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1631172980 aflatoxin


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food sources and toxicological effects

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1631172980 aflatoxin

  1. 1. FOOD SCIENCE AND TECHNOLOGY AFLATOXINS FOOD SOURCES, OCCURRENCE AND TOXICOLOGICAL EFFECTS No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.
  2. 2. FOOD SCIENCE AND TECHNOLOGY Additional books in this series can be found on Nova‘s website under the Series tab. Additional e-books in this series can be found on Nova‘s website under the e-book tab.
  4. 4. Copyright © 2014 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. Library of Congress Cataloging-in-Publication Data Published by Nova Science Publishers, Inc. † New York ISBN: (eBook)
  5. 5. CONTENTS Preface vii Chapter 1 Bio-Prevalence, Determination and Reduction of Aflatoxin B1 in Cereals 1 Jelka Pleadin, Ksenija Markov, Jadranka Frece, Ana Vulić and Nina Perši Chapter 2 Aflatoxin Occurrence 35 Elham Esmaeilishirazifard and Tajalli Keshavarz Chapter 3 Aflatoxins in Food and Feed: Contamination Exposure, Toxicology and Control 63 Marta Herrera, Antonio Herrera and Agustín Ariño Chapter 4 Immunosuppressive Actions of Aflatoxin and Its Role in Disease Susceptibility 91 Johanna C. Bruneau, Orla Hayden, Christine E. Loscher and Richard O’Kennedy Chapter 5 Aflatoxins Hazards and Regulations Impacts on Brazil Nuts Trade 107 Otniel Freita-Silva, Renata Galhardo Borguini and Armando Venâncio
  6. 6. Contentsvi Chapter 6 Polymorphisms of DNA Repair Genes and Toxicological Effects of Aflatoxin B1 Exposure 125 Xi-Dai Long, Jin-Guang Yao, Qian Yang, Cen-Han Huang, Pinhu Liao, Le-Gen Nong, Yu-Jin Tang, Xiao-Ying Huang, Chao Wang, Xue-Ming Wu, Bing-Chen Huang, Fu-Zhi Ban, Li-Xia Zeng, Yun Ma, Bo Zhai, Jian-Jun Zhang, Feng Xue, Cai-Xia Lu and Qiang Xia Chapter 7 Incidence of Aspergillus Section Flavi and Interrelated Mycoflora in Peanut Agroecosystems in Argentina 157 María Alejandra Passone, Andrea Nesci, Analía Montemarani and Miriam Etcheverry Chapter 8 Toxicological Effects, Risk Assessment and Legislation for Aflatoxins 191 Marina Goumenou, Dimosthenis Axiotis, Marilena Trantallidi, Dionysios Vynias, Ioannis Tsakiris, Athanasios Alegakis, Josef Dumanov and Aristidis Tsatsakis Chapter 9 Food Sources and Occurrence of Aflatoxins: The Experience in Greece 233 Ioannis N. Tsakiris, Elisavet Maria Renieri, Maria Vlachou, Eleftheria Theodoropoulou, Marina Goumenou and Aristides M. Tsatsakis Chapter 10 Aflatoxins As Serious Threats to Economy and Health 259 Lipika Sharma, Bhawana Srivastava, Shelly Rana, Anand Sagar and N. K. Dubey Index 287
  7. 7. PREFACE Progress in understanding the biology of Aspergillus has greatly improved with the new techniques in genome sequencing and the developed molecular tools that enable rapid genetic analysis of individual genes. Particularly, the genetics of aflatoxin synthesis is regarded as a model to gain insight into fungal secondary metabolism. This compilation discusses topics that include the prevalence of aflatoxin B1 in cereals; contamination exposure, toxicology and control of aflatoxins in food and feed; immunosuppressive actions of aflatoxin; hazards and regulations; toxicological effects, risk assessment and legislation for aflatoxins; and the threat aflatoxins have on the economy and health. Chapter 1 - Moulds of Aspergillus genus are among the most important causes of food and feed spoilage and can produce mycotoxins as toxic secondary metabolites when under adverse conditions. Aflatoxins are a group of mycotoxins that commonly contaminate maize and groundnuts, and are categorized by the International Agency for Research on Cancer under Class 1A human carcinogens. From the food safety standpoint, one of the most important mycotoxins is aflatoxin B1 (AFB1). Due to its potent carcinogenic, teratogenic and mutagenic effects dependent on the level and length of exposure, the presence of this contaminant in food and feed should be kept as low as achievable. In order to investigate the occurrence of AFB1, determine its concentrations and explore the possibility of its reduction using different methods, samples of maize, wheat, barley and oat were collected from different cultivation fields during a three-year period. The immunoassay (ELISA) as a screening method and high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) as a confirmatory method were used to determine AFB1 concentrations. Maize contamination seen with AFB1
  8. 8. Adina G. Faulknerviii concentrations higher than permitted was associated with climate conditions established in the period of concern, which was extremely warm and dry, and might had favored mould production and AFB1 formation. Substantial to almost absolute AFB1 reduction in the maize samples was achieved using gamma radiation. A strong antifungal effect was also obtained upon the use of essential oils and lactic acid bacteria as biological AFB1-reduction alternatives. As the presence of AFB1 in cereals could be dangerous for human and animal health, in order to prevent its harmful effects and huge economic problems, the prevention of formation of this contaminant and consistent control over it are of major interest. Based on these substantiated grounds, possibilities of implementing new methods of AFB1 determination and reduction within the frame of safe food production are virtually countless. Chapter 2 - Toxigenic fungi in crops have been divided historically into two groups, field and storage fungi. Mycotoxins are produced by toxigenic fungi at the fields and in the storage. Although many compounds are termed as ―mycotoxin‖, there are only five agriculturally-important fungal toxins: deoxynivalenol, zearalenone, ochratoxin A, fumonisin and aflatoxin. Penicillium and Aspergillus species are the most important storage fungi. However, they can also invade stressed plants in the field. The main mycotoxins produced by Aspergillus species are aflatoxins, citrinin and patulin. The word ‗aflatoxin‘ comes from ‗Aspergillus flavus toxin‘, based on the fact that A. flavus and A. parasiticus are the predominant species responsible for aflatoxin contamination of crops prior to harvest or during storage. Aflatoxins B1, B2, G1, and G2 are the four major isolated aflatoxins from food and feed commodities. A. flavus and A. parasiticus have distinct affinity for nuts and oilseeds including peanuts, maize and cotton seed. Cereals are a general substrate for growth of A. flavus but, unlike nuts, small grain cereal spoilage by A. flavus is the result of poor handling. Moreover, aflatoxin M1 as a milk contaminant has potential risk for animal and human health. The character of the aflatoxin problem varies by region. For instance, aflatoxin accumulation in stored maize in subtropical Asia has risen rapidly in post-harvest conditions whereas in the US, the issue is pre-harvest condition of maize. Therefore, the exposure to aflatoxins differs between countries particularly due to different diets. Food contamination with Aspergillus is associated with warm and dry climates. However, in variable environmental conditions, the aflatoxin contamination may differ from one year to another at the same location. Progress in understanding the biology of Aspergillus has greatly improved with the new techniques in genome sequencing and the developed molecular
  9. 9. Preface ix tools that enable rapid genetic analysis of individual genes. Particularly, the genetics of aflatoxin synthesis is regarded as a model to gain insight into fungal secondary metabolism. Well-designed research on production of the aflatoxin precursor sterigmatocystin with the genetic model A. nidulans, has contributed greatly to our knowledge of the aflatoxin pathway and the global regulatory mechanisms. According to the recent studies, fungal pathogenesis is related to lipid-mediated fungal-host crosstalk, suggesting that secondary metabolism may be controlled by oxylipins at the transition level. Also, some oxylipins have been reported to be engaged in the signalling mechanism like quorum sensing responses in Aspergillus. Quorum sensing molecules and their genes which are responsible for intra and inter kingdom communications could be applied in the future aflatoxin bio-control strategies. Chapter 3 - Aflatoxins (AFs) are secondary metabolites produced by various fungal species of the genus Aspergillus such as Aspergillus flavus and Aspergillus parasiticus. The most important compounds are aflatoxins B1, B2, G1 and G2, as well as two metabolic products secreted in milk, M1 and M2. The worldwide occurrence of aflatoxins contamination in raw agricultural products has been well documented; such contamination occurs in a variety of food and feed, such as cereals, nuts, dried fruits, spices and also in milk as a consequence of the ingestion of contaminated feed. However, pistachios, peanuts and corn are the most frequently contaminated food items reported in the Rapid Alert System for Food and Feed (RASFF) of the European Union. The occurrence of aflatoxins is mainly affected by environmental factors such as climatic conditions, geographic location, agricultural practices, and susceptibility of the products to fungal growth during harvest, storage and processing. High contamination levels of aflatoxins are mainly associated with post-harvest growth of Aspergillus moulds in poorly stored commodities. Aflatoxins can cause adverse effects to the health of animals and humans. These toxins have been reported to be associated with acute liver damage, liver cirrhosis, induction of tumors and teratogenic effects. Aflatoxin B1 (AFB1) is usually predominant and the most toxic among aflatoxins because it is responsible for hepatocarcinoma in animals and strongly associated with the incidence of liver cancer in humans. AFB1 is a genotoxic and mutagenic chemical, and it has been classified by the International Agency of Research on Cancer (IARC) as human carcinogen (group 1). The toxic effects of the ingestion of aflatoxins in both humans and animals depend on several factors including intake levels, duration of exposure, metabolism and defense mechanisms, and individual susceptibility. Aflatoxins affect not only the health of humans and animals but also the economics of agriculture and food.
