryptic hydrolysis are spiked with known amounts of standard AQUA peptides and both native and surrogate peptides are monitored by LC-MS operating in multiple reaction monitoring (MRM) mode. The peptide amount is determined by the ratio of the ion intensities of AQUA peptide and its native cognate (Brun et al. 2009; Kretschy et al. 2012).

LMW toxins include either peptides of ribosomial synthesis, often generated through maturation of longer precursors (examples are the microbial curvacin, sakacin, nisin and many other classes) or non-ribosomial peptides (NRP). These last are produced by a variety of micro-organisms including bacteria, cyanobacteria (blue-green algae), yeasts and fungi. They exhibit a multitude of structures (cyclic, branched, non-α-amide bonds, lipid or glycosyl moieties) but their peculiarity is the occurrence of ‘unusual’ amino acids (not belonging to the 20 commonly found in proteins) also belonging to the stereochemical (D) series. These unusual structural features make their characterization and quantitative analysis a very complex task, which can be faced only by a combination of peptidomic strategies based on the integrated use of high-performance chromatographic techniques with identification techniques based on MS/MS, nuclear magnetic resonance (NMR), spectroscopic and biological platforms. The possible occurrence in contaminated foods and water reservoirs makes their sometimes extreme toxicity – typical is the case of the cyanobacterial hepatotoxic microcystins – and makes the development of accurate and sensitive detection strategies a priority to ensure food safety.

1.4.1 Bacterial Toxins

Traditional means of controlling microbial spoilage and safety hazards in foods include freezing, blanching, sterilisation, curing and use of preservatives. However, the developing trend of consumer's demand for ‘naturalness’, as indicated by the strong growth in sales of organic and chilled food products, has resulted in a move towards milder food preservation techniques. This raises new challenges for the food industry. Proteomic approaches have been directed to the development of methods for bacterial profiling through MALDI–TOF–MS and ESI–MS/MS fingerprinting of bacterial proteins in order to distinguish among different species and, in some cases, among strains. Through this profiling method, it was possible to conduct fast and sensitive detection of pathogens and spoilage microorganisms affecting food quality and safety during processing and storage.

The development and use of appropriate analytical tools that allow comprehensive and robust metabolic analyses for various chemical structures in a reasonable time is key to understanding the impact of the gut microbiome on the host's overall metabolic signature. Metabonomic/metabolomic analyses involve the combination of high-density spectral data generated from biofluids and tissues in combination with a computer-based pattern recognition strategy. Currently, several analytical techniques, such as GC-MS (Fiehn et al. 2000; Hall et al. 2002; Kell, 2004; Jonsson et al. 2004; Clish et al. 2004). HPLC-MS and LC-NMR-MS are used to generate spectral profiles from which information that pertains to physiology and latent disease can be extracted.

Each technology has its specific applications, as well as its own advantages and drawbacks. For example, the inherent sensitivity of GC-MS is useful in the detection of low concentrations of volatile micro-metabolites to trace the presence and identity of food spoilage micro-organisms (Argyri et al. 2015). Differential ionization and ion suppression of metabolites in the case of LC-MS can limit the sensitivity and practicality of this approach. Recently, ultra-high-performance liquid chromatography (UPLC) has become available, with greatly improved resolution characteristics and capabilities (Plumb et al. 2004). Using MS detection, UPLC-MS allows the separation of unprecedented numbers of bacterial metabolites in food samples and the possible production of harmful peptide toxins.

Although 1H NMR spectroscopy is less sensitive, it requires little or no sample pre-treatment. Furthermore, it is now possible to statistically reconstruct latent biomarker information from stochastically varying positional noise from a series of individual spectra with extensive peak overlap (Cloarec et al. 2005). The aromatic region of a typical 1H NMR spectrum of urine provides a particularly clear window for visualization of the metabolic signature of microbial products, dietary metabolites, parasite-related metabolites and many drug metabolites. Whereas the NMR approach to monitoring microbial presence and activity has been directed at generating a global view of the entire microbiome, several LC-MS and GC-MS studies have been directed at identifying targeted microbial metabolites. For example, certain gut micro-organisms such as Clostridium tetani and yeasts such as Candida albicans have been associated with the onset or exacerbation of autism (Bolte, 2002; Martirosian, 2004). GC-MS methods have been developed to detect metabolites of a C. tetani-related tyrosine derivative, which has been shown to be elevated in the urine of children with various conduct disorders (Bolte, 1998).

