Ebook fruit and cereal bioactives sources, chemistry, and applications – part 2

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[.]

Part III Mycotoxic Bioactives of Fruits and Cereals 12 Mycotoxic Bioactives in Cereals and Cereal-Based Foods Anuradha Vegi 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 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 serious 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 253 254 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications food 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 Cerealbased 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 filamentous 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 information of these fungi is given in the following sections 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 not produce DON were studied to determine the role of DON in plant pathogenesis (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 causing major losses (McMullen et al 1997b) Fusarium adversely affects malting 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 producing various mycotoxins such as beauvericin, moniliformin, gibberellic acid, nivalenol (NIV) and DON (Desjardins et al 2000) Mycotoxigenic Aspergillus in Cereals Aspergillus species infect and grow in stored cereal grains and produce mycotoxins such as aflatoxins and ochratoxin A (OTA; Madhyastha et al 1993; Pitt 1987) Species known to produce OTA in Mycotoxic Bioactives in Cereals and Cereal-Based Foods 255 cereals 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 carbonarius 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 ochratoxin 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 contents 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 capability 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 256 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications several 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 mycotoxigenic Aspergillus flavus and A parasiticus in cereals and cereal-based foods (Figure 12.1A; Brase 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) (a) O O O O O CH3 O O (b) N H O H N O O H3C N H CH3 O N CH3 H N H (c) OH O OH H3C O OH CH3 O OH CH3 O O HO O CH3 OH NH2 O HO O Figure 12.1  Structure of (A) aflatoxin B1, (B) ergotoxine, and (C) fumonisin B1 (From ChemID, Aflatoxin B1, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; ChemID, Ergotoxine, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; Chem ID, Fumonisin B1, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009.) Mycotoxic Bioactives in Cereals and Cereal-Based Foods 257 When 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 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 mycotoxins known as fumonisins in cereal grains such as corn, sorghum, and rice (CAST 2003) Their structure 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, fumonisins 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 cornbased foods, the maximum levels for total fumonisins in those foods were set by the FDA at 2–4 µg/g (depending on the type of corn product) and European countries adopted a stricter limit of 0.2 µg/g of 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 and the most important mycotoxin in cereals (Figure 12.2A) Ochratoxin A is produced by two main fungal species, A ochraceus and P verrucosum in stored cereal grains (Pitt 2000) A liquid chromatographic 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 samples (Trucksess et al 1999) Four samples of wheat and one sample of barley were contaminated with >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 immunomodulatory 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 ng/g are set for OTA in cereal-based foods (FAO 2004) 258 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications (a) O OH O OH O O N H CH3 Cl (b) OH CH3 O O HO O Figure 12.2  Structure of (A) ochratoxin A and (B) zearalenone (From Chem ID, Ochratoxin A, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; Chem ID, Zearalenone, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009.) Trichothecenes Trichothecene mycotoxins are bioactive secondary metabolites produced in infected grains by many fungi such as Myrothecium, Stachyobotrys, Fusarium, Trichoderma, and Trichothecium (Figure 12.3; 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 including 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 a 12,13-epoxy-trichothec-9-ene ring Trichothecenes are macrocyclic or nonmacrocyclic depending on the presence of a macrocylic ester or an ester-ether bridge between C-4 and C-15 (Chu 1998) The nonmacrocyclic