IIIPart Mycotoxic Bioactives of Fruits and Cereals 253 12 Mycotoxic Bioactives in Cereals and Cereal Based Foods Anuradha Vegi Introduction Mycotoxic bioactives are secondary metabolites synthesized b[.]
Trang 1III Part Mycotoxic Bioactives of Fruits and Cereals
Trang 312
Mycotoxic Bioactives in Cereals
and Cereal-Based Foods
Anuradha Vegi
Introduction
Mycotoxic bioactives are secondary metabolites synthesized by toxigenic fungal species A wide variety
of mycotoxins are produced by various fungi, often a single fungal species can synthesize more than one type of the mycotoxic bioactive under optimal conditions These fungi and their mycotoxins pose a seri-ous threat to not only the plant species such as cereals on which they survive and grow, but also are toxic
to animal and human health who consume mycotoxin-contaminated cereal-based foods The detrimental effects of these toxic bioactives to plants and animals have been researched extensively
Some of the toxigenic fungal species and a variety of mycotoxins that they produce in some cereals and cereal-based foods will be discussed in this chapter The mycotoxin presence during some cereal-based
CONTENTS
Introduction 253
Mycotoxigenic Fungi in Cereals and Cereal-Based Foods 254
Mycotoxigenic Fusarium Species in Cereals 254
Mycotoxigenic Aspergillus in Cereals 254
Penicillium Species in Cereals 255
Mycotoxins in Cereals and Cereal-Based Foods 255
Aflatoxins 256
Ergot Alkaloids 257
Fumonisins 257
Ochrotoxins 257
Trichothecenes 258
Zearalenone 259
Occurrence of Mycotoxic Bioactives during Cereal-Based Food Processing 259
Barley Malting 260
Baking and Extrusion 260
Corn Milling 260
Worldwide Distribution of Mycotoxic Bioactives 261
Analytical Methods to Detect and Quantify Mycotoxins in Cereals 261
Microbiological (Culture) Methods 261
Gaseous Chromatography Methods to Detect Aspergillus, Fusarium, and Penicillium Volatiles 262
Polymerase Chain Reaction (PCR) Based Methods 262
Immunological Detection Methods 263
Summary 265
References 265
Trang 4food processing will also be included in the chapter Occurrence, worldwide distribution, and toxicities
of important mycotoxic bioactives will also be highlighted The chapter will also focus on some of the detection and quantification methods for mycotoxigenic fungal species and their toxins in cereals and cereal-based foods
A myriad of toxigenic fungal species and mycotoxins have been studied and even as you read this chapter, there are many more mycotoxic bioactives being discovered and studied worldwide The author
of this chapter would strongly encourage the readers to find literature that focus on new fungal bioactives and updated information on known mycotoxins
Mycotoxigenic Fungi in Cereals and Cereal-Based Foods
Cereal grains are the staple food sources worldwide for both animal and human consumption Cereal-based foods have high nutritional value including carbohydrates (45–76%), proteins (6–17%), and fat (1–7%), depending on the type of the cereal grains such as wheat, barley, oat, corn, and rice (Butt et al 2008; Shewry 2007; Sugiyama et al 2003; Thondre and Henry 2009; Zhao et al 2009) Many filamen-tous plant pathogenic fungi are known to attack cereal crops in the farm (field fungi) as well as during
storage (storage fungi) These fungi belong to many genera including Aspergillus, Fusarium, Penicillium
(Abarca et al 2001; Burgess et al 1987; Castella et al 2002; Muller et al 1997; Pitt 2000) More informa-tion of these fungi is given in the following secinforma-tions
Mycotoxigenic Fusarium Species in Cereals
Fusarium species belong to the field fungi group that attack cereal grains in the field Fusarium head
blight (FHB) is a disease of cereal grains like wheat, corn, oats, rye, and barley (Burgess et al 1987;
Muller et al 1997) In the United States, FHB is mainly caused by F graminearum (Stack 2003) The
yield and quality of grain are reduced due to FHB, and grain that is harvested is usually contaminated with trichothecene mycotoxins such as deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON; Cook 1981a; Kommedahl and Windels 1981) The trichothecene
mycotoxins produced by F graminearum affect grain safety and are also hazardous to human and animal