More accurate description of microorganisms contaminating food have been achieved by integration of proteomics with peptidomics and metabolomic methodologies able to provide either structural or quantitative identification of specific metabolites produced by the various spoilage micro-organisms. It can be foreseen that these methods are being integrated to design sensitive sensors on a microchip surface for automated detection. The omics technologies can also help scientists to derive better understanding of the life cycles of bacteria. Defining the mode of action of food-borne bacteria and the mechanisms that confer ‘stress resistance’ should enable more rational design of food preservation techniques. In addition, this information can also be used to pinpoint areas of the food chain that are most susceptible to microbial contamination.

In this respect, the analysis of pathogenic micro-organisms deserves particular caution, as the risks associated with their contamination are not limited to their living presence and capacity of infectivity. They can generally release protein/peptide toxins able to survive for long time, even in foods after bacterial cell contamination has been removed, as happens for many of the microbes which cause food-borne diseases, including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Clostridium botulinum A and various Salmonella species. All these pathogenic bacteria excrete a variety of virulence factors into extracellular medium and to the cell surface which have essential roles in the colonization and insurrection of the host cells and thus reflect the degree of bacterial pathogenicity. These toxins, being heat-stable and resistant to proteases, can be a danger to the consumer health.

Bacterial toxins can be divided into two large groups: endotoxins and exotoxins. Endotoxins are structural components of Gram-negative bacteria; they are of polysaccharide nature and have very similar profiles of action. The most representative is lipid A (the inner layer of lipopolysaccharide (LPS), which in turn constitutes the outer portion of the membrane that covers the cell wall). These can be released in case of cell death.

Exotoxins, including both LMW and HMW proteins, are not structural components, but are released by the bacterium; they are produced by both the Gram-positive and Gram-negative bacteria and are typical to each bacterium. They are found in cell extracts and in the culture medium, and unlike endotoxin, are proteins. Their main characteristic is that they have very different profiles of action. Soluble proteins are sometimes glycosylated. They are divided into three groups, depending on the type of action carried out: cytotoxins, which enzymatically attack and kill host cells; neurotoxins, which interfere with the normal transmission of nerve impulses; enterotoxins, which through an abnormal stimulation are responsible for the abnormal functioning of the cells of the gastrointestinal tract.

As already stated, the development of ESI and MALDI-MS instruments have represented a great step ahead in the analysis of bacterial proteins. Further to providing high-sensitivity and specificity determination of microbial protein patterns (Sauer and Kliem, 2010), they constitute the basis of high-throughput platforms for the identification and confirmation of pathogen toxins.

E. coli is a Gram-negative bacterium commonly found in the lower intestine of warm-blooded organisms. E. coli is not always confined to the intestine, and its ability to survive for brief periods outside the body makes it an ideal indicator organism to test environmental and food samples for faecal contamination (Feng et al. 2002). Most E. coli strains are harmless, but some of them can cause gastroenteritis, urinary tract infections and neonatal meningitis (Vogt and Dippold, 2005). Food poisoning caused by E. coli is usually caused by eating unwashed vegetables or undercooked meat (Sheen and Hwang, 2010). Severity of illness varies considerably; it can be lethal, particularly to young children, the elderly or the immune-compromised, but is more often mild. Toxigenic E. coli strains that cause infections in human and domestic animals have been classified into different categories, including enterotoxigenic E. coli (ETEC) (Nataro and Kaper, 1998), which is the most important pathogen of diarrhoea in infants, children, and adults, accounting for 280 million episodes and more than 400,000 deaths annually. Currently, diarrheal disease remains a leading global health problem (WHO, 2005). ETEC is endemic in many developing countries and is frequently encountered by tourists, members of the military or other visitors (Aranda-Michel and Gianella, 1999; Coster et al. 2007). In addition to traveller's diarrhoea, ETEC can cause disease symptoms clinically indistinguishable from cholera caused by Vibrio cholerae (Vicente et al. 2005). These diseases can be extremely debilitating and may be fatal in the absence of treatment (Spangler, 1992). ETEC produces two types of enterotoxins that cause diseases in man and various domestic animals: low weight, heat-stable enterotoxin (HST), and high weight, heat-labile enterotoxin (LT). HST is considered an important cause of diarrhoea in pigs but is rarely associated with humans (Handl and Flock, 1992). LT is the major virulent factor of ETEC (Holmgren and Svennerholm, 1992). The severe losses of water and electrolytes which occur during infection appear to be caused by this toxin, whose action is mediated by stimulation of adenylate cyclase activity in the epithelial cells of the small intestine. LT is also very similar in sequence and structure to cholera toxin (Sharp et al. 1973). Both toxins consist of a B pentamer with five identical B (LTB) subunits and a catalytic A (LTA) subunit (Mudrak and Kuehn, 2010).