trichothecenes are T-2 toxin, diacetoxyscirpenol, and DON (Jarvis 1991) Fusarium sporotrichioides 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 trichothecenes 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 inhibits 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 259 Mycotoxic Bioactives in Cereals and Cereal-Based Foods (a) H (b) H H3C H OH O H O H3C O OH O O O CH3 OH OH HO CH3 HO OH (c) CH3 O O O HO CH3 CH3 CH3 CH3 O O O O O CH3 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 ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009.) states of the United States and Canada, thus grain buyers in those regions temporarily stopped purchasing 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 µg/g for DON in finished wheat products by U.S Food and Drug Administration Zearalenone Zearalenone belongs to benzannulated macrolactones (Figure 12.2B; Brase et al 2009; Winssinger and 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 mycotoxin 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 260 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Barley 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 temperatures during kilning vary over a range of 40°C–50°C to 80°C–90 oC (Hough et al 1971; 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 days of germination there was a significantly (p 150°C), very high 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 of barley meal—reported that higher residence time (70 s) and medium temperature level (160 oC) 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 mycotoxins (Castells et al 2008) Thus, in the corn milling process, the mycotoxins are not completely eliminated; however, the concentrations of these toxins differ in various fractions of corn and cornbased products Mycotoxic Bioactives in Cereals and Cereal-Based Foods 261 Worldwide Distribution of Mycotoxic Bioactives Cereals and cereal-based foods are attacked by mycotoxigenic fungi and their mycotoxins worldwide Depending on the type of staple food used in various parts of the world, the type of mycotoxin attacking cereals and cereal-based foods also vary In some parts of the world such as North America, cereals such as wheat and barley and their products are mostly affected by the mycotoxins, whereas in Asian regions of the world, cereals such as rice and rye are contaminated with mycotoxins (Desjardins et al 2008; Ng et al 2009) Deoxynivalenol producing Fusarium in cereals is common in many regions of the world However, in some countries of Asia (such as Nepal) there has been a higher prevalence of virulent nivalenol producing Fusarium graminearum in cereals such as corn (Desjardins et al 2008) Cereal grains, such as wheat and barley in Poland, were contaminated with OTA that was produced by both P verrucosum and A ochraceus, of which Penicillium species was the major source of OTA produced (93%; Czerwiecki et al 2002) A survey of the on-farm stored cereal grains such as wheat, barley, and oats in the United Kingdom showed that 21% among 306 samples were contaminated with OTA, and barley was reported to be more susceptible to OTA contamination than wheat (Scudamore et al 1999) A recent survey conducted in Canadian dry pasta samples (n = 274) indicated more than 0.5 ng/g of OTA present in 21, 18, and 66%, respectively, of pasta samples, collected during years 2004, 2005, and 2006 The degree of contamination with mycotoxins such as OTA is thus variable, depending on the wheat crop year (Ng et al 2009) A study conducted in corn tortilla and masa flour samples from California, had fumonisins in all samples (n = 38) Corn-based foods are thus very susceptible to fumonisin contamination and daily consumption of highly contaminated (>1000 ng/g of fumonisins) cornbased products can be prevented to reduce the risk of disease in potentially pregnant women and their offspring (Dvorak et al 2008) Analytical Methods to Detect and Quantify Mycotoxins in Cereals Many methods are available that can detect and quantify mycotoxigenic bioactives in cereals Some methods are specific for a single mycotoxin (Kabak 2009), whereas recent studies focus on methods that can analyze multiple mycotoxins in cereals and cereal-based foods (Frenich et al 2009; Garon et al 2006) There are different methods for analyzing mycotoxins in cereals including but not limited to microbiological, chromatographical, and polymerase chain reaction (PCR) based assays for the detection of mycotoxigenic fungi in cereals, and immunoaffinity clean-up/fluorescence detection methods for mycotoxins However, the method of choice for many