health (Prelusky et al 1994) Trichothecenes are also potent phytotoxins (Desjardins and Hohn 1997) Warm, wet weather conditions (like continuous wetness at 25°C) at the time of wheat anthesis were important factors for severe FHB in wheat (Bai and Shaner 1994; McMullen et al 1997a) Mutants of
F graminearum that do not produce DON were studied to determine the role of DON in plant
pathogen-esis (Proctor et al 1995) Additional studies confirmed that production of DON plays an important role in the spread of FHB within a spike of wheat (Bai 2001) Barley is also affected by FHB, which is endemic
in Northeast Asia (Cook 1981b) and has affected the Red River Valley region of the United States
caus-ing major losses (McMullen et al 1997b) Fusarium adversely affects maltcaus-ing barley Fusarium reduces
the kernel plumpness of infected malting barley as well as wort color during brewing process (Flannigan 1996; Schwarz et al 2001)
Corn, a staple food source for people worldwide including Mexico where human consumption of corn
products is as high as 60% of the total corn produced, is also susceptible to Fusarium species (Garcia
and Heredia 2006; Sydenham et al 1990) Fumonisins are a group of mycotoxins that are produced by
F verticillioides in corn, and they are highly toxic to both animals and human (Sydenham et al 1990) Rice, an important component of the diet in many Asian countries, is also plagued by the Fusarium species In Nepal, the common species found on rice were F verticillioides and F graminearum
(Desjardins et al 2000)
Mycotoxigenic Aspergillus in Cereals
Aspergillus species infect and grow in stored cereal grains and produce mycotoxins such as
aflatox-ins and ochratoxin A (OTA; Madhyastha et al 1993; Pitt 1987) Species known to produce OTA in
Trang 5cereals include A auricomus, A melleus, A ochraceus, A ostianus, A petrakii, A sclerotiorum, and
A sulfureus (in the A ochraceus group); A alliaceus and A albertensis (in section Flavi); A carbonar-ius and A niger (in section Nigri); A glaucus (in section Aspergillus), and P nordicum (Abarca et al
2001; Bayman et al 2002; Castella et al 2002; Dalcero et al 2002)
The chemical composition of cereal grains is reported to be changed due to colonization by ochra-toxin A-producing fungi When autoclaved barley and wheat samples were separately inoculated with
A alutaceus and P verrucosum and incubated at 28°C for 7, 15, and 30 days, the OTA production of
P verrucosum on both barley and wheat increased significantly over time (Madhyastha et al 1993) Higher OTA levels were produced by Aspergillus alutaceus on wheat than on barley The lipid con-tents of both barley and wheat decreased due to colonization by A alutaceus and P verrucosum, and
a small decrease in their starch content was observed Also, a higher concentration of protein in wheat was observed (Madhyastha et al 1993) Ochratoxin A has been reported to affect cereal grains and to
be nephrotoxic in animals that consume infected grain (Keblys et al 2004; Madhyastha et al 1993; Pitt 1987)
Aflatoxins are another set of mycotoxins that are produced by A flavus and A parasiticus in corn
and corn-based food products (Garcia and Heredia 2006) These mycotoxins are highly carcinogenic, cause liver disease, and in extreme cases of exposure even cause death (Azziz-Baumgartner et al 2005, Bandyopadhyay et al 2007, Williams et al 2004) These aflatoxigenic fungal species also affect other
crops such as peanuts and cottonseed (Horn 2007; Payne et al 1998) Aspergillus species also produce
other bioactive toxins such as citrinin in cereal grains such as wheat (Abramson et al 1990; Betina 1984)
Penicillium Species in Cereals
Many stored cereals and cereal-based food products are contaminated with mycotoxins produced
by Penicillium species Various factors are involved in the contamination of stored cereal grains by mycotoxigenic Penicillium Mechanical damage can occur at harvesting and also by rodents, birds,
and insects that facilitate infection by fungi Increasing moisture content, grain temperature, fungal spore content, and free fatty acids are the main factors involved in fungal spoilage of cereal grains (Abramson 1991)