  10. 10. Adina G. Faulknerx Because of the multiple adverse health effects to humans and animals caused by aflatoxin consumption, many nations worldwide have regulatory standards on aflatoxin in food and feed. The European Union (EU) regulation on aflatoxins in foodstuffs is among the strictest in the world (Commission Regulation (EC) nº 1881/2006 and successive amendments). Maximum contents of aflatoxins in feeds are also established by Commission Regulation (EU) nº 574/2011 on undesirable substances in animal feed. Throughout the world there are many advisory bodies concerned with food safety, including the World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO), the Codex Alimentarius Joint Expert Committee for Food Additives and Contaminants (JECFA), and many others, which regularly assess the risk from mycotoxins, advise on controls to reduce consumer exposure and establish different regulations for these toxins in different countries. Chapter 4 - Aflatoxins are secondary metabolites produced by fungi of the Aspergillus species. They occur as contaminants in a variety of food and feed stuffs that have been infected with the producing fungi. Aflatoxin exposure is known to cause a number of acute and chronic effects in both humans and animals, including immunosuppression, liver and other cancers, and failure of vaccination regimens. The immunomodulatory effects of the aflatoxins have been shown to affect cell-mediated immunity more than humoral immunity. In particular, aflatoxin exposure modulates secretion of inflammatory cytokines and phagocytic function. Decreases in phagocytosis and inflammation observed following aflatoxin exposure may reduce the effectiveness of the host immune response to infection, thereby increasing susceptibility to infection in individuals exposed to these toxins. The aim of this chapter is to summarise the immunomodulatory effects of aflatoxin exposure in order to better understand its potential immunosuppressive effects in humans and animals. The relationship between these immunosuppressive actions and susceptibility to infection will also be discussed. Chapter 5 - Brazil nut is an important non-timber forest product produced in Amazon region. This nut is used as food with high value in the international market, due to its high nutritional and flavor characteristic and to their association with environmental conservation and alleviation of poor people living from Amazonia. Annually, several hundred tons of Brazil nuts are produced in Brazil. However, they are susceptible to aflatoxins (AF) contamination. Because of the detection of unacceptable level of AF in Brazil nuts consignments arriving in European Union ports, in 2003, special conditions were imposed on Brazil nuts entering the European Union,
  11. 11. Preface xi decreasing the acceptable levels of AF. In 2010, the European Union revised AF regulation on nuts; these new limits are more adequate when considering the complexity of Brazil nut chain and the low risk related to its low consumption. This chapter points data on the occurrence of AF in Brazil nuts, as reported by the Rapid Alert System for Food and Feed (RASFF), and evaluates the efforts made by all sectors involved in the agribusiness of Brazil nuts, in Brazil, in order to contribute to protection of both domestic and international consumers from possible health hazard caused by AF. Chapter 6 - Aflatoxin B1 (AFB1) is an important genic toxin produced by the moulds Aspergillus parasiticus and Aspergillus flavus. AFB1 is metabolized by cytochrome P450 enzymes to its reactive form, AFB1-8,9- epoxide (AFB1-epoxide), which covalently binds to DNA and induces DNA damage. DNA damage induced by AFB1, if not repaired, may cause such genic tox toxicological Effects as DNA adducts formation, gene mutations and hepatocellular carcinoma (HCC). During the repair process of DNA damage produced by AFB1, DNA repair genes play a central role, because their function determines DNA repair capacity. In this study, the authors investigated the association between seven polymorphisms (including rs25487, rs861539, rs7003908, rs28383151, rs3734091, rs13181, and rs2228001) in DNA repair genes XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and XRCC3, and toxicological effects of AFB1 using a hospital-based case-control study. Toxicological effects of AFB1 were analyzed by means of the levels of AFB1-DNA adducts, the mutant frequency of TP53 gene, and the risk of AFB1-related HCC. The authors found that the mutants of XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and XRCC3 had higher AFB1-DNA adducts levels, compared with the wilds of these genes (3.276 vs 3.640 μmol/mol DNA for rs25487, 2.990 vs 3.897 μmol/mol DNA for rs861539, 2.879 vs 3.550 μmol/mol DNA for rs7003908, 3.308 vs 3.721 μmol/mol DNA for rs28383151, 3.229 vs 3.654 μmol/mol DNA for rs3734091, 2.926 vs 4.062 μmol/mol DNA for rs13181, and 3.083 vs 3.666 μmol/mol DNA for rs2228001, respectively). Furthermore, increasing risk of TP53 gene mutation and HCC was also observed in these with the mutants of DNA repair genes. These results suggested that polymorphisms of DNA repair genes might modify the toxicological effects of AFB. Chapter 7 - Studies in typical and new Argentinean peanut areas showed that toxigenic Aspergillus section Flavi strains are widely distributed in soils and seeds, with high probability of being transferred to the storage ecosystem. Mycological analyses of soil showed that Aspergillus section Flavi population were present in the two areas at similar counts (3.2x102 cfu g-1 ). Within this
  12. 12. Adina G. Faulknerxii section, two fungal species were frequently isolated with isolation percentages of 73 and 90% for A. flavus and of 27 and 9% for A. parasiticus in soil samples from traditional and new areas, respectively. The percentages of the different A. flavus phenotypes from both peanut-growing areas showed that L strains were recovered in the highest percentage and represented 59 and 88% of the isolates with variable ability to produce aflatoxins (AFs). Peanut kernels collected at harvest time from different localities of Córdoba and Formosa provinces showed A. flavus and A. parasiticus contamination. The 42.8 and 70% were classified as type L and the percentages of aflatoxigenic A. flavus strains were 68.6 and 80.0% in samples from traditional and recent peanut- growing areas, respectively. Highly toxigenic A. flavus S strains were isolated with major frequency from soil and kernel samples coming from traditional peanut-growing area. Aflatoxin contamination was detected in peanut kernels from typical peanut growing area. Harvested peanut were stored during 5 months in three storage systems (big bags, wagons of conditioning and drying and stockpiled warehouse) and mycological population succession was analyzed. Fungal isolation was greater from pod (95%) than from kernel tissues. The most common fungi identified included Penicillium, Aspergillus, Eurotium and Fusarium spp. Within Aspergillus genus, the section Flavi had the greatest mean counts of 1.4x104 , 9.4x102 , 5.2x102 cfu g-1 for big bags, wagon and warehouse, respectively. A. flavus and A. parasiticus strains with variable ability to produce AFs were isolated from peanut kernels stored in the three systems at all sampling periods in the order of 1.5x102 , 2.3x102 and 4.5 cfu g-1 , respectively. .A. flavus S and L strains contributed to silo community toxigenicity during all storage period. Total AF levels ranging from 1.1 to 200.4 ng g-1 were registered in peanuts conditioned at the higher aW values (0.94–0.84 aW) and stored in big bags. Despite the water stress conditions registered in the stockpiled warehouse throughout the storage period, AFB1 levels ranging between 2.9 and 69.1 ng g-1 were registered from the third sampling. Therefore, the interaction between biological and abiotic factors and substrate may promote the Aspergillus contamination and the subsequent AF accumulation in peanut from sowing to storage, highlighting the need to promote good practices in order to avoid the risk of these metabolites contamination in peanut food chain. Chapter 8 - Aflatoxins are toxic metabolites produced by the fungus Aspergillus. The main representatives are aflatoxins B1, B2, G1, G2. Their occurrence in food like nuts, cereals and cereal-derived products is a result of fungal contamination before harvest and during storage. Milk can also be
  13. 13. Preface xiii contaminated by aflatoxin M1 (main metabolite of B1) as a result of animals‘ exposure to feed contaminated by the aflatoxin B1. Aflatoxins manifest acute and chronic toxicity. Evidence of acute aflatoxicosis in humans involving a range of symptoms from vomiting to death has been reported mainly in Third World Countries. In relation to chronic toxicity aflatoxins are well known for their genotoxic and carcinogenic properties while recent studies evident a series of other possible effects like reprotoxicity, impaired growth in children, intestinal functions, chronic fatigue syndrome, compromise immunity and interfere with protein metabolism and multiple micronutrients that are critical to health. The critical step for aflatoxins‘ risk assessment is the estimation of the real exposure. For this reason a number of surveys are conducted globally using tools like biomarkers of exposure and modeling. In addition new parameters like the climate change are now taken into consideration in order to predict possible current and future changes of exposure to aflatoxins. As aflatoxins are compounds of natural origin and their presence in food cannot be totally eliminated the risk management is based on keeping the total exposure as low as reasonably achievable taking into account the social-economic impact of crop and livestock losses. Exposure reduction is achieved mainly by reducing the number of highly contaminated foods reaching the market by regulatory control but also applying detoxification strategies. According to the EU regulatory framework minimization of the exposure to aflatoxins is based on setting maximum levels of aflatoxins in different foodstuffs (4 – 10 µg/kg total aflatoxins) and feed (EC/1881/2006, Directive 2002/32/EC). Products exceeding the maximum levels should not be placed on the EU market. Methods of sampling and analysis for the official control of aflatoxins, are also set (EC/401/2006) in order to ensure common sampling criteria to the same products and that certain performance criteria are fulfilled. The United States Food and Drug Administration (FDA) has established the action levels for aflatoxin present in food to the 20 µg/kg (0.5 µg/kg for milk) and up to 300 µg/kg for feed. Finally an action level of 10 µg/kg total aflatoxins is also used from Japan authorities. Chapter 9 - This paper presents a review of the occurrence of aflatoxins in different food commodities in Greece, based both on results represented in literature as well as results derived from monitoring programs of the Center of Toxicology Science & Research, Medical School, University of Crete. Aflatoxins, can pose a severe threat to food safety, since they are characterized carcinogenic to humans, IARC Group 1. They may be formed or developed in any stage of the agricultural production (primary production, processing and