researchers depends on how quick, sensitive, reliable, specific, and cost-effective the method Microbiological (Culture) Methods Cereal grains if infected can be positively identified using culture methods Validation of many modern assays to identify and quantify mycotoxigenic fungi is done primarily using traditional culture methods (Bluhm et al 2002, 2004) To determine the mycological inhabitants on the cereal grains, direct plating of the grains can be done on the growth media Also, surface sterilization before plating of the cereal grains as an initial step can help enumerate the internal fungi (Samson et al 2000) Selective media can help isolate and identify specific mycotoxigenic fungi from cereal grains and cereal-based food products Ochratoxin producing P verrucosum can be isolated from cereal grains using dichloran yeast extract sucrose glycerol agar (DYSG; Frisvad et al 1992; Samson et al 2000) For isolating aflatoxin producing A flavus and A parasiticus species, aspergillus differential medium (ADM) is very helpful (Bothast and Fennel 1974) Similarly for Fusarium species, czapek iprodione dichloran agar (CZID) can be selectively identified in cereal foods (Abildgren et al 1987; Samson et al 2000) These microbiological methods are very useful in isolating and detecting 262 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications mycotoxigenic fungi, however they are labor intensive and time-consuming Thus, other methods have been developed that can be performed in less-time and give reliable results Gaseous Chromatography Methods to Detect Aspergillus, Fusarium, and Penicillium Volatiles Fungal deterioration of stored cereals can be detected at an early stage Volatile compounds are produced by fungi in stored grains (Abramson et al 1980) These volatiles can be detected by several chromatographic techniques such as gas chromatography–mass spectrometry (GC–MS) and other methods For detection of the volatiles produced by the storage fungi in cereals, a sample collection is the primary step Various methods can be applied to collect the samples Headspace methods include direct collection of gaseous volatiles released by the fungi, onto an adsorbent such as activated carbon (Abramson et al 1980) Sometimes the volatiles, which are released into the growth medium, should be extracted and this can be done using steam distillation and extraction or supercritical fluid extraction (Kaminski et al 1972) Another important method known as headspace solid-phase microextraction (SPME) can be used to extract the volatiles The volatiles from the headspace can be extracted onto a fused silica fiber coated with a polymeric organic liquid and then can be directly transferred to a gas chromatography (GC) machine and analyzed (Nilsson et al 1996) Volatile fungal metabolites have been used as indicators of fungal growth in stored cereal grains (Borjesson et al 1992; Tuma et al 1989) Various volatiles found include 3-octanone, 1-octen-3-ol, and 3-methyl-1-butanol, and 3-methylfuran (Abramson et al 1980; Borjesson et al 1989, 1990; Tuma et al 1989) The 3-methylfuran was produced by many fungi such as Penicillium brevicompactum, P glabrum, P roqueforti, Aspergillus flavus, A versicolor, and A candidus during early stages of growth on wheat and oats (Borjesson et al 1992) Some volatiles are unique to some fungal species such as thujospene, which is produced by Aspergillus, and not produced by Penicillium species Penicillium glabrum produces 3-octanone and P brevicompactum is reported to produce high amounts of acetone (Borjesson et al 1992) These volatiles were reported to be correlated positively with accumulated carbon dioxide and ergosterol in cereals (Borjesson et al 1992) Pasanen et al (1996) showed that P verrucosum can be differentiated into toxigenic and nontoxigenic isolates based on volatile production High amounts of ketones are produced by ochratoxin-producing P verrucosum species when compared to nontoxigenic isolates of P verrucosum Fusarium species also produce various volatiles from stored grain Fusarium sambucinum produced sesquiterpenes such as β-farnesene, β-chamigene, β-bisabolene, α-farnesene, and trichodiene on wheat kernels (Jelen et al 1995) These volatiles produced by fungi in stored grain were also correlated with mycotoxin production F sporotrichioides produced volatile terpenes that correlated with the trichothecene mycotoxins including T-2 toxin, neosolaiol, diacetoxyscirpenol, HT-2 toxin, and T-2 tetraol (Pasanen et al 1996) Olsson et al (2002) reported that mycotoxin contamination in grains can be detected by determining