Heat treatment of the toxin breaks down the pentameric LTB ring into monomers and releases LTA. The enzymatically active A subunit is responsible for the toxicity, and the B subunit binds the receptor and facilitates the entry of the A domain into cells of the intestinal epithelium (Mudrak and Kuehn, 2010). Importantly, the results of numerous investigations have shown that LTB could be a promising candidate to produce a vaccine antigen against LT-producing ETEC (Svennerholm and Tobias, 2008). The development of an effective vaccine for ETEC would have a significant global impact on reducing morbidity due to diarrheal episodes in both developed and developing countries. For this reason, it would be important to have a sensitive, specific, fast and simple method for the characterization of LTB. Most of rapid in vitro tests for the recognition of E. coli enteropathogens available are ELISA-based methods and, although they are rapid and sensitive, are susceptible to produce false positives or false negatives. Where positives are observed, a confirmatory method that can make a direct measurement of the analyte is needed.

For these reasons, the possibility of characterizing the LT enterotoxin of ETEC using omic approaches has been investigated (Sospedra et al. 2012). The B subunit from recombinant E. coli was characterized by LC/ESI-MS, MALDI-TOF analysis. Confirmatory analysis was carried out by detection of most of the tryptic fragments of the B subunit by MALDI-TOF-MS, obtaining total coverage of the protein sequence. Possible bio-variations in the toxin (disulphide bonds, O-GlcNAc-1-phosphorylation) among strains used for vaccine production can easily be determined by MS analysis.

For the exploration of virulence factors expressed in the secreted proteome fraction, in a very recent study different S. aureus strains were analysed using gel-based bottom–up proteomic approach. A still more complex situation is expected for analysis of foods, mostly constituted by a highly complex matrix of proteins, lipids, carbohydrates and many other molecular species which interfere with detection of the predictable toxin amounts (in the order of parts per billion). For this reason, the combination of MS methodologies with advanced immunochemical, chromatographic and electrophoretic isolation procedures must be applied. One such study has been carried out to define the toxin contamination levels of two ripened Protected Designation of Origin (PDO) status Italian cheeses, Grana Padano and Pecorino Romano. A procedure combining proteomic approach with immunochemical, chromatographic and electrophoretic techniques and MS/MS analyses was developed to monitor production and levels of enterotoxin A (SEA) and B (SEB) of S. aureus and Shiga-like toxins produced by E. coli O157:H7. By producing cheese samples using milk purposely contaminated with bacteria, it was possible to monitor 10–100 ppb contamination level, and analysis of randomly collected market samples allowed excluding toxin contamination in the two cheese types.

Very recently, UPLC-ESI-MS/MS has allowed high-sensitivity quantification of SEA and SEB in complex matrices such as milk and shrimp (Muratovic et al. 2015). The analysis was based on the monitoring of a tryptic peptide from each of the toxins (proteotypic peptide) as the target analyte, together with the corresponding ¹³C-labeled synthetic internal standard peptide. Comparison to the reference ELISA method showed good coherence for the two methods, but the major advantage of the developed method is that it is multiplexed and allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label- and antibody-free approach. Therefore, this method might represent an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

1.4.2 Fungal NRP Toxins

The various classes of fungal peptides, depsipeptides, and peptaibiotics have been comprehensively reviewed recently (Degenkolb et al. 2008). Fungal and bacterial NRP composition was compared based on an analysis of the Norine database (Caboche et al. 2008; Caboche et al. 2009) that catalogues more than 1000 peptides representing more than 10 000 monomer occurrences and >500 different monomer types (Caboche et al. 2010).