The food substrate plays an important role on the type of mycotoxin produced in Penicillium species Penicillium verrucosum produced OTA and citrinin on bread, but the same strain produced only citrinin
when yeast extract agar (YES) was used as a substrate, and produced no mycotoxins when cheese was
used as a substrate (Kokkonen et al 2005) Penicillium nordicum, when tested on the same substrates, produced OTA on all three substrates used Penicillium crustosum produced another mycotoxin named
roquefortin C on all three substrates, and produced a mycotoxin, penitrem A, only on cheese (Kokkonen
et al 2005) Citrinin and OTA had nephrotoxic effects, whereas penitrem A and roquefortin C were neurotoxic to mammals such as swine (Keblys et al 2004)
Corn or maize silage, which is an important animal feed, is also reported to be contaminated with
the mycotoxigenic Penicillium species The researchers have reported that not only the ensiled corn
but also freshly harvested silage was contaminated with mycotoxins (patulin, mycophenolic acid,
cyclopiazonic acid, and roquefortine C) produced by Penicillium (Mansfield et al 2008) In a study
conducted during 2003–2005 among bakery mills of Lithuania, freshly milled rye flour were mostly
contaminated with Penicillium species including P biforme, P brevicompactum, P chrysogenum,
P cyclopium, P expansum, P roqueforti, and P velutinum These Penicillium species have the
capa-bility to produce mycotoxins such as citrinin, cyclopiazonic acid, OTA, patulin, and roquefortin C (Lugauskas et al 2006)
Mycotoxins in Cereals and Cereal-Based Foods
Bioactive mycotoxins are secondary metabolites produced by fungal pathogens in cereal grains and cereal-based foods under optimal conditions in the field, during storage, or in processing There are
Trang 6several types of mycotoxins that plague the cereal food industry Some of these mycotoxins are described
in the following sections
aflatoxins
Cereals are commonly contaminated with aflatoxins A wide variety of aflatoxins are produced by
et al 2009; Garcia and Heredia 2006) Structurally aflatoxins are polyketide-derived furanocoumarins and they are mainly divided into six types: aflatoxin B1, B2, G1, G2, M1, and M2 Aflatoxin B1 is the most common form as well as very carcinogenic (Brase et al 2009; IARC 1993)
O
O
O
O
O
O
O
O
O O O
OH OH
OH
CH3
CH3
CH3
H3C
H
.
HO HO
O
N N
N
N
N
NH2
O
CH3
CH3
CH3
H3C
H H
H
H
(a)
(b)
(c)
Figure 12.1 Structure of (A) aflatoxin B1, (B) ergotoxine, and (C) fumonisin B1 (From ChemID, Aflatoxin B1, Chem
National Library of Medicine, Betheseda, MD, 2009; Chem ID, Fumonisin B1, Chem ID Plus Advanced, National Library
of Medicine, Betheseda, MD, 2009.)
Trang 7When cereal-based food products contaminated with aflatoxins are consumed, they cause cancer, liver disease, and other health problems in animals and humans West African countries such as Nigeria are plagued by aflatoxin contamination where corn is an important part of the diet (Azziz-Baumgartner et al 2005; Bandyopadhyay et al 2007) Because of health concerns caused by aflatoxin containing foods, the United States and a few other countries have limited the dietary intake of aflatoxin to 20 ng/g and in Europe it is 4 ng/g (FAO 2004)
ergot alkaloids
Structurally ergot alkaloids belong to indole alkaloids derived from a tetracyclic ergoline ring system (Figure 12.1B; Brase et al 2009) Ergot disease caused by Claviceps purpurea can have up to 10% yield
loss in wheat and almost 5% in rye crops, as reported by McMullen and Stoltenow (2002) Barley is also reported to be affected by ergot alkaloids (Schwarz et al 2006) One of the first known diseases caused
by a mycotoxin in human populations and animals is ergotism Loss of limbs, gangrene, and an effect
on the central nervous system are attributed to ergot alkaloids in animals and humans (De Costa 2002; van Dongen and de Groot 1995) Even though ergot alkaloids are known to cause damage to both cereal crops and to animals and humans who consume cereal-based foods, no regulatory limits are set as yet for ergot alkaloids in cereals
Fumonisins
Fusarium group of fungi, especially F verticillioides and F proliferatum produce another set of