the volatiles produced by the storage fungi in barley They found that barley samples with a normal odor had no detectable OTA, whereas the samples that had off-odor had an average OTA of 76 µg/kg and 69 µg/kg of DON The samples with more OTA produced higher amounts of ketones such as 2-hexanone, 3-octanone (Olsson et al 2002) Polymerase Chain Reaction (PCR) Based Methods The PCR is an assay that can be used to amplify a specific deoxyribonucleic acid (DNA) fragment of a fungal species and can be used to identify the species (Niessen and Vogel 1997; White et al 1990) Many PCR assays have been developed to detect and quantify mycotoxigenic fungi in cereal grains Assays involving DNA can help in quick, reliable, and specific detection and quantification of fungi in cereal grains Fusarium graminearum was detected and quantified using a PCR assay (Niessen and Vogel 1998) Fungal ribosomal DNA (rDNA) genes have been used to design DNA primers for PCR reactions These genes are highly conserved and are species-specific (White et al 1990) Primers Mycotoxic Bioactives in Cereals and Cereal-Based Foods 263 used in PCR can be used for species-specific ­detection of mycotoxigenic fungi F graminearum was detected by developing a PCR assay utilizing the primers specific to the galactose oxidase gene, which is produced by very few fungal species including F acuminatum, F subglutinans, and F graminearum (Barbosa-Tessmann et al 2001; Niessen and Vogel 1997) Fusarium culmorum, F graminearum, and F avenaceum were detected and differentiated by using species-specific primers for the internal transcribed spacer (ITS) region of the rDNA genes (Schilling et al 1996) Edwards et al (2001) developed a quantitative PCR assay for the trichodiene synthase gene (Tri5) of trichothecene-producing Fusarium The researchers found a positive correlation between Tri5 DNA and DON produced by F culmorum and F graminearum in winter wheat Fungal species have also been grouped using their rDNA genes and results have been correlated with mycotoxigenicity An A niger species aggregate (92 isolates) was tested for OTA production These isolates were grouped into the two species, A niger and A tubingensis, by using their ITS-5.8S rDNA restriction fragment length polymorphism (RFLP) patterns Only six out of the 92 isolates studied produced OTA, and these OTA isolates were A niger isolates (Accensi et al 2001) Traditional PCR assays can be used to detect and quantify a single gene as well as more than one gene in a single reaction (multiplex PCR; Nicholson et al 1998; Niessen and Vogel, 1998) Group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium was done using multiplex PCR assays in cornmeal (Bluhm et al 2002) However, these assays are time-consuming as they involve post-PCR processes such as gel electrophoresis Real-time PCR with SYBR Green I dye or TaqMan probes quantify PCR products in less time and not involve gel electrophoresis (Bluhm et al 2004; Reischer et al 2004; Schnerr et al 2001) Real-time PCR involving TaqMan probes has been used for specific detection and reliable quantification of fungal pathogens (Bluhm et al 2004; Geisen et al 2004; McDevitt et al 2004) Figure 12.4 depicts the TaqMan probe mechanism of real-time PCR assays These probes are oligonucleotides with a reporter dye on the 5′ end and a quencher dye on the 3′ end that attach to specific DNA sequences during the PCR cycles (Geisen et al 2004; Heid et al 1996; McDevitt et al 2004) When the quencher dye is in close proximity to the reporter dye, there is no fluorescence emission However, when the DNA polymerase enzyme comes in close contact with the probes during PCR, the 5′ nuclease activity of the enzymes cleaves the probes, separating quencher and reporter dyes on the probes and thereby increasing the fluorescence emission (Heid et al 1996) The accumulation of PCR products is detected by monitoring the increase in fluorescence of the probe (Bluhm et al 2004) TaqMan probes only attach to specific sequences of DNA and different quencher-reporter dye combinations can be used for different genes to be detected and quantified, thus they can be used in multiplex real-time PCR to detect more than one fungal pathogen The amplification products formed during real-time PCR can be quantified by performing standard curve analysis Also, correlation studies can be done to validate the real-time PCR assay data Aspergillus flavus was detected and quantified using real-time PCR for the nor-1 gene involved in the aflatoxin biosynthetic pathway There was a positive correlation between the copy number of the nor-1 gene