Most of these peptides are not normally found in foods, but occasional outbreaks have been reported. More importantly, their biochemical properties make them promising candidates for the development of novel tools for pest control in food packaging, for instance. Those most intensively investigated properties are formation of pores in bilayer lipid membranes as well as antibacterial, antifungal, occasionally antiviral, insecticidal, and antiparasitic activities. These new research fields have stimulated the investigation on the methods for their analytical research in biological samples. Recently, the terms ‘peptaibiome’ and ‘peptaibiomics’ have been proposed (Krause et al. 2006) to describe – in analogy to the proteome and proteomics – the approach to analyse the entirety and dynamics of peptaibiotics produced by a fungal strain under defined conditions. For instance, by a rapid and selective solid-phase extraction (SPE) method, the peptaibiotic-containing fraction was selectively absorbed, and the eluate was subsequently analysed by HPLC coupled to ESI-IT-MS/MS. The advantage of IT-MS for screening is the generation of a genealogy of the diagnostic product or daughter ions. For instance, every MS2 product ion generated by collision with inert gases such as He and Ar, or high-purity N2, can be fragmented separately, thus generating MS3 product ions. The further fragmentation of these ions will result in MS4 product ions, and so on (Degenkolb et al. 2003).

Some Pseudomonas species are able to produce cyclic NRPs containing non-protein amino acids and linked to a long lipid chain, called lipodepsipeptides (LDPs). One group is formed by nonapeptide lactones acylated by a long-chain, 3-hydroxy fatty acid and includes the syringomycins (SRs) (Ballio et al. 1988; Segre et al. 1989; Fukuchi et al. 1990a); the syringotoxins (STs) (Ballio et al. 1990; Fukuchi et al. 1990b, 1994a; Flamand et al. 1996), the syringostatins (Fukuchi et al. 1990c, 1992), and the pseudomycins (Harrison et al. 1991; Ballio et al. 1994b; Coiro et al. 1998). The other group contains lipopeptides formed by a long and highly hydrophobic peptide chain, C-terminated by a polar lactonized penta- or octa-peptide moiety, this group includes tolaasins (Nutkins et al. 1991), syringopeptins (SPs) (Ballio et al. 1991, 1995; Isogai et al. 1995), fuscopeptins (FPs) (Ballio et al. 1996; Baré et al. 1999), and corpeptins (Emanuele et al. 1998). These molecules are able to alter the permeability of biological membranes, thereby exerting a strong antibiotic action. Many producing strains actually have a double meaning: for some plant species they are pathogenic, but for others act as biocontrol agents protecting against pathogens. MS procedures (Monti et al. 2001) have been set up for screening LDP-producing bacterial strains and for identifying and assessing individual LDPs. These could be of practical value in view of potential applications, e.g. biocontrol of post-harvest fungal diseases (Gallo et al. 2000; Monti et al. 2001).

1.4.3 Other Fungal Toxins and LMW Mycotoxins

The term mycotoxin is usually reserved for a heterogeneous group of LMW toxins produced by fungi that readily colonize crops and forages. Mycotoxin contamination is largely influenced by climatic and geographical conditions, from farming practices and conservation, and the type of substrate concerned, since some products are more susceptible than others to fungal growth. Mycotoxins are developed on plants pre-harvest (on field contamination), in the plant foodstuff post-harvest, during storage (in warehouses, silos, etc.), processing and transport. They are produced by the secondary metabolism of fungi and highly toxic for animal and humans. Due to their detrimental action on cellular functions, they exert nephrotoxic (ochratoxins), hepatotoxic (aflatoxins), immunotoxicity (aflatoxins, ochratoxins), mutagenic (aflatoxins), teratogenic (ochratoxins) and carcinogenic (aflatoxins, ochratoxins, fumonisin) actions. They are thermostable and are not fully eliminated by normal cooking procedures or by the different treatments that foodstuffs normally undergo during their preparation. Therefore, mycotoxins or their active derivatives can persist after the death of the fungus and be present even in apparently non-contaminated materials.

Known mycotoxin structures are numerous, although most have only been characterized in laboratory studies and have a very low probability of being found as natural contaminants. In fact, only a few have so far been found in foodstuffs, and only very few of them have been conclusively associated with mycotoxicosis. Because of the toxicity of mycotoxins, it is necessary to have reliable and accurate screening and confirm methods, as well as an accurate quantification at very low concentration levels. For this goal, GC-MS and LC-MS are the most suitable analytical approaches. Due to the scarce volatility of mycotoxins, GC-MS determination is possible after a suitable derivatisation step (Schollenberger et al. 2008). In consequence, LC-based methods, using several analysers such as single Q (Silva et al. 2009), TOF (Tanaka et al. 2006), and mainly MS/MS (Spanjer et al. 2008) have been explored.

A sensitive method was developed for simultaneous detection of 12 mycotoxins in corn, nuts, biscuits and breakfast cereals based on a single extraction step followed by UHPLC–MS/MS (Pastor-Montoro et al. 2007). The selectivity of the MS/MS detection allowed the elimination of time-consuming cleanup steps (Frenich et al. 2009).

In a recent work, Malachová et al. (2014) evaluated the performance of a LC-MS/MS multi-analyte method for mycotoxins and other fungal as well as bacterial metabolites. Furthermore, a validation procedure has been developed and applied to four model matrices. Based on these results it was possible to conclude that quantitative determination of mycotoxins by LC-MS/MS based on a ‘dilute and shoot’ approach was also feasible in case of complex matrices (Malachová et al. 2014).

With the exception of neoefrapeptins, peptaibiotics have only been reported and sequenced in three families, Hypocreaceae, Clavicipitaceae, and Bionectriaceae of the order Hypocreales. Reports on peptaibiotics from basidiomycetes are most likely due to infection of an asco- or basidiomycetes fruiting body with mycoparasites might not be visible (Degenkolb et al. 2007).

However, it is well known that poisonous mushrooms produce toxins which not only vary along species but also within the same species, according to the periods of the year. These toxic substances are essentially of four types: cytotoxic toxins, neurotoxic toxins, toxins irritating the gastrointestinal system, toxins that cause poisoning if associated with alcohol consumption. Severe intoxication or death, caused by the consumption of mushrooms collected by inexperienced people, is a great concern for public health.

Toxins produced by poisonous fungi are produced by Amanita phalloides, Amanita virosa and Amanita verna and are fatal in many cases. These substances (amanitins and phalloidins) act on cellular enzymes; their danger lies in the fact that often recognition of the poisoning is not immediate and therefore appropriate treatments are late or antidotes are not available. For these reasons, screening methods for toxins possibly present either in foods or in suspected poisoned subjects would be of great importance. In contrast, although the structural details of the main toxins have been defined many years ago, only recently the availability of peptidomics MS-based tools has stimulated the development of analytical procedures. Ahmed et al. (2010) have reported a procedure for simultaneous analysis of α-amanitin, β-amanitin, and phalloidin in mushrooms by LC-ESI-TOF-MS. This method can be applied for the measurement of the Amanita toxins in human specimens, such as urine and blood of intoxicated subjects (Ahmed et al. 2010).

1.4.4 Marine and Cyanobacterial Biotoxins

Microalgae, together with the simplest animal organisms, form the plankton dispersed in seawater. Shellfish and fish feed themselves with plankton by filtering huge volumes of sea water. Normally, the amount of filtered water varies according to the season, the temperature of the water and to the amount of insolation. Nowadays, because of sea eutrophication and global climate warming, algae of tropical origin have become part of the plankton present in every sea: they are potentially toxic because they can produce toxic compounds which accumulate in the tissues (pulp) of shellfish. The increase in trade volumes and naval traffic and the opening of new waterways have altered the biological balance of the sea. Therefore, the issues linked to the presence of algal biotoxins are acquiring great importance for food health and hygiene. Multiple in vivo and in vitro bioassays have been coupled to the purification process to search for the biological activity of interest.

Recently, reliable methods, MS/MS-based, have been proposed for surveillance purposes both to elucidate toxin algal fragmentation pathways and for quantitative analyses at trace levels. Novel approaches include the use of a μHPLC–ESI-MS/MS system for the analysis of toxins in seafood and the evaluation of the matrix effect in shellfish extracts on accurate quantitation of toxins. As for GC-MS, research has been focused on the development of new and simple sample treatment procedures like diphasic dialysis extraction followed by GC-MS detection and on the use of ion trap MS (IT-MS) with EI and CI modes for screening purposes. However, undoubtedly methods involving MS techniques such as biomolecular interaction analysis MS (BIA-MS) represent the novelty in MS determination of toxins in foods, this approach combining rapidity, sensitivity and selectivity in detection of low toxin amounts in foodstuffs. A recent exhaustive review collects MS/MS and MSn behaviours of the major marine toxins (Pengyuan, 2014).

Cyanobacterial peptide toxins relevant to food analysis are those from lake and river waters. Organisms from genera such as Mycrocystis, Anabaena, Planthotrix and many others may produce very potent toxins also contaminating water, fish and seafood. Among contaminants along the food chain, algal toxins produced by cyanobacteria in water reservoirs are a special concern. The occurrence of toxin-producing blooms in freshwaters has been implicated in several animal and human poisoning outbreaks worldwide. The analysis of algal toxins is therefore of growing interest for water surveillance authorities. MALDI-TOF-MS and ESI-Q-TOF-MS/MS have been useful for fast screening and quantitative determination, as well as for structural characterization of known and unknown toxin structures (untargeted analysis) (Ferranti et al. 2008, 2009, 2011; Gallo et al. 2009). MS analysis has then been extended to the setup of a method for quantitative determination of microcystins in food integrators for human and animal nutrition, as well as to the analysis of biological fluids in patients to diagnose food toxin poisoning. In this way, qualitative and quantitative information can be obtained to monitor rivers, lakes and water reservoirs and to improve current knowledge on algal contamination in the food, feed and drinking water chain, and also to detect protein marker to discover the use of unsafe water in fish and seafood aquaculture.

Also CE-MS has been proposed as a methodology to study proteins (phycobiliproteins) from the edible microalga like Spirulina platensis, which is a natural source of functional ingredients. Reliable and reproducible CE-MS analysis of the main proteins found in this microalga have been reported (Simò et al. 2005). It was demonstrated that both analysers, CE-IT-MS and CE-TOF-MS, allowed the characterization of these proteins with coherent results.

On the other side, several marine organisms, for instance Crustacea and Mollusca, are an immense potential source of new biologically active compounds. These compounds are unique because the aqueous environment requires a high demand of specific and potent bioactive molecules. Diverse peptides with a wide range of biological activities have been discovered, including antimicrobial, antitumor, and antiviral activities and toxins among others. Purification techniques used to isolate these peptides include classical chromatographic methods such as gel filtration, ion exchange and reverse-phase HPLC.

1.5 Phytotoxins

A number of different plant-derived toxic substances, known as phytotoxins, may be present in fruits and vegetables that are part of human diet. The toxic compound can be distributed throughout the plant or accumulated only in certain parts (leaves, drupes, crops, skin). Knowledge of substances possibly occurring in these plants is of great importance for the prevention of many intoxications. It is therefore necessary to know the chemical nature, mechanism of action and the toxic effects of these substances, the raw materials that are present, and their concentrations because the toxicological risk is most often due to the effects of long-term consumption of contaminated foods.

Vegetal toxins include alkaloids, glycosides, saponins, resin, oxalates, tannins, phenolic compounds and amines. Amino acids, peptides and protein toxins constitute one of the most heterogeneous classes. Analytical omic-based methods have provided important contributions either in the structural definition of novel vegetal toxins or in the development of reliable procedures for monitoring their presence in vegetal-derived foods and their persistence during food processing. In this regard, it must be underlined that the technological processes of production and sanitization, conservation and cooking can neutralize, reduce or eliminate toxins but can also increase risk when incorrectly applied.

This is the case for phytohaemagglutinins (PHAs), members of the lectin family of lectins, able to interfere in the absorption of amino acids, lipids, carbohydrates and vitamins of the diet (anti-nutritional activity), to cause red cell agglutination, and to inhibit thyroid function by interfering with the metabolism of iodine. They are present in soy, peanut, bean, lentil, pea, castor bean, chickpea and grass pea. PHAs, however, are thermolabile and are easily eliminated by the usual conditions used either for home or restaurant cooking or for industrial production of dried powders and soups, ready-to-eat meals and salads. Therefore, the toxic effects occur when the food is not correctly cooked or subjected to other appropriate thermal treatments able to deactivate these molecules (Bora, 2014). Bean-derived alpha-amylase inhibitor preparations (Phaseolus vulgaris) are specifically used as a food supplement to decrease starch digestion in subjects affected by chronic diabetes and obesity (starch-blockers, weight-blockers). Inaccurate heat treatments of legumes have caused severe poisoning outbreaks over the years. PHAs present in uncooked legumes can cause damage to the intestinal mucosa and inhibit digestion and absorption of nutrients. The best-studied plant lectins are wheat germ agglutinin and red bean PHA.

For all these reasons, MS-based strategies have been applied and optimized to set up proteomic