myco-toxins known as fumonisins in cereal grains such as corn, sorghum, and rice (CAST 2003) Their struc-ture is very similar to that of the backbone of sphingolipids (Figure 1C; ApSimon 2001; Brase et al 2009) The fumonisin group consists of fumonisins A1-A4, B1-B4, C1-C4, and P1-P4; however, fumoni-sins B1, B2, and B3 are of major concern in cereals and cereal-based foods
Fumonisins are the causative agents in the brain damage of horses known as leukoencephalomalacia (Marasas et al 1988) The fumonisins in corn also affect human populations as they are reported to be involved in esophageal cancer (Rheeder et al 1992) In experimental animals, such as rodents, fumonisin B1 is considered to be hepatotoxic (Domijan et al 2008) Because of their prevalence in corn and corn-based foods, the maximum levels for total fumonisins in those foods were set by the FDA at 2–4 µg/g
fumonisins in processed corn baby food (FAO 2004)
Ochrotoxins
Two main types of ochratoxins are present including ochratoxin A (OTA) and B (OTB) Ochratoxin A
is a fungal secondary metabolite that is a chlorinated isocoumarin derivative linked to L-phenylalanine
fungal species, A ochraceus and P verrucosum in stored cereal grains (Pitt 2000) A liquid
chromato-graphic method was used to test for the presence of OTA in wheat, barley, green coffee, and roasted coffee in the United States Ochratoxin A contamination (>0.03 ng/g) was found in 56 of 383 wheat samples, 11 of 103 barley samples, nine of 19 green coffee samples, and nine of 13 roasted coffee
>5 ng/g OTA, indicating that cereal grains are more susceptible to OTA producing fungi (Trucksess
et al 1999)
Ochrotoxin has been classified as a possible carcinogen for humans and it is a potent teratogen and hepatotoxin (Lindsey 2002; Petziner and Ziegler 2000) It has been established that OTA has an immu-nomodulatory effect on a human monocyte/macrophage cell line (Muller et al 2003) and also is involved
in human Balkan endemic nephropathy (Castegnaro et al 2006) Because of their toxigenicity in animals and humans, there have been strict rules in European countries where maximum levels of 3 ng/g are set for OTA in cereal-based foods (FAO 2004)
Trang 8Trichothecene mycotoxins are bioactive secondary metabolites produced in infected grains by many
Jarvis 1991; Sharma and Kim 1991) These are toxic to humans as well as animals (Prelusky et al 1994;
Wache et al 2009) Mycotoxigenic fungi such as F graminearum produces 8-ketotrichothecenes
includ-ing deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV), and 4-acetyl nivalenol (4-ANIV), as well as the estrogenic mycotoxin zearalenone (ZEN; Mirocha et al 1989; Seo et al 1996) The trichothecenes are all tricyclic sesquiterpenes with
non-macrocyclic trichothecenes are T-2 toxin, diacetoxyscirpenol, and DON (Jarvis 1991) Fusarium sporo-trichioides is the primary species that produces mycotoxins such asT-2 toxin and diacetoxyscirpenol in
cereal grains (Abramson et al 1993)
Fusarium graminearum isolates can be characterized as chemotypes based on the type of trichoth-ecenes they produce There are three main chemotypes (Ia, Ib, and NIV chemotypes) of F graminearum
The chemotype Ia produces DON and 3-ADON; chemotype Ib produces DON and 15-ADON; and the NIV chemotypes produce NIV and 4-ANIV (Ichinoe et al 1980; Moss and Thrane 2004) The NIV chemotypes are not found in North America but are reported in Africa, Asia, and Europe (Ichinoe et al
1980, 1983)
Deoxynivalenol, an important trichothecene is produced by many species of Fusarium including Fusarium graminearum, F avenaceum, F crookwellense, F culmorum, F poae, and F sporotrichioides
(Abramson et al 1993) This trichothecene mycotoxin is phytotoxic to many cereal grains such as corn, wheat, and barley (Cosette and Miller 1995; Salas et al 1999; Wakulinski 1989) Deoxynivalenol inhib-its germination and root growth in wheat (Wakulinski 1989) Trichothecene mycotoxins such as DON, 3-ADON are also toxic to animals Corn was contaminated with DON and ZEN in the northeastern
O
O
O
O
O
OH
HO
OH
OH
CH3
CH3 Cl
N H (a)
(b)
Figure 12.2 Structure of (A) ochratoxin A and (B) zearalenone (From Chem ID, Ochratoxin A, Chem ID Plus
Library of Medicine, Betheseda, MD, 2009.)
Trang 9states of the United States and Canada, thus grain buyers in those regions temporarily stopped purchas-ing corn in 1991 (Bergstrom 1991) The neurotoxic effects of DON lead to feed refusal and reduced weight gain in swine (Prelusky 1997) A reduction in the uptake of sugars (glucose) and minerals was observed in mice fed with DON contaminated food (Hunder et al 1991) Human immune defense cells such as macrophages are reported to be affected by DON Immunosuppression and inhibition of cell surface activation markers of human macrophages were observed when exposed to low doses (150 µM)
of DON (Wache et al 2009) The maximum limit is set at 1 µg/g for DON in finished wheat products by U.S Food and Drug Administration
Zearalenone
Barluenga 2007) This mycotoxin was first isolated from Fusarium graminearum in 1962 It coexists with other Fusarium mycotoxins (CAST 2003) Hyperestrogenism is caused by ZEN due to its similarity
with 17-estradiol in the binding to cytosolic estrogen receptors (Kuiper-Goodman et al 1987) This myc-otoxin has been reported to be affecting male (decreased spermatozoa) and female reproductive systems (early puberty) in animals and human populations (Etienne and Dourmad 1994; Shier et al 2001; Yang
et al 2007) The European Union has set regulatory limits of ZEN from 20 to 200 ng/g in unprocessed and processed cereal-based foods (FAO 2004)
Occurrence of Mycotoxic Bioactives during Cereal-Based Food Processing
Fungi infect, survive, grow, and produce mycotoxins in cereal-based foods while being processed under optimal conditions The mycotoxin levels in various cereal-based food processing stages can increase or decrease depending on the type of processing step This section of the chapter gives brief information on
a few mycotoxins and their fate during various stages of cereal-based food processing
O
O O
OH
OH
OH
CH3
H3C
O
O O
HO HO
OH
OH
CH3
H3C
O
O
O
O O O
HO
CH3
CH3
CH3
CH3
CH3
CH3 (c)
Figure 12.3 Structure of important trichothecene mycotoxins (A) deoxynivalenol, (B) nivalenol, and (C) T-2 toxin
(From Chem ID, Deoxynivalenol, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; Chem
ID, Nivalenol, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; Chem ID, T-2 toxin, Chem
Trang 10barley Malting
Barley is an important cereal crop, used in the malting and brewing processes for beer production and used as livestock feed (Noots et al 1998; Schwarz et al 1995, 2001) The malting process is divided into three main steps including steeping, germination, and kilning (Karababa et al 1993; Noots et al 1998) During the steeping process, the barley is soaked in water at 12–20°C for 36–52 hours to elevate the moisture content of the barley to 42–45% (Noots et al 1998; Schwarz 2001) During the steeping process, the grain is allowed to have brief air-rests The germination process follows steeping and lasts for 4–5 days at 15°–20°C with controlled humidity After germination, the green malt is subjected to higher temperatures during the kilning process for 18–24 hours The
Schwarz 2001)
Researchers have reported that Fusarium infection of barley kernels increased 15–90% during the
steeping step of malting (Douglas and Flannigan 1988; Flannigan 1996) There was an increase in
Fusarium species, colony forming units (CFU) from 300 cfu/g to 8000 cfu/g during malting (Flannigan
et al 1984) Schwarz et al (1995) have reported that after 5 days of germination there was a significantly
(p <.05) higher level of DON in germinated barley than during steeping Steeping decreased the
prehar-vest formed DON concentration, as DON is water soluble The DON levels increased by 18%–114% after five days of germination (Schwarz et al 1995)
The kilning process did not affect the DON concentration, as DON is heat stable (Niessen et al 1991;
Schwarz et al 1995) Deoxynivalenol and diacetoxyscirpenol produced by Fusarium affected the
malt-ing process, and FHB infected barley resulted in gushmalt-ing of beer (Munar and Sebree 1997; Schapira
et al 1989) Mycotoxins were found in the beer after contaminated barley was used for brewing (Munar and Sebree 1997; Schwarz et al 1995)
baking and extrusion
High temperature processing steps such as baking seem to show a varied affect on different myco-toxins Some mycotoxins such as OTA were not destroyed during bread making (Scudamore 2003; Subirade 1996) However, DON—which is supposed to be a heat-resistant mycotoxin—decreased
in concentration during baking of bread, biscuits, and cookies (Scott et al 1983) In a study on the effect of wheat bread making on aflatoxins, the total aflatoxins concentration were reduced by 41% after baking (El-Tawila et al 2003) Jackson et al (1997) have shown that the interior regions of baked corn muffins had increased levels of fumonisin B1 when compared to the outer layers of the muffin, indicating destruction of fumonisins under high temperature processing Breakfast cereals
pressures, and severe shear forces are used in extrusion cooking (Bullerman and Bianchini 2007; Harper 1992) In a study by Castells et al (2006), extrusion cooking—using a single screw extruder
decreased OTA
Corn Milling
A recent study by Scudamore and Patel (2009) has indicated that in dry corn milling, the endosperm
of the grain contained low levels of Fusarium mycotoxins, such as deoxynivalenol, zearalenone,
and fumonisins However, embryo and outer grain layers (used mostly as animal feed) had up to five times more concentration of mycotoxins (Scudamore and Patel 2009) In a study by Castells et al (2008), a similar trend was observed where the outer layers of corn (used in animal feed flour and corn flour) had relatively high levels of mycotoxins such as fumonisins B1, B2, and B3, and aflatoxins B1, B2, G1, and G2 Also, they observed that corn meal and flaking grits had lower levels of myco-toxins (Castells et al 2008) Thus, in the corn milling process, the mycomyco-toxins are not completely eliminated; however, the concentrations of these toxins differ in various fractions of corn and corn-based products