FOOD SCIENCE AND TECHNOLOGY
FOOD SOURCES, OCCURRENCE
AND TOXICOLOGICAL EFFECTS
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FOOD SCIENCE AND TECHNOLOGY
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FOOD SCIENCE AND TECHNOLOGY
FOOD SOURCES, OCCURRENCE
AND TOXICOLOGICAL EFFECTS
ADINA G. FAULKNER
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
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
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
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
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
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.
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,
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
). Within this
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
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
, 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
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
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
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
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.
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.
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
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
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
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
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
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
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
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.,
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
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.
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
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
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;
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.
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
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).
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
Jelka Pleadin, Ksenija Markov, Jadranka Frece et al.14
Table 1. Results of the ELISA method validation
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
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
184.108.40.206. 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.
220.127.116.11. 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
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
Operator Analyst 1 / Analyst 2
Cereal Maize / Barley
Extraction 2h / 3h
Storage of extracts
24 hours, +4 °C before injection/
RC filter Producer 1- Agilent Technologies/
Producer 2 - Phenomenex
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
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
18.104.22.168. 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.
22.214.171.124. 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
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. Concentrations of AFB1 in cereals harvested during 2010-2012
period on different fields
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
AFB1 is detected (>LOD).
Mean AFB1 concentrations determined using ELISA and LC-MS/MS.
In 32/25 maize samples, AFB1 concentrations were higher than MRLs applicable to
food / feed.
In 2 wheat/1 barley sample, AFB1 concentrations were slightly higher than MRL
applicable to food.
Minimal AFB1 concentration determined among the positive samples.
Maximal AFB1 concentration determined among the positive samples.
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
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.
Table 5. Concentrations of AFB1 in maize before and after γ-irradiation
AFB1 level in
Mean AFB1 level
Dose of 5 kGy Dose of 10 kGy
20-40 4 28.1 n.d.b
100 n.d. b
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
Maize samples in which AFB1 concentrations were higher than MPL set for feed (20 µg/kg).
After maize samples‘ irradiation, AFB1 was not detected.
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
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
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%.
Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 23
Table 6. Inhibitory effects (%) of essential oils on mould growth
and AFB1 production
Wild thyme Cinnamon Lavender Sage Rosemary
A. parasiticus 2999
87 100 61 47 25
77 100 70 62 48
A. flavus 305
89 100 72 53 27
80 100 68 58 43
A. niger 388
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
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)
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
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
The results are expressed as the average values ± SDs obtained with triple assays.
0-h sample collected after centrifugation.
Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 25
The highest level of cereal AFB1 contamination was observed with maize
in comparison to wheat, barley and oat (in which the lowest AFB1 levels were
observed). Radiation-based technology could be used as an effective method
of mould growth & development prevention and the reduction of AFB1 in food
and feed. The results pointed towards the possibility of essential oils usage as
an alternative method of AFB1 reduction in agro-industries. Lactic acid
bacteria, characterized as functional cultures and proven to bind mycotoxins,
could also be used for human and animal protection against harmful effects of
mycotoxins. The toxicity of AFB1 and its seemingly unavoidable occurrence
in cereals later used as food and feed components, make the prevention and
detoxification of this mycotoxin the most challenging toxicological problem
that needs further studying and the establishment of an effective control using
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