  14. 14. Adina G. Faulknerxiv storage) as a result of transitional weather conditions or of poor storage. Studies, monitoring programs and surveys, which have been carried out in Greece, are mainly focused in milk and dairy products. In this context, several studies have been conducted in animal feeds as well, since there is notable evidence that they are potential sources of aflatoxins in milk production. Additionally, both black and green olives have been examined for possible contamination by aflatoxins, due to the fact that they are damaged during harvest and processing and thus providing a substrate for aflatoxin development. Finally, a limited number of studies investigate the presence of aflatoxins in different processed products like breakfast cereals. The above foodstuffs have been studied on account of their high nutritional value and the fact that they are consumed by different population groups. Results indicate that residue levels of aflatoxins which are presented in fresh as well as processed agricultural products, do not pose any considerable risk for the Greek population groups. The most important factors influencing the levels of aflatoxins in major agricultural products appear to be the growing and cultivation techniques, as well as the food safety parameters during harvesting, storage and processing. An additional issue, which seems to raise concern internationally, is the fact that climate change in combination with modifications in the cultivation techniques may affect the frequency and severity of aflatoxin residues in agricultural products. Chapter 10 - This review deals with the aflatoxins especially with their food sources, wide occurrence and toxicological effects on animals and humans. Aflatoxins are highly oxygenated, heterocyclic, difuranocoumarin compounds and are an important group of mycotoxins produced by the fungi. There are almost 20 different types of aflatoxins identified till now; among these AFB1 is considered to be the most toxic. Aflatoxins persist to some extent in food even after the inactivation of the fungi by food processing methods, such as ultra-high temperature products, due to their significant chemical stability. Aflatoxins can affect a wide range of commodities including cereals, oilseeds, spices, and tree nuts as well as milk, meat, and dried fruits. Twenty-five percent of the world‘s crops are affected with mycotoxins. On a worldwide scale, the aflatoxins are found in stored food commodities and oil seeds. Some of the foods on which aflatoxin producing fungi grow well include cereals (maize, sorghum, pearl millet, rice, wheat, corn, oats, barley), oilseeds (peanut, soybean, sunflower, cotton), spices (chile peppers, black pepper, coriander, turmeric, ginger), and tree nuts (almond, pistachio, walnut, coconuts), sweet potatoes, potatoes, sesame, cacao beans, almonds, etc., which on consumption pose health hazards to animals, including
  15. 15. Preface xv aquaculture species of fish, and humans. Food commodities affected by aflatoxins are also susceptible to other types of mycotoxins and multiple mycotoxins can co-exist in the same commodity. Various cereals affected by aflatoxins are also susceptible to contamination by fumonisins, trichothecenes (especially deoxynivalenol), zearalenone, ochratoxin A and ergot alkaloids. More than 5 billion people in developing countries worldwide are at risk of chronic exposure to naturally occurring aflatoxins through contaminated foods. Aflatoxin is a potent liver toxin causing hepatocarcinogenesis, hepatocellular hyperplasia, hepatic necrosis, cirrhosis, biliary hyperplasia, and acute liver damage in affected animals. Effects of aflatoxins in animals depend on age, dose and length of exposure, species, breed and nutritional status of the animal. Health effects occur in fish, companion animals, livestock, poultry and humans because aflatoxins are potent hepatotoxins, immunosuppressants, mutagens, carcinogens and teratogens. Aflatoxin– B1 has been shown to cause significant morphological alterations along with reduced phagocytic potential in chicken and turkey macrophages. Aflatoxin- B1 exposure to chicken embryos causes significant suppression in macrophage phagocytic potential in chicks after hatch. Aflatoxin intercalates into DNA and alkylates the DNA bases through its epoxide moiety resulting in liver cancer. Other effects include mutagenic and teratogenic effects. Exposure of biological systems to harmful levels of aflatoxin results in the formation of epoxide, which reacts with proteins and DNA leading to DNA-adducts, thus causing liver cancer. The primary target of aflatoxins is the hepatic system. Acute effects include hemorrhagic necrosis of the liver and bile duct proliferation while chronic effects include hepatocellular carcinoma (HCC). HCC is the sixth most prevalent cancer worldwide with a higher incidence rate within developing countries. Preliminary evidence suggests that there may be an interaction between chronic aflatoxin exposure and malnutrition, immunosuppression, impaired growth, and diseases such as malaria and HIV/AIDS. Outbreaks of acute aflatoxin poisoning are a recurrent public health problem. The discussion of this problem and its remedies must be held in the context of the associated question of food insufficiency and more general economic challenges in developing countries. Aflatoxin constitutes a serious health concern to the entire food chain, necessitating a multidisciplinary approach to analysis, action, and solution.
  16. 16. In: Aflatoxins ISBN: 978-1-63117-298-4 Editor: Adina G. Faulkner © 2014 Nova Science Publishers, Inc. Chapter 1 BIO-PREVALENCE, DETERMINATION AND REDUCTION OF AFLATOXIN B1 IN CEREALS Jelka Pleadin1, , Ksenija Markov2 , Jadranka Frece2 , Ana Vulić1 and Nina Perši1 1 Croatian Veterinary Institute, Laboratory for Analytical Chemistry, Zagreb, Croatia 2 Faculty of Food Technology and Biotechnology, Zagreb, Croatia ABSTRACT Moulds of Aspergillus genus are among the most important causes of food and feed spoilage and can produce mycotoxins as toxic secondary metabolites when under adverse conditions. Aflatoxins are a group of mycotoxins that commonly contaminate maize and groundnuts, and are categorized by the International Agency for Research on Cancer under Class 1A human carcinogens. From the food safety standpoint, one of the most important mycotoxins is aflatoxin B1 (AFB1). Due to its potent carcinogenic, teratogenic and mutagenic effects dependent on the level and length of exposure, the presence of this contaminant in food and feed should be kept as low as achievable. In order to investigate the occurrence of AFB1, determine its concentrations and explore the  Corresponding Author: Tel: +38516123626; Fax: +38516123670; E-mail:
  17. 17. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.2 possibility of its reduction using different methods, samples of maize, wheat, barley and oat were collected from different cultivation fields during a three-year period. The immunoassay (ELISA) as a screening method and high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) as a confirmatory method were used to determine AFB1 concentrations. Maize contamination seen with AFB1 concentrations higher than permitted was associated with climate conditions established in the period of concern, which was extremely warm and dry, and might had favored mould production and AFB1 formation. Substantial to almost absolute AFB1 reduction in the maize samples was achieved using gamma radiation. A strong antifungal effect was also obtained upon the use of essential oils and lactic acid bacteria as biological AFB1-reduction alternatives. As the presence of AFB1 in cereals could be dangerous for human and animal health, in order to prevent its harmful effects and huge economic problems, the prevention of formation of this contaminant and consistent control over it are of major interest. Based on these substantiated grounds, possibilities of implementing new methods of AFB1 determination and reduction within the frame of safe food production are virtually countless. 1. INTRODUCTION Cereal grains may become contaminated by moulds that produce mycotoxins as toxic chemical compounds while in the field and during storage. This group of compounds represents a significant food safety issue and poses as a risk to health and wellbeing of humans and animals. Food and feed contamination with mycotoxins, as toxins of frequent incidence in agricultural goods, has a negative impact on economies of the affected regions, especially in the developing countries where harvest and post-harvest techniques of mould growth prevention are not adequately implemented (Rustom, 1997). Cereals such as maize, wheat, barley and oat represent a significant part of not only human, but also animal diet, and play a role in industrial food & feed processing. Cereal grains balance the nutrition by virtue of providing a low-fat diet that has a number of advantages, especially when whole-grain foodstuffs are consumed. However, grains are a common source of contaminants, especially mycotoxins, which, under favorable temperature and humidity conditions, may produce mycotoxins before and/or during harvest, handling, shipment and storage. The most important mycotoxins are aflatoxins B1, B2, G1 and G2, fumonisin B1, T-2 toxin, zearalenone, ochratoxin A and
  18. 18. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 3 deoxynivalenol. Maize and maize products are known to be prone to contamination by fungi that produce secondary metabolites such as aflatoxins (Groopman and Donahue, 1988). Among food & feed contaminants, aflatoxins are of current concern and have received a great deal of attention during the last three decades. They were first heavily researched and truly understood after the death of more than 100,000 young turkeys on poultry farms in England that was found to be related to the consumption of Brazilian peanut meal (Goldblatt, 1969). Aflatoxins are known to be produced by two species of Aspergillus genus, specifically Aspergillus flavus and Aspergillus parasiticus, and represent highly toxic, mutagenic, teratogenic and carcinogenic compounds that exhibit an immunosuppressive activity, causing both acute and chronic toxicity in humans and animals (Eaton and Gallagher, 1994; Massey et al., 1995; EFSA, 2004; Meggs, 2009). Among them, aflatoxin B1 (AFB1) is the most potent liver carcinogen known in mammals, and is classified by the International Agency for Research on Cancer (IARC) as Group 1 carcinogen (IARC, 1993). Factors that promote fungal infection and AFB1 production are inoculum availability, weather conditions and pest infestation during crop growth, maturation, harvesting and storage (Lopez-Garcia and Park, 1998). Generally speaking, crops stored for more than a few days become a potential target for mould growth and mycotoxin formation (Turner et al., 2009). In general, mycotoxins, aflatoxins included, are stable compounds not destroyed during most of the food processing operations, which might lead to the contamination of cereals and their final products. However, aflatoxin presence can sometimes be reduced by making improvements in farming practices, such as providing better storage conditions or using modified seeds, or by making improvements in manufacturing processes. Due to the fact that aflatoxins represent the type of mycotoxins most commonly found in cereals, many studies have attempted to define multiple aspects of contamination of human food and animal feed chains, and still do so, so that the topic is a very hot one. Such a contamination is often unavoidable and still poses as a serious problem associated with important agricultural goods, which emphasizes the need for suitable processing capable of inactivating the toxin. Maize, as the most widely grown crop extensively used for animal feeding and human consumption, represents a particular problem. Due to its nutritional value, a high percentage of the world maize production is destined for animal feeding. The European Food Safety Authority document (EFSA, 2013), prepared based on analytical data on four aflatoxins (B1, B2, G1, and G2) recovered in
  19. 19. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.4 food samples collected between 2007 and 2012, reports that the collection of data on the occurrence of aflatoxins in relevant foodstuffs should be continued in order to gather a representative number of samples in different food categories; in addition, the document draws attention to the need for harmonizing the reporting formats across the European countries. This chapter presents the results of AFB1 determination in four types of commonly used cereals intended for food and feed, collected during a three- year period from different cultivation fields, as well as the results of an investigation into the possibilities of contamination reduction and/or avoidance. For the sake of AFB1 determination, the immunoassay (ELISA) as a screening method and high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) as a confirmatory method were used. Gamma radiation and essential oils & lactic acid bacteria, on the other hand, were used to investigate the possibilities of AFB1 reduction in contaminated maize samples. 1.1. Exposure to AFB1 through the Food Chain The Food and Agriculture Organization (FAO) estimated that 25% of the world food-intended crops are contaminated with mycotoxins, and that aflatoxins, as the most toxic among them, are the trickiest to deal with because of their widespread occurrence in maize, peanuts and its products, cottonseed, chilies, peppers, pistachio nuts and some other foodstuffs (Scholthof, 2003). Contaminated feed also represents the main source of AFB1 infestation in farm animals, which get to be contaminated through parasites living on plants even prior to harvesting or on stored harvested crops (Huwing et al., 2001; Gareis and Wolff, 2000). As fodder, cereals and seeds used for dairy cattle feeding are inevitably in contact with yeasts and filamentous fungi, contamination of these raw materials frequently occurs already in the field. AFB1 contamination can also occur during harvesting, transport and storage of cereals and their products, as well as due to post-harvest mishandling that can lead to rapid feed spoilage (Alonso et al., 2011). In animals intended for meat production that had consumed contaminated feed, the ingestion of AFB1 leads to substantial degradation of meat quality (Bonomi et al., 1994). Cattle exposure to mycotoxins generally occurs through the consumption of contaminated feed. Nelson et al.. (1993) described mycotoxicoses arising on the grounds of exposure to mycotoxin-contaminated rations. Ruminants, such as cattle and sheep, are generally more resistant to
  20. 20. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 5 mycotoxins than most animals, especially pigs, as ruminal microbial population plays a role in detoxification process. This assumption is based on the finding that rumen flora is able to convert a number of mycotoxins into metabolites that are less potent or even biologically inactive at common exposure levels (Kiessling et al., 1984). The first identified source of mycotoxins in ruminant diets was the contamination of feed concentrates with aflatoxins. AFB1 occurs in many typical energy-rich concentrates such as grain maize, sorghum, pearl millet, rice, soybean products, peanuts, sunflower & cotton seeds, palm kernels and copra (Vargas et al., 2001; Abbas et al., 2002; Attala et al., 2003). Humans are exposed to AFB1 either directly through the consumption of contaminated food or indirectly through the consumption of animal products (i.e. milk and eggs) coming from animals that had consumed contaminated feed (Rustom, 1997; Bennett and Klich, 2009; Markov et al., 2013). Since it was first observed that dairy animals consuming feeds contaminated with AFB1 excrete aflatoxin M1 (AFM1) in their milk, studies have established that variations in carry-over rates are significant both at high- and low-level AFB1 feed contamination (Prandini et al., 2009). 1.1.1. AFB1-Related Effects Seen in Humans and Animals Although animal species may vary in their susceptibility to aflatoxins, toxic effects of the latter, known as aflatoxicoses, can generally be divided into acute and chronic, based on some determinants such as the duration and level of exposure, entry route, environmental factors, age, health, nutritional status, and other factors such as stressors affecting the animal (Leeson et al., 1995; FDA, 2002). In case of humans, exposure to AFB1 occurs mainly through the consumption of contaminated food such as corn, peanuts, sorghum, copra and rice, cashew, hazel, peanuts, walnuts, pistachios and almonds (Busby and Wogan, 1984; Abdel-Gawad and Zohri, 1993; Mahoney and Rodriguez, 1996). AFB1 also exhibits its toxicity through the metabolite AFM1, which was first determined in human urine while elucidating the etiology of liver cancer caused by AFB1 (Campbell et al., 1970). It has been reported that about 1.3 to 1.5% of the ingested AFB1 converts into AFM1 that gets to be excreted in human urine (Zhu et al., 1987). As the contamination of foodstuffs and feedstock with aflatoxigenic moulds and their toxins is very common, toxic effects of AFB1 on animal health are encountered worldwide (FAO, 2004). Many animal species such as turkeys, ducklings, rainbow trout, guinea pigs, rabbits, rats and dogs show high susceptibility to aflatoxins. AFB1 can cause liver and other cancers in
  21. 21. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.6 humans and livestock; this has been well established in several animal species including rodents, nonhuman primates and fish, the first symptoms thereby being a lack of appetite and weight loss (Busby and Wogan, 1984; Eaton and Gallagher, 1994). Several research reports have agreed that AFB1 is more toxic for young animals (IARC, 1993, Vainio et al., 1994). It has been observed in many parts of the world that AFB1 poses a major etiological factor in the development of hepatocellular carcinoma in individuals infected with hepatitis B virus (Wild and Hall, 2000). Particularly high incidences of AFB1 contamination have been seen in tropical and subtropical regions, where warm and humid weather provides for conditions optimal for mould growth. Chronic ingestion of AFB1 causes various adverse effects such as increased susceptibility to diseases, loss of reproductive performance and, in case of dairy cattle, a decrease in quantity and quality of milk production. Animal exposure to AFB1 leads to a decrease in feed consumption or even to feed refusal, as well as to the reduction in nutrients‘ absorption, metabolic impairments, decreases in protein synthesis, and endocrine and immune system suppression. Acute intoxication is often fatal for both humans and livestock. In poultry and livestock, severe and sudden anorexia, convulsions, feed refusal, weight loss, discolored liver, reduced egg production, reduced energy conversion rate and milk contamination can be encountered. On top of that, the consumed feed loses its common nutritional value and efficiency, leading to reduced livestock growth rates (Waliyar et al., 2007). 1.1.2. Conditions under Which AFB1 Gets to Be Produced in Cereals Accumulation of mycotoxins before and after cereal harvesting largely reflects actual climate conditions. Fusarium toxins are known to be produced during cereal harvesting under high moisture conditions (Munkvold and Desjardins, 1997), whereas pre-harvest aflatoxin contamination of crops, including peanuts (Dorner, 2008) and maize (Payne, 1998), is associated with high temperatures, insect-mediated damage and prolonged drought. Chronic contamination occurs in warm, humid, tropical, and subtropical maize- growing environments (Widstrom, 1996). The degree of moisture mostly depends on the water content available at the harvesting point, but also on the frequency and extensiveness of drying, aerating, and turning of the grain before and during storage, and the respiration of insects and microorganisms harbored by stored grain (Bryden, 2012). Since Aspergillus can tolerate lesser water activity than Fusarium, these contaminations may occur both pre- and post-harvesting, whereas Fusarium contamination is more specific to the pre- harvesting period (Abramson, 1998). Stored cereals may become infested with
  22. 22. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 7 fungi and insects; such an infestation is also affected by climatic factors such as temperature and humidity, geographical location, type of storage container, and handling & transport procedures (Chelkowski, 1991; Jayas et al., 1995). Climate changes can alter the dynamics of insect populations that facilitate fungal crop infections (Wu et al., 2011). Earlier studies have pointed toward significant dependence of AFB1 occurrence on country or region in which the cereals are grown, as well as on high AFB1 concentrations found in maize, peanuts, tree nuts, rice and cottonseed (Rustom, 1997; Reddy et al., 2009). It has been pointed out that the growth of A. flavus and the production of aflatoxins in various biological materials are influenced by many factors including the type of substrate, its moisture content, ―culpable‖ fungal species, presence of minerals, relative humidity of the surroundings, temperature, and physical damage of the kernels (Viquez et al., 1994). It has been shown that the type of mould and its conidial concentration, as well as maize moisture content, play critical interactive roles in the initiation of mould infestation, spoilage and AFB1 production in maize (Oyebanji and Efiuvwevwere, 1999). Limitation of AFB1 occurrence in crops before harvest can be achieved through the reduction of drought and temperatures, weed control, insect damage reduction, effective harvesting techniques and Aspergillus spore reduction in soil by virtue of crop turnover. Genetic engineering and the development of hybrids resistant to Aspergillus spp infection (Widstrom, 1996) may offer new ways of limiting pre-harvest aflatoxin contamination of certain crops. Post-harvesting control of AFB1-susceptible crops can be achieved through the control of factors that affect fungal growth, e.g. water activity, temperature, gas atmospheres, and through the use of insecticides or food preservatives. The prime concern relative of the storage of grains and nuts should be to maintain water activity below the limit favoring fungal growth (which is achievable by virtue of moisture control) (IARC, 2002). The risk of kernel damaging and consequent AFB1 production can be reduced by harvesting solely grains having the moisture of around 24% (Prandini et al., 2009). 1.1.3. The Occurrence of AFB1 in Feed In many cases, the levels of AFB1 naturally occurring in feeds intended for dairy cows have been shown to exceed the regulation limits. The contents of feed intended for milking cows slightly vary dependent on the season and geographical area; some 10% of feed is commonly intended for these purposes. Rye, oats, mocha, wheat and sorghum are selected on dairy farms
  23. 23. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.8 based on the acreage and selected pasture; the use of commercial pelleted feed is not uncommon either (Alonso et al., 2011). Given the fact that in geographical regions having a tropical or sub- tropical climate the risk of AFB1 contamination has generally been acknowledged as high, monitoring of feed ingredients for the presence of AFB1 has been focused on imported feeds such as extracted copra, peanut cake, sunflower cakes, corn gluten, rice bran, cottonseed, palm kernel and soy beans, which seem to be the major carriers of AFB1. In some countries, contamination levels above legal limits were linked to high contamination of locally grown maize that was used as animal feed (EFSA, 2004). In different countries AFB1 has been found to be a contaminant of dairy, cottonseed, barley, soy bean, pellet wheat, peanut shells, corn silage and sorghum silage (Decastelli et al., 2007; Sassahara et al., 2005). Certain cases pointed toward an outbreak of acute aflatoxicosis, with high levels of AFB1 observed in maize stored under high humidity conditions (Lewis, 2005). As for dairy cattle, the problem does not end with animal diseases or production losses, since AFB1 presence in feed leads to the presence of its metabolic product AFM1 in milk and dairy products, possibly affecting human health as well (Boudra et al., 2007; Veldman et al., 1992). 1.2. Current EU Legislation Since the discovery of aflatoxins in the 1960s, regulations have been enforced in many countries so as to protect the consumers from harmful effects of these toxins that may contaminate both foodstuffs and feedstuffs. Various factors play a role in defining permissible mycotoxin levels. These include evidence-based data underpinning the risk assessment, such as the availability of toxicological data, food consumption data, data on the level and distribution of mycotoxins in goods intended for human and animal consumption, and data on analytical methodology. Economic factors such as commercial and trade interests and food safety issues also have an impact (FAO/WHO, 2008). Compared to other regions of the world, the European Union (EU) has the most extensive and most detailed regulations governing AFB1 presence in various types of food and feed. Also, many of the EU candidate member states have mycotoxin presence-governing regulations covering the topic as much in depth as the regulations currently in force across the EU itself.
  24. 24. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 9 Methods of sampling and analysis used within the frame of the official mycotoxin control, AFB1 included, are laid down under the Commission Regulation No 401/2006, amended by the Commission Regulation (EU) No 178/2010. This ensures that the same sampling criteria are applied for the same products by the competent authorities throughout the EU and that certain performance criteria, such as recovery and precision, are fulfilled. Maximum permitted levels (MPLs) of aflatoxins in food, including those of AFB1 and total aflatoxins, are laid down under the Commission Regulation (EC) No 1881/2006, amended by the Commission Regulation (EU) No 165/2010. Legal limits for AFB1 in feedstuffs currently adopted by the EU member states and set under the Commission Directive 2003/100/EC that amends the Directive 2002/32/EC, are substantially different from those in other countries that have enforced AFB1 MPLs for animal feeding stuffs. As AFB1 is a genotoxic carcinogen and a strong acute toxin that affects various animal species, it is the only individual mycotoxin whose MPLs are set under the Directive. Some countries have a number of limits, often dictated by the destination of the feedstuff. From the human health‘s point of view, the most stringent criteria apply to feedstuffs intended for dairy cattle because of AFB1‘s conversion into AFM1 that takes place in milk and dairy products (MPL= 5 µg/kg across the EU). 1.3. Analytical Methods of AFB1 Determination For the purpose of AFB1 determination, different screening and confirmatory analytical methods are used. Most of these analytical methods have to be performed using the appropriate cleanup procedures, except perhaps for the immunological assay called the ELISA, with which this step might be skipped. The development of semi-quantitative ELISA as a screening method was a major step forward in the development of rapid, repeatable and sensitive assays suitable for AFB1 determination. Gaur et al. (1981) produced and characterized AFB2-antiserum, equally specific for AFB2 and AFB1, and used it within the ELISA for AFB1 quantification. In the recent years, the ELISA has been described to have advantages over other methods when it comes to AFB1 determination; these advantages mostly lie within its rapidity, high specificity, simplicity of use, low cost and the use of safe reagents (Pestka, 1994; Zheng et al., 2005; Goryacheva et al., 2007, Ayejuyo et al., 2011). Commercially available ELISA kits suitable for the detection of mycotoxins are based on the competitive assay format that uses
  25. 25. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.10 either a primary antibody specific for the target molecule or an enzyme conjugate and the required target. The advantage of micro-titer plate-based immunoassays lies within the fact that these can be used to analyze a large number of samples simultaneously. The complex formed on the occasion then interacts with a chromogenic substrate to give a measurable result. The disadvantage of the ELISA mostly comes as a result of its limited detection range consequential to the narrow scope of the antibodies‘ sensitivity (Turner et al., 2009). Other methods for AFB1 quantification require sophisticated laboratory equipment, including high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography/mass spectrometry (LC/MS) or gas chromatography/mass spectrometry (GC/MS) (Xiang et al., 2006; Krska et al., 2008; Rahmani et al., 2009; Stephard et al., 2011). HPLC has a high efficiency, sensitivity and resolution (Herzallah, 2009; Peiwu et al., 2011). Modern analysis of components heavily relies on HPLC that employs various adsorbents depending on physical and chemical structure of different components. The most commonly used HPLC detectors are fluorescence detectors (FLDs). In order to widen the detection limits, HPLC is used in combination with mass spectrometry (MS) (Li et al., 2009). MS represents a method that allows for a highly accurate and specific detection of AFB1, with limiting factors such as high cost of equipment, complex laboratory requirements, and limitations in the type of solvents used for extraction and separation (Turner et al., 2009). 1.4. Methods of AFB1 Reduction As the presence of moulds and/or mycotoxins in food can be dangerous for human health and represents a huge economic problem, one has all the reasons to allow for the implementation of new methods providing for a safe food production. Methods of control can be classified into two categories: (1) prevention of mould contamination and growth, and (2) detoxification of contaminated products (Riley and Norred, 1999; Mishra and Das, 2003). The prevention of mould growth can be achieved either through pre- or post- harvesting strategies. The applied AFB1-reduction procedure must effectively inactivate or remove the toxin, maintaining at the same time both nutritional and technological properties of the product, while not generating reactive toxic products (López-García and Park, 1998). These methods can be divided into chemical, biological and physical (Kabar et al., 2006). Investigation into the
  26. 26. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 11 methods of AFB1 inactivation in contaminated food and feed has revealed that pre-harvest contamination can be reduced by virtue of proper curing, drying, sorting and storage, all of the aforementioned limiting the growth of aflatoxigenic fungi. However, the implementation of unique, totipotent method of aflatoxin reduction, capable of effectively performing in any given biological material, is virtually impossible. The efficiency of methods of AFB1 inactivation depends on many parameters such as the nature of food and feed, their moisture content and composition, and the level of contamination. Some studies have attempted to achieve detoxification of, or toxin inactivation in, AFB1-contaminated feedstuff using gamma irradiation, thermal inactivation, physical separation, microbial degradation and different chemical treatments (Piva et al., 1995; Rustom, 1997). 1.4.1. Biological Reduction Many microorganisms including bacteria, yeasts and acid-producing moulds can metabolize and inactivate AFB1, Flavobacterium aurantiacum being the most active among them. AFB1 production is also inhibited by lactic acid bacteria, Bacillus subtilis and many moulds. As shown in the fermenting industry settings, aflatoxins do not degrade during fermentation, but have been proven absent from alcohol fraction after distillation. Aflatoxins are usually concentrated in spent grains. When contaminated products are used for fermentation, it is important to determine the end-use of the contaminated by- products. A specific compound found to be a good decontaminating agent is usually more biologically- and cost-efficient if added directly. Literature has revealed that true efficacy of biological methods demonstrating effective decontaminating properties is usually dependent on specific compounds produced by selected microorganisms (Waliyar et al., 2007), as well as on the competition for nutrients required for toxin production, space competition and the production of anti-aflatoxigenic metabolites coming from coexisting microorganisms. Studies have suggested that certain fungi, including A. parasiticus, degrade AFB1, possibly through fungal peroxidases (López- Garcia and Park 1998). 1.4.2. Physical Reduction Inactivation of AFB1 using physical methods involves extraction with solvents, adsorption, and heat- or irradiation-based inactivation. AFB1 levels can be reduced in stored goods using physical procedures such as color sorting, density flotation, blanching and roasting.
  27. 27. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.12 Despite the debate on safety of irradiated foods, food irradiation is becoming a technique of a commercial-scale application, employed so as to render food products sterile (Diehl, 1990). Gamma radiation as a sterilizing treatment with a high penetrating power passes through materials without leaving any residues and causes a direct damage to cell DNA through ionization, inducing mutations and cell killing. There exist a number of reports on increased, decreased or even unaffected production of mycotoxins after irradiation of fungi under various conditions. The Joint FAO/IAEA/WHO Expert Committee pointed out that the irradiation of any food up to the average total dose of 10 kGy poses no toxicological hazard and no special nutritional or microbiological problem (WHO, 1991; Mariotti et al., 2011). In light of the foregoing, in 1999 the European Community authorized this dose as the maximum total radiation dose allowed to be absorbed by irradiated food on average. 1.4.3. Chemical Reduction The use of chemicals to inactivate, bind or remove aflatoxins has been studied extensively using different chemicals such as propionic acid, ammonia, copper sulfate, benzoic acid, urea, citric acid and some other chemicals capable of reacting with aflatoxins (Gowda et al., 2004). These chemicals convert AFB1 to less toxic and less mutagenic compounds including acids, bases, oxidizing agents, bisulfites and gases. Where approved, AFB1 levels in goods destined for animal feeds can be reduced by agents such as adsorbent clays, as well as by ammonization. The main purpose of ammonization is the elimination of AFB1 from feed intended for dairy cows (IARC, 2002). As for the chemical methods of AFB1 reduction, they have generally been labeled as impractical as they call for drastic conditions in terms of temperature and pressure, as well as unsafe because of toxic residues, and unfavorable since leading to degradation of nutritional, sensory and functional properties of the product (Rustom, 1997). To date, chemical methods have been approved only for the reduction of AFB1 in animal feed. Techniques other than the use of chemical sorbents and ammonization have achieved reduction in AFB1 bioavailability that comes as a result of hydrated sodium calcium aluminosilicate binding (Phillips et al., 1988). Ammonization is the only chemical inactivation process that has been shown to efficiently destroy AFB1 in cottonseed and cottonseed meal, peanuts and peanut meal, and maize (Park et al., 1988; Park and Price, 2001).
  28. 28. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 13 2. SURVEY OF AFB1 BIO-PREVALENCE IN CEREALS 2.1. Samples under Study In order to investigate the bio-prevalence, i.e. the occurrence of AFB1 in cereals, a total of 792 samples of maize (388), wheat (155), barley (148) and oat (101) were collected during a three-year period (2010-2012) from different fields situated in northwestern and eastern part of Croatia. Sampling and preparation of the test samples were performed in line with ISO 6497:2002 and ISO 6498:1998, respectively. Determination of moisture content in the sampled materials was performed as well. Prepared test portions were ground into a fine powder having a particle size of 1.0 mm using an analytical mill (Cylotec 1093, Tecator, Sweden) and stored at +4 ºC prior to AFB1 analysis that made use of ELISA and LC-MS/MS methods. 2.2. Implementation of the ELISA Method 2.2.1. Validation of the ELISA Method Validation parameters of the ELISA method were determined using control maize and wheat samples. AFB1 standards employed with the validation of analytical methods were provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany). The limit of detection (LOD), calculated from the mean value of ten determinations of blank maize and wheat samples plus three standard deviations, was 1 µg/kg in both cases. The recovery rate was determined at four different levels (2, 5, 10 and 50 µg/kg) by virtue of spiking the control maize and wheat samples with the prepared standard mycotoxin working solution (100 µg/L) adopted for in-house use (six replicates per concentration level per day). For the determination of intermediate precision, the same steps were repeated on two additional occasions by two independent analysts within a three-month period and under the same analytical conditions. Validation results (given in Table 1) proved the applied ELISA method to be efficient and suitable for the determination of AFB1 in cereals under consideration.
  29. 29. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.14 Table 1. Results of the ELISA method validation Material Spiked concentration (μg/kg) Mean recovery (%) CV (%) Intermediate precision (%) CV (%) Maize 2 85.4 6.1 88.5 8.4 5 90.7 5.7 93.2 7.3 10 92.2 6.3 93.6 7.1 50 95.5 4.9 95.9 6.7 Wheat 2 86.7 4.6 82.6 6.7 5 88.5 5.8 88.9 7.7 10 93.6 7.4 94.6 8.2 50 96.8 6.8 95.2 8.8 2.2.2. Employment of the ELISA Method Sample Preparation Samples were prepared using 5 g of the homogenized sample supplemented with 25 mL of 70%- methanol and shaken vigorously head- over-head on a shaker for three minutes. The extract was then filtrated (Whatman, black ribbon); in the further course, 1 mL of the obtained filtrate was diluted with the appropriate volume of deionized water. When calculating the final AFB1 concentration in the analyzed sample, the applied dilution factor was duly taken into account. ELISA Assay All study samples were first analyzed for AFB1 concentration using the ELISA method that made use of AFB1 ELISA Ridascreen kits provided by R- Biopharm (Darmstadt, Germany). ELISA was also used after the implementation of AFB1 reduction methods. Each kit contains a micro-titer plate with 96 wells coated with antibodies against AFB1, aqueous solutions of AFB1 standard (0, 1, 5, 10, 20, and 50 μg/L), peroxidase-conjugated AFB1, substrate/chromogen (urea peroxide), a stop-reagent (1 N-sulfuric acid), and the washing buffer (10 mM-phosphate buffer, pH=7.4). All other chemicals used for the analysis were of an analytical grade. The ELISA assay employed with the determination of AFB1 in the analyzed cereals, was performed in full line with the kit manufacturer‘s instructions, and made use of an auto-analyzer ChemWell 2910 (Awareness Technology, Inc, USA). The obtained AFB1
  30. 30. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 15 concentrations were calculated from a six-point calibration curve and corrected for recovery. 2.3. The Implementation of LC-MS/MS Method 2.3.1. LC-MS/MS Validation LC-MS/MS validation was carried out according to the Commission Decision 2002/657/EC using an alternative approach of matrix-comprehensive in-house factorial design validation. The software used for the factorial design and calculation was InterVal Plus (quo data, Gesellschaft für Qualitätsmanagement und Statistik GmbH, Dresden, Germany). Within the frame of the validation process, decision limit (CCα), detection capability (CCβ), precision, recovery, repeatability, in-house reproducibility, matrix effects, specificity and ruggedness of the method were studied. The validation process started with the factorial design (Table 2). For the determination of AFB1 in cereals, 8 runs, each at 6 concentration levels, were done within 8 days using different factor combinations. In total, 48 measurements were performed. Within each run, blank samples were fortified at six concentration levels: 2.5, 5, 7.5, 15, 30, and 60 μg/kg. In addition, a blank matrix sample, blank reagent sample and a fortified matrix sample were included into each run. For maize, CCα and CCβ of 5.86 μg/kg and 6.70 μg/kg were determined, respectively. Validation results observed with maize (Table 3), as also the results of other validation parameters determined with both cereals under study, proved LC-MS/MS suitable for AFB1 determination. Table 2. Factors of interest and their levels used for the determination of AFB1 in cereals Factor Level Operator Analyst 1 / Analyst 2 Cereal Maize / Barley Extraction 2h / 3h Storage of extracts (injection solution) 24 hours, +4 °C before injection/ without RC filter Producer 1- Agilent Technologies/ Producer 2 - Phenomenex
  31. 31. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.16 Table 3. Repeatability (sr), in-house reproducibility (sWR) and recovery established for LC-MS/MS used for the analysis of AFB1 in maize Spiked AFB1 concentration (μg/kg) sr (μg/kg) RSD (%) sWR (μg/kg) RSD (%) Recovery (%) 2.5 0.43 17.2 0.43 17.3 101.1 5.0 0.44 8.9 0.44 8.9 100.5 7.5 0.47 6.2 0.47 6.2 100.3 15 0.57 3.8 0.57 3.8 100.1 30 0.86 2.9 0.88 2.9 100.0 60 1.56 2.6 1.63 2.7 99.9 2.3.2. LC-MS/MS Implementation Sample Preparation To 25 g of the sample, a 100 mL of the extraction solution (ACN/H2O=80/20) were added. The mixture was shaken for 2 hours on a horizontal shaker and afterwards filtrated through the Whatman black ribbon filter paper. One mL of the obtained filtrate was diluted with 3 mL of ultrapure water, mixed and filtrated through 0.45µm-RC filter. Forty µL of the sample were injected into the HPLC system. Conditions under Which LC-MS/MS Was Implemented LC-MS/MS method was used to confirm the presence of AFB1 in the samples in which this mycotoxin was initially determined at levels higher than MPLs using the ELISA method (that is to say, in the maize samples only). The HPLC (LC) system (1260 Infinity, Agilent Technologies, Santa Clara, USA) consisted of a degasser, a binary pump, an auto-sampler and a column compartment, and was coupled with a 6410 QQQ-mass spectrometer (MS) (Agilent Technologies, Santa Clara, USA). HPLC separation was performed on XBridge BEH C18 columns (150x4.6, particle size 2.5 μm) at the flow rate of 0.80 mL/min and the temperature of +40 °C. The mobile phase consisted of the constituent A (0.1%-formic acid dissolved in water) and the constituent B (acetonitrile). A gradient elution program was employed as follows: 0-3 min: 90%-A, 18 min: 10%- A, 18.1 min: 90%-A, with the post-run time of 4 min and the injection volume of 40 μL. The conditions under which the mass spectrometry was performed were as follows: electro-spray ionization, positive polarity, capillary voltage of 6 kV, source temperature of +350 °C, nebulizer operating pressure of 45 psi, and the gas flow rate of 9 L/min. The mass
  32. 32. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 17 spectrometer was operated in the multiple reaction monitoring mode, the protonated molecular AFB1 ion at m/z = 313 being the precursor ion. Two product ions at m/z = 285 and m/z = 241 were monitored. The quantification was performed during the most intense transition (m/z 313 → 285) by virtue of extrapolation from six-point calibration curves. 2.4. AFB1 Concentrations Determined in Cereals Statistical analysis of data on AFB1 concentrations obtained by the two methods, was performed using the Statistica Software Ver. 10.0 (StatSoft Inc. 1984-2011, USA), with the statistical significance set at 95%-level (p=0.05). AFB1 presence detected using ELISA was confirmed by virtue of LC-MS/MS, indicating a high concordance between these two methods when employed to the effect of AFB1 determination. The results of AFB1 analyses per each investigated cereal harvested during 2010-2012 period on different fields, together with the determined number (No) and percentage of positive samples, the average (mean), as well as minimal and maximal concentrations and the accompanying standard deviations (SDs) obtained within this investigation, are summarized in Table 4. Table 4. Concentrations of AFB1 in cereals harvested during 2010-2012 period on different fields Cereal No. of total samples No. of positive samplesa Percentage of positive samples (%) Mean of positive samplesb (μg/kg) SD (μg/kg) Mine (μg/kg) Maxf (μg/kg) Maize 388 63 16.2 18.5c 20.3 1.9 97.5 Wheat 155 11 7.1 2.2d 1.0 1.1 3.0 Barley 148 8 5.4 1.5d 0.5 1.2 2.4 Oat 101 2 2.0 1.1 0.1 0.9 1.2 a AFB1 is detected (>LOD). b Mean AFB1 concentrations determined using ELISA and LC-MS/MS. c In 32/25 maize samples, AFB1 concentrations were higher than MRLs applicable to food / feed. d In 2 wheat/1 barley sample, AFB1 concentrations were slightly higher than MRL applicable to food. e Minimal AFB1 concentration determined among the positive samples. f Maximal AFB1 concentration determined among the positive samples.
  33. 33. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.18 Among the investigated cereals, maize was proven to be most contaminated, with AFB1 determined in 16.2% of samples, as compared to 7.1% AFB1-positive wheat, 5.4% AFB1-positive barley, and 2.0% AFB1- positive oat samples. Taking into account the contamination level of AFB1 in cereal samples detected in this research, and given the MPL for cereals intended for foodstuffs, which is 2 μg/kg for all cereals except for maize (to which the MPL of 5 μg/kg applies), it can be concluded that 32 maize samples (8.2%), 2 wheat samples (1.3%) and 1 barley sample (0.7%) had AFB1 concentrations over the MPL, whereas all oat samples met the stipulated value. Comparing the obtained AFB1 level to the MPL of 20 μg/kg, applicable to all cereals intended for feed, it can be concluded that levels higher than MPL were determined in 25 maize samples (6.4%), whereas all wheat, barley and oat samples had satisfied the given criterion. The maximal AFB1 level detected in the maize samples was 97.5 μg/kg, which is around 5 times higher than allowed for feed and even 20 times higher than allowed for food. The lowest number of positive samples and the lowest average concentration of AFB1 were observed with oat, AFB1 thereby being determined in only two samples at concentrations approximating to, or being slightly higher than, the ELISA limit of detection. In general, AFB1 levels higher than maximally allowed were exclusively found in the maize samples of 2012 genus, sampled mostly from fields in the eastern part of the country, i.e. the part known to have the largest grain production and the most developed farming in Croatia. The results of the analysis of variance (ANOVA) revealed statistically significant differences (p<0.05) in AFB1 concentrations between various types of samples under investigation (significantly higher in maize), but no differences (p>0.05) either in AFB1 concentrations determined across the same cereal group (barley, wheat or oat), or between the sampling regions, except for maize under any given scenario. Given the fact that elevated mycotoxin concentrations are usually associated with humidity and temperature as the factors most critical for mould formation and thus also mycotoxin production (Pleadin et al., 2013), the explanation of the results of this investigation could also be sought among these factors. In conclusion, such a high cereal contamination, especially that of maize, could likely be associated with climate conditions established in the investigated regions in the period of concern, which was extremely warm and dry (data obtained from the Croatian Meteorological and Hydrological Institute), which might had favored mould production and AFB1 formation. Therefore, an inadequate food/feed control could result in the consequent contamination of food and feed, which is even more worrying should one bear
  34. 34. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 19 in mind that the affected region is famous for its production of cereals, particularly that of maize, and its wide-scale use of the latter. 3. INVESTIGATION INTO THE POSSIBILITIES OF AFB1 REDUCTION IN MAIZE 3.1. Reduction of AFB1 Using Gamma Radiation The use of gamma (γ) radiation to inactive aflatoxins has already been investigated on some other materials; the results have shown that fungi that produce AFB1 can successfully be deactivated in paper, wood and soil using irradiation doses ranging from 6 to 15 kGy (da Silva et al., 2006). It has also been observed that doses higher than 10 kGy inhibit seed germination (Chiou et al., 1990). Aziz et al. (1997) reported that the dose required for the complete inhibition of fungi in different food and feed range from 4 to 6 kGy. After gamma irradiation with the dose of 1 and later on of 10 kGy, the toxicity of a peanut meal contaminated with AFB1 was reduced by 75% and 100%, respectively (Temcharoen and Thilly, 1982). The presence of water plays an important role in γ ray-based AFB1 destruction, since the radiolysis of water leads to the formation of highly reactive free radicals. These radicals can readily attack AFB1 at the terminal furan ring, yielding the material of lower biological activity (Rustom, 1997). Van Dyck et al. (1982) established the mutagenic activity of AFB1 in an aqueous solution (5 μg/mL water) to be reduced by 34%, 44%, 74% and 100% after the exposure to 2.5, 5, 10, and 20 kGy γ-rays, respectively. The dose of 10 kGy completely inactivated AFB1, and destroyed 95% of AFG1 in dimethyl-sulphoxide-water (1:9, v/v) solution (Mutluer and Erkoc, 1987). AFB1 degradation in range from 37% to 100% was observed after the addition of 1 mL of 5%-hydrogen peroxide to an aqueous AFB1 solution (50 μg/mL) under 2 kGy γ-irradiation. As the prevention of pathogenic fungi growth and the production of AFB1 in agricultural goods represents a very important issue, this study included the investigation into possibilities of reducing AFB1 detected in maize samples using γ-irradiation at the doses of 5 and 10 kGy (which were applied to 25 maize samples containing AFB1 in concentrations over MPLs set for feed). The radiation source was the 60 Co γ-irradiation chamber situated at Rudjer Boskovic Institute, Zagreb, Croatia.
  35. 35. Table 5. Concentrations of AFB1 in maize before and after γ-irradiation Range of AFB1 level in maize (μg/kg) Number of samplesa Mean AFB1 level before irradiation (μg/kg) Dose of 5 kGy Dose of 10 kGy Mean AFB1 concentration (μg/kg) Reduction (%) Mean AFB1 concentration (μg/kg) Reduction (%) 20-40 4 28.1 n.d.b 100 n.d. b 100 40-60 6 53.1 8.71 83.6 1.87 96.5 60-80 6 67.6 15.3 77.4 5.01 92.6 80-100 9 93.0 32.5 65.1 16.4 82.4 a Maize samples in which AFB1 concentrations were higher than MPL set for feed (20 µg/kg). b After maize samples‘ irradiation, AFB1 was not detected.
  36. 36. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 21 The exposure time was calculated based on the natural decay rate (the half- life) of the source and the location of the sample. The absorbed dose was measured using a dosimeter. The results obtained in our earlier preliminary studies showed that the dose of 2, 3 and 5 kGy can effectively stop the germination of aflatoxicogenic mould spores both in vitro and in situ (unpublished data). After γ-irradiation with the doses of 5 and 10 kGy, AFB1 level in the contaminated maize samples was determined using the ELISA method, as described earlier. The mean reduction of AFB1 achieved in the contaminated maize samples under this investigation using γ-radiation doses of 5 kGy and 10 kGy, ranged from 65.1% to 100%, and from 82.4% to 100%, respectively. As can be seen from the obtained results, gamma irradiation yielded a significant AFB1 reduction with both applied doses, especially with that of 10 kGy. It was also observed that the level of AFB1 reduction depends on the level of maize contamination, i.e. the higher the level of maize contamination, the lower the rate of AFB1 reduction, irrespective of the radiation dose applied (Table 5). 3.2. The Reduction of AFB1 Using Essential Oils and Lactic Acid Bacteria A novel way of reducing the proliferation of microorganisms and/or the production of their toxins is the use of essential oils. These oils are natural products extracted from plant materials, which have been proven to inhibit a wide range of food-spoiling microorganisms and the Aspergillus (Bluma et al., 2005). Essential oils applied to that effect insofar have shown a significant impact on AFB1 accumulation, their ultimate effect thereby being dependent on water activity, AFB1 concentration, and the length of incubation (Bluma and Etcheverry, 2008). In the study by Bluma et al. (2009), the effects of essential oils added to maize grains, in terms of their influence on mould growth rate, lag phase and AFB1 accumulation by Aspergillus section Flavi, were evaluated under different water activity conditions. The results showed that essential oils do influence the lag phase length and the mould growth rate, their efficacy thereby being dependent mainly on their concentration and water activity of the substrate; a significant impact on AFB1 accumulation was demonstrated as well. For the purpose of this investigation, the essential oils extracted from wild thyme, cinnamon, sage, lavender, and rosemary were used to examine the potential of controlling the aflatoxigenic fungi A. parasiticus 2999, A. flavus
  37. 37. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.22 305, A. niger 388 and their AFB1 production. Essential oils obtained from a local pharmacy were dissolved in 96 % (by volume) - ethanol (Kemika, Croatia) to the final concentration of 100 µL/mL. The inhibition of mould colonies‘ growth was determined on a PDA supplemented with an essential oil. The results showed that the growth and survival of food/feed-spoiling and AFB1-producing Aspergillus species can be controlled using essential oils, particularly that of wild thyme and cinnamon, which were the most effective in their inhibiting action. In the descending order of efficiency, these were followed by lavender, sage and rosemary essential oils. Wild thyme essential oil inhibited mould growth by about 85%, while cinnamon essential oil completely (100%) inhibited the growth of all tested moulds (Table 6). Soliman and Badeaa (2002) reported a complete inhibition of A. flavus, A. parasiticus and A. ochraceus by thyme and cinnamon essential oils added in concentrations lower than 500 mg/kg. In their research, inhibitory effects of essential oils or their components on mould growth were proportional to their concentration in the cultivation medium. It has been suggested that the mode of antifungal activity of essential oils could include their attack on the fungal cell wall and the retraction of hyphal cytoplasm, ultimately resulting in the mycelium‘s death. Montes-Belmont and Carvajal (1998) investigated the effect of eleven plant essential oils used for the protection of maize against A. flavus and found that the essential oils of cinnamon (C. zeylanicum), peppermint (Mentha piperita), basil (Ocimum basilicum), thyme (Thymus vulgaris), oregano (Origanum vulgare), flavoring herb epazote (Teloxys ambrosiodes) and clove (Syzygium aromaticum) caused a total inhibition of fungal development in maize kernels. In this investigation, the verification of AFB1 production was performed after 21 days of mould incubation in the YES broth (yeast extract 2%, sucrose 20%, and distilled water up to 1 L) into which essential oils were added in pre- defined concentrations. The results showed that only cinnamon oil completely inhibited the production of AFB1 in all tested moulds (Table 6). The addition of wild thyme essential oil significantly inhibited AFB1 production (about 75%) by A. parasiticus 2999, A. flavus 305 and A. niger 388. Approximately 68% of AFB1 production inhibition was attained by the addition of lavender essential oil. Rosemary and sage essential oils showed similar results, their addition inhibiting from 45 to 57% of the toxin production. The obtained results are in agreement with the data published by Atanda et al. (2007). These authors showed that essential oils of the aforementioned plant species can reduce the concentration of the produced AFB1 by about 90%.
  38. 38. Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 23 Table 6. Inhibitory effects (%) of essential oils on mould growth and AFB1 production Moulds/ AFB1 Inhibition (%) Wild thyme Cinnamon Lavender Sage Rosemary A. parasiticus 2999 AFB1 87 100 61 47 25 77 100 70 62 48 A. flavus 305 AFB1 89 100 72 53 27 80 100 68 58 43 A. niger 388 AFB1 81 100 68 58 42 74 100 65 51 43 The results presented in this section suggest that wild thyme, cinnamon and lavender essential oils could be efficiently used against fungi growth and AFB1 production in food and feed during the storage period. Several lactic acid bacteria have been found to be able to bind AFB1 in vitro and in vivo, their efficiency dependent on the bacterial strain. The inhibition of AFB1 accumulation was not related to the pH-decrease, but rather to the occurrence of low molecular weight metabolite produced by the lactic acid bacteria at the beginning of the exponential growth phase (Dalié et al., 2010). The investigation by El-Nezami et al. (1998) showed that probiotic strains such as Lb. rhamnosus GG and Lb. rhamnosus LC-705 are very efficient in removing AFB1, with more than 80% of the toxin trapped in a 20 μg/mL solution (Haskard et al., 1998). It has also been shown that other organisms such as Saccharomyces cerevisiae have the potential to bind AFB1 (Baptista et al., 2004) and are most efficient in AFB1 quenching (Bueno et al., 2006). In order to investigate the possibility of AFB1 reduction, several bacterial strains of lactic acid bacteria (LAB), originally isolated from the traditional Croatian fermented milk and meat products, were tested for their ability to bind aflatoxins. Lactobacillus delbrueckii S1, Lactococcus lactis subsp. lactis SA1, L. plantarum B and L. plantarum A1 were isolated from milk products, while L. plantarum 1K, Leuconostoc mesenteroides K5, Lactoc. lactis subsp. lactis 5K1 and L. acidophilus K6 were isolated from meat products and stored in the Collection of Microorganisms kept by the Laboratory of General Microbiology and Food Microbiology of the Faculty of Food Technology and Biotechnology, University of Zagreb (Croatia). Lactic acid bacteria were cultivated in 5 mL of the de Mann-Rogosa-Sharpe (MRS) broth at +37 °C for 24 h. Bacterial growth was determined using MRS agar plates after a 24 hour- incubation at +37 °C by virtue of traditional plate
  39. 39. Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.24 counting (CFU/mL). Ten mL of the MRS broth were inoculated with 10%- inoculums of each bacterial strain and artificially contaminated with AFB1 in the final concentration of 5 μg/mL. The bacteria and AFB1 introduced into the MRS broth were incubated (at +37o C) for 48 h. After centrifugation (3,500 x g for 10 min), the sample supernatants were collected at 12-, 24-, and 48-h time points. The unbound AFB1 was quantified using the ELISA method. Many studies have suggested that significantly different binding abilities of the LABs can be attributed to different cell – wall structures. In our study, L. plantarum A strain (isolated from cow cheese) exhibited a weaker binding ability (25.1 to 34.3%) than L. plantarum B (isolated from sheep cheese) in spite of their equal genetic structure, which could be explained by differences in their biological activities (Peltonen et al., 2001). Among eight LAB strains, L. delbrueckii S1 and L. plantarum 1K appeared to be the most efficient binders of AFB1, removing approximately 70% of the latter from the liquid media after 0 hours of incubation, which implies that the binding process runs swiftly. The inter-strain differences in AFB1 binding can probably be explained by different bacterial cell wall and cell casing structure. AFB1 is bound to LAB surface components; it appears that this binding involves a number of components (Haskard et al., 2001). In summary, the obtained results clearly show that probiotic strains L. delbrueckii S1, L. plantarum B, L. plantarum 1K and Leuco. mesenteroides K5 bind over 50% of AFB1 present in the MRS broth after a 48-h incubation (Table 7). Table 7. AFB1 binding by lactic acid bacteria AFB1 bound ± SDa (%) Incubation period (h) LAB 0b 12 24 48 L. delbrueckii S1 67.8±0.5 48.3±0.6 53.2±0.3 59.1±1.3 Lactoc. lactis subsp. lactis SA1 21.6±0.2 18.1±0.3 27.5±1.1 28.2±0.5 L. plantarum A 25.1±0.2 21.1±0.4 30.1±2.1 34.3±1.3 L. plantarum B 29.7±1.6 45.3±0.5 50.1±0.5 56.6±0.5 L. plantarum 1K 78.3±0.6 51.6±0.6 60.1±0.4 71.3±0.7 Leuco. mesenteroides K5 47.2±0.5 31.3±0.6 43.2±0.5 51.3±0.8 L. acidophilus K6 22.1±0.4 18.4±0.4 29.2±0.6 32.3±1.1 Lactoc. lactis subsp. lactis 5K1 19.8±0.8 16.3±0.2 25.5±0.6 27.2±0.5 a The results are expressed as the average values ± SDs obtained with triple assays. b 0-h sample collected after centrifugation.
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