determined by real-time PCR and the CFU of A flavus in wheat (Mayer et al 2003) Immunological Detection Methods Enzyme-linked immunosorbent assays (ELISA) and immunoblotting techniques have been used to detect and quantify mycotoxigenic fungi (Iyer and Cousin 2003; Lu et al 1995; Skaug 2003) An indirect ELISA was developed to detect F graminearum and F verticillioides in foods and the detection limits for F graminearum and F verticillioides were 0.1 and µg/ml, respectively (Iyer and Cousin 2003) An ELISA method that was very specific for OTA producing A ochraceus showing no cross-reactivity with Aspergillus, Penicillium, Fusarium, Mucor, and Alternaria exoantigens was developed by using rabbit antibodies that were produced against the exoantigens of A ochraceus (Lu et al 1995) The immunoaffinity column (IAC) can be used for sample clean-up for higher recovery of mycotoxins from cereals and cereal-based foods A cartridge containing solid support such as agarose gel on 264 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Denaturation Annealing Primer Primer Taq polymerase TaqMan probe Reporter Extension Quencher Cleaved quencher Fluorescing reporter Cleaved nucleotides Amplified DNA Figure 12.4  Depiction of TaqMan probe mechanism in real-time PCR used to detect and quantify mycotoxigenic fungi in cereals and cereal-based foods which the anti-mycotoxin antibody that is immobilized is used in IAC Visconti et al (2005) developed a sensitive and accurate method for simultaneous detection of T-2 and HT-2 toxins in cereal grains using immunoaffinity clean-up coupled with high-performance liquid chromatography (HPLC) with fluorescence detection The IAC in the study containing monoclonal anti T-2 antibodies helped in capturing the T-2 and HT-2 toxins of cereals such as wheat, corn, and barley Then these toxins were eluted and quantified by reversed-phase HPLC with fluorometric detection (Visconti et al 2005) Immunologists claim mycotoxins produced by the fungi in cereal foods can be directly detected by ELISA with less cost However, the detection limits (0.05 ng – µg) of these assays to quantify the fungi or their mycotoxins limit their use when compared to PCR assays whose detection limit is as low as pg (Bluhm et al 2004) Mycotoxin ELISA methods are also known to be cross-reactive with interfering substrates in food samples In recent studies, immunological methods are preferred again Mycotoxic Bioactives in Cereals and Cereal-Based Foods 265 as higher recovery rates and lower detection limits of the mycotoxins in cereal grains can be achieved with the IAC clean-up (Brenn-Struckhofova et al 2009; Visconti et al 2005) Summary Cereal grains like barley, corn, rice, and wheat in the field, during harvest, in storage, and while processing are constantly attacked by mycotoxigenic fungi such as Fusarium, Penicillium, and Aspergillus species There are a wide variety of the mycotoxic bioactives that are produced by fungi in cereal foods such as aflatoxins, fumonisins, ochratoxins, and trichothecenes These mycotoxins not only affect the cereal crops by reducing yield, quality, and safety of the cereals but also affect animal and human health if they consume contaminated cereal-based foods Worldwide many regulatory limits are present to help control the entry of mycotoxins in foods meant for human consumption However, lack of regulatory limits on some mycotoxins such as ergot alkaloids can still be very dangerous to both animals and human if they consume very 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R Bushnell, 14 St Paul, MN: APS Press Sydenham, E W., P G Thiel, W F O Marasas, G S Shephard, D J Van Schalkwyk, and K R Koch 1990 Natural occurrence of some Fusarium mycotoxins in corn from low and high esophageal cancer prevalence areas of the Transkei, southern Africa J Agric Food Chem 38:1900–3 Thondre, P S., and C J K Henry 2009 High-molecular-weight barley β-glucan in chapatis (unleavened Indian flatbread) lowers glycemic index Nutr Res 29 (7): 480–6  ... in some cereals and cereal- based foods will be discussed in this chapter The mycotoxin presence during some ? ?cereal- based 25 3 25 4 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications. .. set for OTA in cereal- based foods (FAO 20 04) 25 8 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications (a) O OH O OH O O N H CH3 Cl (b) OH CH3 O O HO O Figure  12. 2  Structure of... in cereal foods (Abildgren et al 1987; Samson et al 20 00) These microbiological methods are very useful in isolating and detecting 26 2 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications