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1-1-2010

Comparing detection methods of aflatoxin and exploring aflatoxin decontamination methods

Rebecca Burgett Singleterry

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COMPARING DETECTION METHODS OF AFLATOXIN AND EXPLORING

AFLATOXIN DECONTAMINATION METHODS

By

Rebecca Burgett Singleterry

A Thesis Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Agricultural Life Science (Biochemistry) in the Department of Biochemistry and Molecular Biology

Mississippi State, Mississippi

December 2010

COMPARING DETECTION METHODS OF AFLATOXIN AND EXPLORING

AFLATOXIN DECONTAMINATION METHODS

By

Rebecca Burgett Singleterry

Approved:

______Ashli Brown Ty Schmidt Assistant Professor of Biochemistry Assistant Professor of Animal and Molecular Biology Animal and Dairy Science (Major Professor) (Committee Member)

______Scott Willard Din-Pow Ma Professor and Head of Biochemistry Professor of Biochemistry and Molecular Biology and Molecular Biology (Committee Member) (Graduate Coordinator of the Department of Biochemistry and Molecular Biology)

______George Hopper Interim Dean of the College of Agriculture and Life Sciences Name: Rebecca Burgett Singleterry

Date of Degree: December 10, 2010

Institution: Mississippi State University

Major Field: Agriculture and Life Science (Biochemistry)

Major Professor: Dr. Ashli Brown

Title of Study: COMPARING DETECTION METHODS OF AFLATOXIN AND EXPLORING AFLATOXIN DECONTAMINATION METHODS

Pages in Study: 81

Candidate for Degree of Master of Science

Ethanol fermentation of highly concentrated aflatoxin-contaminated corn (Zea

mays) was conducted on a lab scale to determine if aflatoxin concentrated in the distilled

ethanol and/or dry distillers grain end-products. Alliquots of fermented mash, distilled

ethanol, stillage, and dry distillers grain (DDG) were analyzed via LC-MS/MS and

immunoassay detection methods for aflatoxin. Results indicate that aflatoxin does not

greatly concentrate during fermentation in the DDGs and is undetectable in distilled

ethanol. Addition of binders, MTB-100®, to aflatoxin-contaminated DDGs showed great

reduction in aflatoxin concentrations when analyzed via LC-MS/MS. Also, an experiment

investigating detoxification of aflatoxin using Clorox® was conducted. Results obtained

from LC-MS/MS showed a positive correlation of decreased aflatoxin levels with

increasing Clorox® levels following a logarithmic trend.

Key words: aflatoxin, ethanol fermentation, decontamination methods

ACKNOWLEDGMENTS

I cannot thank enough the encouragement and prodding of my major professor

Dr. Ashli Brown to whom I am forever humbly indebted for the completion of this degree in her willingness to see me through it. Thank you for your guidance and sense of humor through my years as an undergraduate and graduate student.

Next, I would like to thank Dr. Scott Willard and Dr. Ty Schmidt for advising me through this process. I have so much enjoyed getting to know you. It is professors like you that make a world of difference in shaping the careers of students, and I am so blessed to have been under your guidance.

Also, I would like to say a special thank you to Dr. Brian Baldwin for the use of his lab and sharing his experience in the field. Thank you for taking the time to help me with the set-up of my experiments and for answering all my questions. Your sense of humor made it all worth while.

Next, I would like to thank all the staff from the mass spectrometry lab of

Mississippi State Chemical Laboratory which became my family during my years at the lab. You are great co-workers from whom I learned much about the “art” of science. I now realize that the special atmosphere present at the lab will never be replicated elsewhere. I will miss you guys greatly.

Finally, I would like to thank my family. I am not sure if I would have finished if it were not for your faithful encouragement. Most of all, I would like to

ii thank my heavenly Father and Savior for His love towards me. Thank you for your grace and forgiveness, of which none I deserve.

iii TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... ii

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

CHAPTER

I. LITERATURE REVIEW ...... 1

Aflatoxin ...... 1 Introduction into ...... 4 Introduction into Mycotoxins ...... 7 History of Aflatoxin’s Discovery ...... 9 Biological Activity ...... 10 Factors Influencing Aflatoxin Formation ...... 11 Toxicity ...... 13 International Legislation ...... 18 Sampling Method ...... 18 Detection Methods ...... 20 Ethanol Fermentation ...... 25 Detoxification of Aflatoxin ...... 27 LITERATURE CITED ...... 30

II. INVESTIGATION OF AFLATOXIN INOCULATED CORN (Zea mays) DURING ETHANOL FERMENTATION USING LC-MS/MS AND IMMUNOASSAY DETECTION METHOD, A STRATEGY FOR SALVAGING AFLATOXIN- CONTAMINATED DDGS USING BINDERS ...... 41

Abstract ...... 41 Introduction ...... 42 Materials and Methods ...... 46 Corn Sample Information ...... 46 Preparation and Fermentation of Corn ...... 47 Distillation of Ethanol ...... 49

iv Preparation, Extraction, and Analysis of Samples ...... 50 Corn...... 50 DDGs ...... 51 Fermented Mash ...... 51 Distilled Ethanol ...... 51 Stillage ...... 52 Immunoassay Conditions ...... 52 Immunoaffinity column assay ...... 52 Enzyme-link immunosorbent assay ...... 53 LC-MS/MS Conditions ...... 53 Standards used for LC-MS/MS Analysis ...... 55 Preparation and Analysis of DDGs mixed with Binders ...... 55 Results ...... 56 Discussion ...... 62 Conclusion ...... 65 LITERATURE CITED ...... 67

III. DETOXIFICATION OF AFLATOXIN USING CLOROX® ANALYZED BY LC-MS/MS ...... 72

Abstract ...... 72 Introduction ...... 72 Materials and Methods ...... 74 Standards Information ...... 74 Sample Preparation ...... 74 LC-MS/MS Conditions ...... 75 Results ...... 76 Discussion ...... 77 Conclusion ...... 78 LITERATURE CITED ...... 79

v

LIST OF TABLES

1.1 Acute toxicity of aflatoxin B1 expressed as a single oral dose LD50. (taken from Newberne and Butler, 1969) ...... 14

1.2 FDA Action Levels for Aflatoxin. (www.fda.gov, 2010) ...... 17

2.1 Gradient method used for the mobile phase in separation of samples for the LC-MS/MS method ...... 54

2.2 Distribution of aflatoxin B1 in products from ethanol fermentations ..... 58

3.1 Gradient method used for the mobile phase in separation of samples used for the LC-MS/MS method ...... 75

vi

LIST OF FIGURES

1.1 Chemical structures of common aflatoxins B1, B2, G1, and G2 produced by A. flavus and A. parasiticus ...... 2

1.2 Chemical structure of aflatoxin metabolites ...... 3

1.3 Aspergillus flavus. A. Colony formed in culture. B. Conidiophore. C. Scanning electron micrograph of conidia. D. Conidia shown under a light microscope at 100x. (Revised from Klich, 2007) ...... 6

1.4 Characteristic conidiophores of Aspergillus. (revised from Klich, 2007) .. 7

1.5 Schematic of ethanol fermentation process for dry grind and wet mill. (revised from Bothast and Schlicher, 2005) ...... 26

2.1 Schematic showing experimental set-up of a lab scale ethanol fermentation for negative and aflatoxin-contaminated corn samples ...... 48

2.2 Picture showing the micro-scale set-up for ethanol collection ...... 50

2.3 Chromatogram obtained from LC-MS/MS analysis showing positive controls using aflatoxin-contaminated DDGs (14000 ppb total aflatoxin) and samples containing sorbents mixed in with contaminated DDGs ...... 61

3.1 Graph showing seven different Clorox®: water solutions ranging from 0.001 to 0.1% Clorox®, each mixed with (500 ng/mL) of aflatoxin B1 The data follows a logarithmic equation (y = -120.71Ln(x) - 261.56 with a correlation coefficient, R2, of 0.9953, showing that the more concentrated the Clorox® solution the lower the aflatoxin concentration. Error bars for a 0.1% Clorox® replicate sample is shown ...... 76

vii CHAPTER I

LITERATURE REVIEW

Aflatoxin

Aflatoxin is a very toxic secondary metabolite produced from some species of

Aspergillus commonly found in agricultural commodities and feed. It can be classified as

a mutagenic, carcinogenic, difuran-containing, and polyketide-derived compound where

the liver is the primary target. (Bennett and Klich 2003; Klich, 2007). Aflatoxin’s toxicity has classified it as a Group I carcinogen by the International Agency for Research on

Cancer (IARC) (IARC, 1985). The four main aflatoxin metabolites commonly

encountered and studied are aflatoxins B1, B2, G1, and G2 (Figure 1.1), which were first isolated and characterized after the 1960 Turkey “X” disease in England (Blount, 1960).

A. flavus mainly produces aflatoxin B1 and B2, and A. parasiticus produces B1, B2, G1,

and G2 (Wei and Jong, 1986; Klich and Pitt, 1988; Klich, 2007). Now, more than 16 aflatoxin metabolites have been characterized (Goldblatt, 1969; Newberne, 1974; Bennett and Klich, 2003) with most of them being products of biotransformation from the major metabolites (Figure 1.2). Aflatoxins M1 and M2, metabolized from B1 and B2 respectively

in the body, are also a main concern since they are expressed in milk of animals that have

consumed aflatoxin contaminated feed (van Egmond, 1989; Chun Pei et al., 2009).

Consequently, this can poison their suckling offspring and can contaminate dairy

1

Figure 1.1 Chemical structures of common aflatoxins B1, B2, G1, and G2 produced by A. flavus and A. parasiticus

2

Parasiticol (B ) B1 R0 3 B2

G1 G2 1-Methoxy-aflatoxin B2 2-Methoxy-aflatoxin B2 3

1-Ethoxy-aflatoxin B 1-Ethoxy-aflatoxin G M 2 2 M1 2

1-Acetoxy-aflatoxin B B2a GM1 2 G2a

Figure 1.2 Chemical structure of aflatoxin metabolites

products for the consumer market. First isolated from lactating cows, M1 and M2 showed the same toxicity as their derived chemical compounds (Allcroft and Carnaghan, 1963).

Although aflatoxins B and G are structurally similar, in terms of potency, B1 is the most

toxic followed by G1, B2, and G2 (Wogan, 1966; Maggon et al., 1970; Squire, 1981).

Aflatoxin B1 is also the quantitatively predominant aflatoxin of toxigenic producing

strains (Wogan, 1966; Bennett and Klich, 2003). In pure form aflatoxins B1, B2, G1, and

G2, are an odorless crystalline solid, pale-white to yellow in color, and they have

relatively high melting points (Maggon et al., 1970). Aflatoxins strongly fluoresce under

ultraviolet light. Aflatoxins B1 and B2 fluoresce blue; Aflatoxins G1 and G2 fluoresce

green (Bennett and Klich 2003). Aflatoxins are soluble in methanol, chloroform, acetone,

and acetonitrile. These solvents are used in many AOAC® Official MethodsSM for

extracting aflatoxin from a variety of matrices. Aflatoxins are light sensitive and break

down over time in the presence of light (Klich 2007).

Introduction into Aspergillus

Aspergillus (Figure 1.3) can be classified as a common saprobic, filamentous fungi (Scheidegger and Payne, 2003), sometimes also referred to in literature as a mould.

Aspergillus was named by Pier Antonio Micheli in 1729 for its characteristic aspergillum like shape (see Figure 1.3, insert B), an implement used for sprinking “holy” water in religious activities. Typically, fungal species are classified by their morphological and cultural differences (Raper and Fennel, 1965; Samson et al., 2000), but molecular means

of differentiating fungi is becoming more popular since there are overlapping features

4

among species (Peterson et al., 2001). Colonies of different Aspergillus strains differ in

diameter size, growth rate, and color ranging from green, yellow-green, brown, and black

(Klich, 2002). All Aspergillus species share a common spore-bearing aspergillum

structure consisting of a foot cell, stipe and vescicle (Figure 1.4). Stipes are rough to

smooth, and vesicles are globose to elongated in shape. Phialides, spore-producing cells,

cover the entire vesicle, pointing out in all different directions. Seriation may be

uniseriate, containing just phialides or biseriate, containing metulae, which are additional

short outgrowths from the vesicle. The conidia formed may be globose to ellipsoidal in

shape (Hedayati et al., 2007). In general, Aspergillus’ primary form of reproduction is considered asexual (Klich, 2002). The genus of Aspergillus is divided into sections with

many aflatoxin-producing species belonging to section Flavi, which includes Aspergillus

flavus and A. parasiticus, the two predominant and economically important aflatoxin-

producing species (Bennett and Klich, 2003; Yu et al., 2004). Other aflatoxin-producing

species less commonly encountered are A. nomius, A. pseudotamari, A. bombysis and A.

parvisclerotigenus (Bennett and Klich, 2003; Klich, 2007). Some other aflatoxin-

producing species not included within the section Flavi are A. ochraceoroseus (Bennett

and Klich, 2003), A. rambellii, Emericella venezuelensis, and E. astellata; the latter two species have anamorphs (asexual states) similar to Aspergillus. (Frisvad et al., 2005).

Section Flavi also includes nonaflatoxin-producing species that are of economic importance in food fermentation, A. oryzae and A. sojae (Wood, 1977; Hedayati et al.,

2007; Klich 2007). The genome of A. flavus (Payne et al., 2006) and A. oryzae (Machida

et al., 2005) has recently been sequenced and will provide insight into the underlining

5

workings of this and serve as a model for similar species. Aspergillus is mostly found in areas that are hot and humid. For fungi growth, environmental conditions must be favorable with humidity having the greatest impact (Gibson et al., 1994; Wilson and

Payne, 1994).

Taxonomy

ƒ Kingdom: Fungi

ƒ Phyllum:

A B ƒ Order:

ƒ Class:

ƒ Family:

ƒ Genus: Aspergillus

CDC

Figure 1.3 Aspergillus flavus. A. Colony formed in culture. B. Conidiophore. C. Scanning electron micrograph of conidia. D. Conidia shown under a light microscope at 100x. (Revised from Klich, 2007)

6

Conidia Phialides Metulae

Vesicle

Stipe

Foot cell

Figure 1.4 Characteristic conidiophores of Aspergillus. (revised from Klich, 2007)

Introduction into Mycotoxins

Mycotoxins are defined as naturally occurring toxic secondary metabolites produced by filamentous fungi (Bennett and Klich, 2003). The name mycotoxin comes from the Greeks word ‘mykes’ meaning fungus and the Latin word ‘toxicum’ meaning poison. It was coined in 1962 after the devasting 1960 Turkey “X” disease epidemic.

There are several hundred recognized mycotoxins (Bennett and Klich, 2003) produced from approximately 200 fungi. However, only about 20 mycotoxins occur frequently in agricultural crops. It should be noted that not all toxic compounds produced by fungi are classified as mycotoxins; the determining factor is the metabolite’s primary target and its 7

concentration. For example, the fungal metabolite is usually considered an antibiotic if it

is toxic to bacteria. When a fungal metabolite is toxic to vertebrates and animals at low

concentrations, it is usually classified as a mycotoxin. Individual mycotoxins may be

produced by one or more species of fungi, and one species of fungi may produce several

mycotoxins. Mycotoxins differ in toxicity and are expressed in varying amounts.

Mycotoxins show great diversity in their chemical structures, biosynthetic pathways,

biological effects, level of toxicity, primary target, and are produced by a variety of fungi

species (Bennett and Klich, 2003). The production of mycotoxins is a natural fungi

process, but its production is not necessary for the growth of the fungi. In plant

pathology, many mycotoxins are known to enhance and aid in pathogenic invasion of fungi on a host. Production of mycotoxin may also be a mechanism of survival by warding off surrounding factors that may compete with its food source and therefore

helps in the establishment of the growth of its colony. Mycotoxicoses, diseases caused by

mycotoxins, is mainly procured from eating contaminate food and less commonly by

respiratory, dermal or other means of contact with the toxin. (Bennett and Klich, 2003).

As described by Bennett and Klich (2003), mycotoxicoses are examples of ‘poisoning by

natural means’, which is comparable with poisonings to pesticides or residues from heavy

metals. Mycotoxins are not a major human and animal health threat in developed cities

where they are heavily regulated, but are more of a health concern in third world

countries where, for example, many aflatoxin-contaminated foods are staple foods, and

preventative means (i.e. farming practices and regulatory measures) are not in place.

8

History of Aflatoxin’s Discovery

Before aflatoxin’s discovery in 1960, mycotoxins were not given much attention and little was devoted in understanding and studying their physical and chemical properties or their purpose for existence. However, when over 100,000 turkeys died within a few months in England from aflatoxin poisoning, the era of mycotoxin awareness and study began in earnest (Blount, 1961). At first, the cause of death was unknown and the epidemic was labeled Turkey “X” disease. The syndrome was then reported in ducklings, chickens (Asplin and Carnaghan, 1961), swine (Loosmore and

Harding; 1961; Harding et al., 1963), and calves (Loosmore and Markson; 1961). The majority of the cases happened within a relatively small radius around London with no reported cases in neighboring countrysides. The acute onset and rapid demised of the

Turkey “X” disease confused investigators. Healthy turkeys were unaffected when mingled with the diseased birds, the sick turkeys did not respond to antibiotic treatments, and they were dying by the droves. Physically, the birds did not appear dehydrated and their bodies were in good conditions, but looked to be poisoned. Autopsies, showed inflammation of small intestines, particularly the duodenum, and the kidneys and liver were seen to be congested and swollen. The liver also showed pale necrotic lesions and affected by hemorrhages. The investigators were finally able to link a common factor among the turkey farms back to a Brazilian grain that was found in all of the grain fed to the turkeys. When looking closer at the grain, the investigators identified a mycotoxin was present from A. flavus that was growing on the grain (Sargeant et al., 1961; Sargeant et al, 1963). The mycotoxin was hence called aflatoxin from the strain of fungus found

9 and its toxic affect. Shortly afterwards, aflatoxins B1, B2, G1, and G2 were identified using chromatographic techniques (Nesbitt et al., 1962; van der Zijden et al., 1962). The toxins were given their names based on the colored they fluoresced, whether blue or green, under ultraviolet light and designated either as one or two based on their relative distance traveled.

Biological Activity

Aflatoxin’s primary target organ is the liver (Bennett and Klich, 2003; Williams et al., 2004). Aflatoxins toxicity is due to its difuran ring (Maggon et al., 1970).

Aflatoxins are oxidized by mixed function oxygenase of the liver P-450 system, resulting in a reactive 8,9-epoxide as the major product. This product attacks the N7 position of guanine residues in double stranded DNA and protein (Eaton and Groopman, 1994;

Bedard and Massey, 2006). Aflatoxin B1-DNA adducts can result in GC to TA transversions and hence induce mutations in the p53 tumor suppressor gene at codon 249

(Aguilar et al., 1993; Lasky and Magder, 1997; Chan et al., 2003). The development of hepatocellular carcinoma is believed to result from inactivation of the p53 tumor suppressor gene. Studies from China and Africa have recorded aflatoxin G2 to also produce sister chromatid exchange in animal cells in culture (Moss, 2002). Aflatoxins

B1, B2, G1, and G2 is derived via oxidative ring opening of an anthraquinone intermediate, which is formed as a product of the polyketide pathway (Dutton, 1988). The commonly accepted biosynthetic pathway and clustered genes responsible are outlined by Yu et al.

(2004).

10

Aflatoxin exposure can be monitored in humans and animals by analyzing the

7 blood, milk, or urine for aflatoxin metabolites. The aflatoxin B1-N - guanine adduct is a

reliable biomarker analyzed in urine to determine aflatoxin exposure but only shows

recent exposure (Bennett and Klick, 2003). Other biomonitoring techniques of aflatoxin

include synchronous fluorescence spectroscopy measuring aflatoxin DNA adducts in

urine (Autrup et al., 1983).

Factors Influencing Aflatoxin Formation

Aflatoxins are detected in corn, peanuts, cottonseed, treenuts, almonds, figs,

spices, and many other foods (Payne, 1983; Klich et al., 1986; Lillard et al., 1970; Price

et al., 1993; Martins et al., 2003; Bircan et al., 2008; Delage et al., 2008; Rahimi et al.,

2008). A. flavus and A. parasiticus are the predominant species responsible for aflatoxin

contamination of crops prior to harvest or during storage. A. flavus is the major

contaminant of agriculture (Bennett and Klich 2003), especially in corn, and A.

parasiticus is a major economic problem in peanuts. While A. parasiticus is found in a

variety of soils (Klich, 2002b) it is rarely isolated in other crops (Diener et al., 1987).

Aspergillus is a robust fungus, and its ability to flourish in harsh environments allows it to overpower other fungi for nutrients in the soil and vegetation (Bhatnagar et al., 2000).

Most strains of A. parasiticus produce aflatoxin while aflatoxin production in A. flavus

varies with strain, substrate, and geographic location (reviewed by Klich, 2007). The

effects of water activity and temperature are critical for mold growth and aflatoxin

production (reviewed by Klich, 2007b). Studies investigating these aspects give a

11

consensus that maximum aflatoxin production occurs when water activities approaches

from 0.96-0.996 (Koehler et al., 1985; Gqaleni et al., 1997) and optimum temperature

being between 24° and 30° C, depending on the variety of strain and substrate present

(Schroeder and Hein, 1967; Shih and Marth, 1974; Northolt et al., 1976; Northolt et al.,

1977; Durakovic et al., 1987; Ogundero, 1987; Barrios-Gonzalez et al., 1990; Gqaleni et

al., 1997). Sclerotia, produced by some Aspergilli isolates, are hyphal masses. High

aflatoxin production was observed when small sclerotia was present (Cotty, 1989;

Mahanti et al., 1996) but the presence of sclerotia in general does not seem to have an

effect on aflatoxin production (Bennett et al., 1979; Lisker et al., 1993). There is a

positive correlation between sporulation and aflatoxin production (Reib, 1982; Guzman-

de-Pena and Ruiz-Herrara, 1997; Kale et al., 2003). Drought stress in plants is usually the

major contributing factor of aflatoxin contamination in crops (Diener et al., 1987; Klich,

1987), along with high humidity which is favorable for mold growth. Crops may also be

introduced to contamination of aflatoxin during storage if conditions are favorable for

mold growth due to moisture content and the relative humidity of the area where the

crops are being stored. It is not completely understood what factors initiate the production

of aflatoxin by certain strains of fungi. Given certain environmental conditions, aflatoxin

may or may not be produced and, if produced, it is in varying amounts. Still, the

occurrence of aflatoxins in field crops is influenced by certain factors; hence the extent of

contamination will vary with geographic location, agricultural practices, and the susceptibility of commodities to fungal invasion during preharvest, storage, and/or

processing periods.

12

Toxicity

Many species have an experimentally determined LD50 (lethal dose it takes to kill

50% of the population) for aflatoxin poisoning. As seen from table 1.1, the values range from 0.3 to 10 mg/kg body weight. Some animal species are more susceptible to aflatoxin poisoning than others, and each animal species respond differently to aflatoxin poisoning, depending on exposure concentration, duration of exposure, sex, age, health, and diet

(Bennett and Klich, 2003). It is not known for sure what the LD50 for humans is, but if for example, we were to use a pig model, which is similar in comparison to the human physiology, from table 1.2, it shows that the LD50 mg/kg bodyweight for a pig is 0.6 mg of aflatoxin. That means in a sample size with each pig weighing 100 kg (220 pounds),

60 mg of aflatoxin is sufficient to kill half of the pigs in the sample size. There is one reported incident where a laboratory worker attempted suicide by ingesting over 40 mg of purified aflatoxin over a six month period with no reported side effects except for rash, nausea, and headache. Fourteen years later, in a follow-up examination, her liver and blood tests were declared

13

Table 1.1 Acute toxicity of aflatoxin B1 expressed as a single oral dose LD50. (taken from Newberne and Butler, 1969)

Species LD50 mg/kg bodyweight Rabbit 0.30 Duckling 0.34 Cat 0.55 Pig 0.62 Dog 1.00 Guinea pig 1.4 Rat 5.5 - 7.20 Mouse 9.00 Hamster 10.20

14 normal (Willis, 1980). Aflatoxin poisoning in humans has been reported in third world countries where regulation of food/feed commodities is not set in place. One of the largest human epidemiological studies conducted in dealing with aflatoxicosis occurred in 1974 in India (Krishnamachari et al., 1975). The outbreak occurred in over 150 villages in two neighboring states. It was reported that 397 people were affected with symptoms including high fever, jaundice, edema of the arms and legs, vomiting, and swollen livers; 108 people died. Autopsies showed proliferation of the bile duct, fibrosis of the liver, and gastrointestinal hemorrhages. It was estimated that a minimum of 2-6 mg of aflatoxin was ingested for an undetermined number of days. Interestingly though, those that survived, showed full recovery with no adverse side effects during a follow-up ten years later. Recently, an aflatoxicosis outbreak happened in Kenya, Africa in 2004

(Probst et al., 2007). No aflatoxin outbreaks in humans has been reported in the United

States though there have been a few reported incidences in animals. One of the more recent, occurred in latter year of 2005 when over 100 dogs consumed aflatoxin- contaminated dog food (Stenske, 2006). The incident was traced back to contaminated corn from South Carolina had been overlooked by routine testing of aflatoxin.

Aflatoxicosis is poisoning resulting from ingestion of aflatoxins in contaminated food or feed. Acute aflatoxicosis (high doses of aflatoxin) results in death and effects from chronic aflatoxicosis (low to moderate doses of aflatoxin) result in carcinoma of the liver, necrosis, cirrhosis, and immune suppression (Williams et al., 2004). The liver is the primary target organ from studies conducted in poultry, fish, primates, pigs, and rodents when fed aflatoxin B1 (Newberne and Butler, 1969). From a few epidemiological studies

15

conducted in humans, some symptoms inclueded jaundice, impaired food conversion and

decreased growth rates. Effects of acute aflatoxicosis (high doses of aflatoxin) produce

acute necrosis, cirrhosis, and carcinoma of the liver as shown in many animal studies

(Newberne and Butler, 1969). Although acute aflatoxicosis has rarely been reported in humans it can be assumed that these same toxic effects found in animal studies would be similar in humans (Williams et al., 2004). In 1987, World Health Organization (WHO) classified the aflatoxins as Group 1 carcinogen. By 1988 aflatoxin B1 was placed on the

list of human carcinogens by the IARC (IARC, 1985). However, there is no worldwide

standard for mycotoxin concentrations. The Food and Drug Administration (FDA) has

taken cautionary measures and established specific guidelines on acceptable aflatoxin

levels in human and animal food commodities (Table 1.1).

16

Table 1.2 FDA Action Levels for Aflatoxin. (www.fda.gov, 2010)

Species Commodity Action Level

Humans Milk 0.5 ppb (M1)

Any food except Humans 20 ppb milk Immature animals Corn and other or when end use is 20 ppb grains not known

Animal feed other All species than corn or cotton 20 ppb seed meal

Breeding beef, Corn and other cattle, swine, or 100 ppb grains mature poultry

Finishing swine of Corn and other 300 ppb 100 lbs. or greater grains

Beef cattle, swine, Cottonseed meal 300 ppb poultry

17

International Legislation

With the inter-trade among countries, it is necessary to have a universal standard

for mycotoxin levels. The Food and Agriculture Organization of the United Nations

(FAO) and WHO established Codex Alimentarius Commission (CAC) in 1961 with the

purpose to elaborate international food legislation, which includes mycotoxin in foods

and feeds and to establish maximum limits (Berg, 2003). A similar process is being

executed in the European Union, known as the European Commission (EC) (Berg, 2003).

Mycotoxin prevention has been successful by codes set by the Codex Committee on Food

Additives and Contaminants (CCFAC), which establishes measures in Codes of Practice that govern conditions during storage and transport and farming techniques. For example a decline in aflatoxin contamination in milk has been observed since CCFAC’s efforts to set guidelines to reduce the occurrence of aflatoxin in animal feed (Brinkhorst, 2003).

Sampling Method

Analysis of mycotoxins in feed meal and food products is a difficult task. The largest source of error in mycotoxin detection is collecting a representative sample from harvest crops and feed (Whitaker, 2003). As it is impractical and impossible to test every kernel harvested in a bulk lot for mycotoxin contamination, a sample from the lot is taken

and analyzed. Unfortunately, obtaining a proper representation of mycotoxins is hindered

by the fact that mycotoxins are heterogeneous throughout the grains in a lot. Though

studies have shown, using raw shelled peanuts, that the majority of the kernels are good and fit for human consumption with only less than 0.1% of the kernels being

18

contaminated (Whitaker et al., 1974), the kernels that are contaminated may have extremely elevated concentrations of mycotoxin (Whitaker and Wiser, 1969). An excellent example was given by Whitaker et al. (1972) of the complexity and complication that occur when trying to estimate the true mycotoxin concentration in a given lot. For this example ten 5000 g samples were taken from the same lot of shelled peanuts and measured for total aflatoxin (ppb). The measured samples are reported in ascending order: 0, 0, 0, 0, 3, 13, 19, 41, 43, 69 ppb for the ten samples tested. The average of the ten samples is 19 ppb, the best estimate of the true concentration. The EU has a maximum limit of 15 ppb for total aflatoxin for peanuts designated for further processing. As Whitaker (2003) pointed out, if the true concentration of the lot for total aflatoxin was 19 ppb then the lot is unacceptable and unfit of export. However, out of the ten samples tested, six were under the 15 ppb maximum limit. The result is that 60% of

the time, the lot would be incorrectly classified as being acceptable and 40% of the time the lot would be correctly classified as unacceptable. Therefore, it is common in this situation to have false negatives (test gives a positive result for aflatoxin when in fact there are false positives. A sample may be taken from a large truckload of feed that tests false positive (test gives a positive result when in fact the lot may be negative for

mycotoxins or there are extremely high levels of mycotoxins present in another area of

the truckload harvested). No standardized sampling method is in place, but sampling

methods and techniques are improving. It is common practice to take many random

subsamples from within a truckload of harvest grain and pool them together as one

sample to be tested.

19

Detection Methods

Detection of mycotoxin levels must meet government regulatory standards.

Excluding milk, the FDA has action levels set for total aflatoxin at 20 ppb and Europe

has action levels set for total aflatoxin at 4 ppb for all food commodities, excluding milk.

Because only trace amounts of aflatoxin may be present within a sample, it is critical to

have high through-put detection methods which are accurate and sensitive.

Gilbert and Anklam (2002) give a comprehensive compilation of official methods

of analysis for aflatoxin, including matrix, detection limits, percent recovery, and

references to each method listed. Growing in popularity for mycotoxin detection is liquid

chromatography coupled to mass spectrometry (LC-MS) for its sensitivity and multi-

analyte analysis capability. A brief look into two Immunochemical and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods are discussed along with their strength and weaknesses.

Immunochemical methods use antibodies to distinguish three-dimensional structures. Over the last two decades, the enzymed-link immunosorbent assay (ELISA) method has gained in popularity for mycotoxin quantification as a high throughput assay

that is rapid and accurate, requiring low sample volumes and little sample clean up

compared to thin layer chromatography (TLC) and high performance liquid

chromatography (HPLC) methods (Zheng et al., 2005; Zheng, et al., 2006). Direct

competitive ELISA (Chu 1996) is commonly used in mycotoxin quantification. The basic

principle of the assay is mycotoxins extracted from the sample and controls are mixed

with a conjugate (enzyme-coupled reaction to mycotoxin of interest). The mixture is then

20

added to microwells coated with antibodies. The mycotoxin from the sample extract

competes with the conjugate for antibody binding sites. The wells are then washed to

rinse any free analytes that are not attached to the antibodies. Enzyme is then added to the

wells to produce a color change when bound to the conjugate. The mycotoxin

concentration is inversely proportional to the color intensity. The samples can be

analyzed by visually comparing them to the controls. In order to make this method more

sensitive a spectrophotometer can be used to measure the optical densities of the controls

to generate a standard curve and the optical densities of the samples can be plotted

against the curve to calculate the concentration of mycotoxin in the sample. ELISA assays for total aflatoxin and those specific for Aflatoxin B1 or M1 are commercially

available and capable of accommodating a variety of matrices (Lin et al., 1998; Lee et

al., 2004). Some are approved by the Grain Inspection, Packers, and Stockyards

Administration (GIPSA), United States Department of Agriculture (USDA), and

Association of Official Analytical Chemists (AOAC International) for official

determination with detection limits as low as 0.1 ppb. The simplicity and high through-

put of ELISA assays make them profitable for mycotoxin quantification, giving results in

minutes rather than hours or days as compared to TLC, HPLC, or MS methods (Zheng et

al., 2006). This makes ELISA a great screening method to quickly analyze and monitor

food safety of large amount of food commodities coming in from the field for

commercial processing, ultimately, saving time and money, as well as, providing the

consumer with a safe product (Zheng et al., 2006). Since skilled technicians are not

21 needed, as with HPLC and MS methods, ELISA techniques are beneficial in third world countries where mycotoxin contamination is a problem.

One of the challenges faced with ELISA methods for sample analysis is matrix interference, causing false positives by decreasing the color change (Lee et al., 2004).

These effects occur when agents extracted from the sample hinder the analyte from binding to the antibody and/or inhibiting enzyme activity (Wilkinson et al., 1988; Dell et al., 1990; Ramakrishna et al., 1990; Figueira et al., 1991; Li et al., 1994). Another major challenge is the variation seen between different assays. Studies have shown consistent greater recovery values in ELISA assays when comparing them with HPLC and LC/MS methods (Samdal et al., 2005; Zheng et al., 2005; Colak et al., 2006; Faupel-Badger et al., 2010) but in all cases the correlation between the compared methods were good.

Immunoaffinity column (IAC) is another immunochemical method (Zheng et al.,

2006) which uses antibodies to distinguish three-dimensional structures. Immunoaffinity column is used as a clean-up step after sample extraction and before analysis and used widely in mycotoxin analysis (Scott and Trucksess, 1997). The IAC is easy to use and convenient consisting of a small plastic cartridge containing a binding material. For mycotoxin analysis, IAC contains antibodies for the mycotoxin of interest, which is immobilized on a solid support. The sample extract is applied to the column where the mycotoxins in the sample will bind to the antibodies. Water is passed over the column to remove any impurities and then the mycotoxins are eluted from the column by washing with a solvent, such as methanol. The fraction containing the mycotoxins is then subjected to chemical modification before measuring on a flurometer or can be directly

22 injected for HPLC analysis. Some of the advantages of IAC are the ability to concentrate the mycotoxin in the column, it is an easy, fast and efficient clean-up step, and the immunoaffinity column allows for high selectivity of analyte of interest. However, IAC columns have a loading limit, as they can only hold a limited amount of the analyte.

Mass spectrometry has become a more popular method for mycotoxin detection over the last decade with its ability to analyze multi-analytes concurrently at low concentration levels (Zollner and Mayer-Helm, 2006; Kokkonen and Jestoi, 2009;

Monbaliu et al., 2010). Mass spectrometry is selective and sensitive in its ability to analyze specific mycotoxins. Each mycotoxin produces a distinct measureable signal

(Krska et al., 2008), making it a confirmatory method for the molecular identity of the mycotoxin(s) of interest. Because of its high sensitivity and suitable interfaces, such as atmospheric pressure ionization, little clean-up is necessary but the trade off is poor accuracy and precision due to the effects of co-eluting matrix on the signal intensity of the analytes (Krska et al., 2008). This challenge can be overcome by using matrix- assisted standards (Kokkonen and Jestoi, 2009). Liquid chromatorgraphy-mass spectrometry or LC-MS/MS is now the method of choice for analyzing aflatoxins due to the polarity of aflatoxins (Vahl and Jørgensen, 1998; Cavaliere et al., 2007; Krska et al.,

2008). The overall theory behind LC/MS methods is a sample is first physically separated by LC and then measured and identified by its molecular weight via mass spectrometry.

LC is based on two components, a mobile phase which is the solvents and a stationary phase consisting of a column. A sample is injected into the mobile phase which is then pumped through a stationary column. The analytes affinity between the mobile phase and

23

stationary phase separates the sample into its individual components. Polar samples, such

as aflatoxins, can be separated by reversed phase liquid chromatography where the

mobile phase is a gradient of water and organic solvent making it hydrophilic, and the stationary phase column is made of silica with attached alkyl chain groups, making it

hydrophobic. As aflatoxins in the mobile phase consisting mostly of water are pumped

across the stationary phase, the aflatoxins interact with the hydrophobic stationary phase.

As the gradient of the mobile phase is changed to consist more of an organic solvent,

such as methanol, the analytes that are more polar (less hydrophobic) will disassociate

from the hydrophobic stationary phase. As the percentage of organic solvent in the mobile phase increases the least polar molecules will elute last. Aflatoxin G1, the most

polar of the four main aflatoxins, will elute from the stationary column first followed by

B2, G1, and B1.

The analytes separated by liquid chromatography are then introduced into the

mass spectrometer. Mass spectrometry is a great tool for being able to identify unknown

compounds and studying molecular structure. It can measure some analytes at parts per

quadrillion. The basic principle behind MS is making a charged particle called an ion and

upon subjecting it into an electric or magnetic field, the mass to charge ratio (m/z) of the

ion effects its motion and hence its detection. There are five main components to the

mass spectrometer, the vacuum system, source region, mass analyzer, detector, and data

system. The vacuum system is necessary to minimize neutralization, scattering, and ions

reacting with molecules. The source region creates a charge on a neutral molecule,

24

making an ion. After a neutral molecule has been ionized, it is introduced into the mass analyzer where the ions are sorted and then detected and analyzed by a data system.

Ethanol Fermentation

With the increasing push for alternative fuel sources and the desire to become more independent from foreign oil, ethanol production industries are now seen throughout America. Within the last ten years alone, Renewable Fuels Association (RFA) reports that the number of ethanol plants has grown from 50 to 183 occupying 26 states

(www.ethanolrfa.org/industry/statistics/, October, 2009). One hundred seventy of these plants as of January 2009 are now in operation producing over 10,500 millions of gallons per year of ethanol (RFA, October, 2009). At least 160 of these ethanol plants use corn as there starting source. In 2003, 10% of the corn crops within the U.S. were used to make ethanol (Bothast, 2005). Corn is commonly used because it is high in starch, which is readily converted into glucose and fermented into ethanol and its co-products carbon dioxide and distillers grains. Fuel ethanol is made by either dry grind process (67%) or a wet mill process (33%) (Figure 1.5). The dry grind process makes more ethanol per bushel of corn (2.8 gal) in comparison to the wet mill (2.5 gal.). However, the wet mill process takes advantage of the nutrients in corn and expends more energy in milling the corn into its various fractions, such as fiber, starch, gluten, and germ. The starch is used to make ethanol and the remaining fractions are further refined to make corn oil and

animal feed. The dry milling process

25

Dry Grind Wet Mill

Corn Mill Corn Steep

Degerm/ Oil Defiber Corn Gluten Gluten Separation Meal

Enzyme Liquefy Enzyme Liquefy

Enzyme Saccharify Enzyme Saccharify

Yeast Ferment Yeast Ferment CO2 CO2

Distill Dry Distill Dry

Dehydrate Dehydrate Distiller’s Dry Corn Gluten Ethanol Grains Ethanol Feed

Figure 1.5 Schematic of ethanol fermentation process for dry grind and wet mill. (revised from Bothast and Schlicher, 2005)

26

(Dien et al., 1997; Saha et al., 1998; Kim et al., 2008) does not separate out the

components of the corn before ethanol fermentation. The entire corn kernel is ground and

water is added to form a “mash.” Enzymes are then added to the mash to break down the

starch from the corn into simple sugar. Yeast is added to the mash and transferred into

fermenters where the yeast converts the simple sugars into carbon dioxide an ethanol.

After fermentation the ethanol is distilled and further refined for industry and the remaining stillage is then centrifuges to separate the course grain from the solubles. The

solubles are concentrated to about 30% solids by evaporation forming a “syrup” that is

added back to the course grain where it is dried together to form dried distiller’s grains

with solubles (DDGS). Dried distiller’s grains with solubles are high in protein and sold

as feed additive for animals. Schingoethe (2004) reports that 100 kg of corn produces

approximately 40.2 L of ethanol, 32.3 kg of DDGS, and 32.3 kg of carbon dioxide.

Detoxification of Aflatoxin

Animal feed contaminated with mycotoxin is a problem for farmers worldwide.

The best method of control is on the front end during preharvest, but this is not

completely feasible. Therefore, many methods for detoxification of aflatoxin have been

researched over the years and have been incorporated for industrial methods of

decontamination of food and feed. Methods include physical, biological, and chemical

means (Peraica et al., 2002). Physical methods of decontamination include sorting,

blending contaminated crop with uncontaminated crop, extraction with organic solvents,

radiation, and binders. Because aflatoxin contamination is not homogenous throughout

27

crops, sorting by removing mould contaminated peanuts (Scott, 1998; Pitt and Hocking,

2006) and pistachios (Pearson et al., 2001) has shown to greatly reduce aflatoxin levels but is not practical for maize and cottonseed (Scott, 1998) and can be labor intensive.

Because of the fluorescent properties of kojic acid, a metabolite commonly produced by fungi that also produce aflatoxin, UV is commonly used for screening purposes to sort maize and cottonseed, with good efficiency but with possible false negatives since the present of aflatoxin is not contingent on the presence of kojic acid (Scott, 1998).

Blending together contaminated and uncontaminated grains to help reduce aflatoxin levels but is illegal in some countries and legalized under extreme circumstances (Kich,

2007). The use of organic solvents is effective but in some cases impractical in cost with the large volumes needed and the difficulty of removing the solvents from the products thereafter (Phillips et al., 1994; Basappa and Shantha, 1996). Radiation via gamma, X- ray, ultraviolet, visible, and microwave have all shown some measure of success (Peraica et al., 2002; Ghanem et al., 2008; Klich, 2007; Herzallah et al., 2008) with sun light seeming to have the greatest impact (Samarajeewa et al., 1990; Herzallah et al., 2008).

Binders added to animal feed are the most commonly used method for protecting animals from mycotoxicosis that are commercially available (Hugwig et al., 2001; Klich, 2007).

The binders are supposed to bind mycotoxins, so as to eliminate possible toxicological effects. Studies have looked into the morphology and structure of binders (Mulder et al.,

2008; Tenorio Arvide et al., 2008; Alimova et al., 2009), possible candidates for use as additives in animal feed (Huwig et al., 2001; Diaz et al., 2002; Diaz et al., 2004;

28

Marroquín-Cardona et al., 2009), and assessing them in animal trials (Schell et al., 1993).

As of to date, no binders are FDA approved for animal use.

Biological methods for the reduction aflatoxin are based on the premise of allowing non-aflatoxin producing fungal strains to displace the toxigenic strains. A number of studies have shown how these strains have reduced aflatoxin in corn, cotton, and peanut crops (Brown et al., 1991; Cotty, 1994; Dorner et al., 1999; Dorner and

Lamb, 2006). Afla-gard®, a commercial product, is available for peanuts to reduce aflatoxin levels through competitive exclusion (Dorner and Lamb, 2006).

Chemical methods include compounds such as formic, ammonium, hydrogen peroxide, ozone, chloride, which are very effective but do not meet Food and Agriculture

Organization (FAO) regulations because due to toxic residues and the reagents reduce the nutritional value of the product (Peraica et al., 2002). Ammoniation is a very effective method, removing over 90% of aflatoxin (Coker 1989). The process involves treating the product with gaseous ammonia or ammonium hydroxide (Bagley 1979; Southern and

Clawson, 1980). Ammoniation is commercially used for the detoxification of aflatoxin in feed, although not FDA approved because of possible toxic products (Peraica et al., 2002;

Klich, 2007).

29

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CHAPTER II

INVESTIGATION OF AFLATOXIN INOCULATED CORN (Zea mays) DURING

ETHANOL FERMENTATION USING LC-MS/MS AND IMMUNOASSAY

DETECTION METHODS, A STRATEGY FOR SALVAGING

AFLATOXIN-CONTAMINATED DDGS

USING BINDERS

Abstract

A lab scale experiment of ethanol fermentation through a dry milling process was

set up to investigate where aflatoxin concentrated during the production. Four corn

samples with high levels of aflatoxin (ranging from 7600 – 14800 part per billion) and

their replicates were compared with a replicated negative control to determine where

aflatoxin concentrated. Fractions were taken from the fermented mash, distilled ethanol,

stillage, and distiller’s dried grains (DDG). These fractions were analyzed using two

different immunoassay methods and LC-MS/MS. Results indicated no aflatoxin was

found in the distilled ethanol. Some aflatoxin was detected in the stillage, but most of the toxin was recovered in the DDGs ranging from 31 to 53%. A second lab scale experiment was conducted to investigate the effect of binders on DDGs detected with high levels of aflatoxin (14000 parts per billion). MTB-100®, a natural feed additive, was mixed with

contaminated DDGs for seven days before extracted and analyzed using LC-MS/MS. A

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significant reduction in aflatoxin levels was seen in comparison to a positive control suggesting that addition of sorbents may be an easy and cost efficient way in salvaging

highly contaminated DDGs for marketable use.

Introduction

Aflatoxin is a potent toxin naturally produced as a secondary metabolite by

certain fungal strains, primarily Aspergillus flavus and A. parasiticus, and is therefore

classified as a mycotoxin (Bennett and Klich, 2003; Yu et al., 2004). Aflatoxin

contaminates a variety of food and crop commodities such as corn, peanuts, spices, tree

nuts, figs, honey, fruit juices, wine, and cotton (Payne, 1983; Klich et al., 1986; Lillard et

al., 1970; Price et al., 1993; Martins et al., 2003; Bircan et al., 2008; Delage et al., 2008;

Rahimi et al., 2008). It is estimated that 25% of the world’s crops are affected by

mycotoxins, according to the evaluation of the Food and Agriculture Organization

(CAST 1980). Aflatoxin is labeled as the most potent carcinogen naturally made in the

environment with its primary mammalian target being the liver (CAST 1979;

International Agency for Research on Cancer, 2002). Aflatoxin was first identified in

1960 in Europe as the culprit behind the Turkey “X” disease which killed over 100,000

turkey poults within a few months of consuming aspergillus-contaminated peanut feed

meal (Blount, 1961). As a result, aflatoxin obtained its name from Aspergillus flavus, and

from the fact that it is toxic. The four main aflatoxins frequently encountered are B1, B2,

G1 and G2 and are designated by the corresponding colors they fluoresce under UV light,

blue or green and the numbers assigned to them are given based by the relative distance

42 traveled on thin layer chromatography (TLC) plate (Wogan, 1966; Bennett and Klich,

2003). A. parasiticus produces aflatoxin B1, B2, G1 and G2 while A. flavus produces aflatoxins B1 and B2 (Wei and Jong, 1986; Klich and Pitt, 1988). Aflatoxin B1 is the most toxic of the four toxins and is the most produced toxin by the fungi (Squire, 1981; Cullen and Newberne, 1994). Upon ingestion, aflatoxin B1 may also be metabolized to make aflatoxin M1, which is expressed in milk of lactating animals, and thereby contaminating dairy products (Van Egmond, 1989; Chun Pei et al., 2009). A. flavus and A. parasiticus are saprobic fungi ubiquitous in nature, commonly found living off of decaying debris in the soil and on crops (Klich, 2002; Klich, 2007). Because of aflatoxin’s carcinogenic nature, it is regulated in animal and human foods in more than 100 countries (Van

Edmond and Jonker, 2005). The U.S. Food and Drug Administration (FDA) has set action levels for aflatoxin at 20 parts per billion (ppb) for all foods intended for human consumption except for milk which is set at 0.5 ppb. The feed limit for finishing beef cattle is 300 ppb and for dairy cattle the feed limit is 20 ppb set by the FDA.

Corn (Zea mays) is currently the main substrate used in ethanol production. The composition of dent corn is 72% starch and 9.5% protein (Watson, 1987). The high starch composition in corn can easily be broken down into simple sugars used to make ethanol.

Consumption of the starch by yeast causes proteins to be concentrated three times in distiller’s dried grains with solubles (DDGS) co-products (Spiehs et al., 2002;

Schingoethe, 2004), which is sold to farmers for animal feed.

A simplistic outline of ethanol production (Dien et al., 1997; Saha et al., 1998;

Kim et al., 2008) using corn starts by grinding entire corn kernels into course flour,

43

known as meal. The meal is then mixed with water to form “slurry.” Alpha-amylase is

added to break down the starch from corn into short chain dextrin sugars, and the slurry is

heated for liquefaction. After liquefaction, the slurry is referred to as “corn mash.” A

second enzyme, glucoamylase, is then added to break down the dextrins to form simple

sugars (glucose) known as saccharification. Finally, yeast is added to the mash and is

transferred to fermenters where the yeast converts the simple sugars into ethanol and

carbon dioxide. In commercial application, carbon dioxide is usually captured and sold

for use in carbonating soft drinks and for use in manufacturing dry ice. After

fermentation, the ethanol is distilled from the fermented mash. The remaining “stillage,”

contains solids from the grain, yeast and water. The distilled ethanol can then be prepared

and sold as fuel. The stillage is then centrifuged, separating the solubles (a liquid

containing 5-10% solids) from the course distiller’s grains (DG) also referred to as “wet cake.” Some of solubles are sent back to the slurry tanks to help recycle water input. The remaining solubles are concentrated by evaporation resulting in a liquid that is 30% solids, referred to as “syrup.” The syrup is then added back to the DG/wet cake and dried forming distiller’s dried grains with solubles (DDGS) that are marketed to farmers as a high protein additive for feed given to cattle, swine, poultry and fish. Schingoethe (2004) reports that 100 kg of corn produces approximately 40.2 L of ethanol, 32.3 kg of DDGS, and 32.3 kg of carbon dioxide.

The economical success of ethanol plants relies in part by the marketability of its

DDGS (Wu and Munkvold, 2008). It is therefore crucial to know how aflatoxin are affected by the fermentation process for the sustainability of ethanol plants, the health of

44

animals fed DDGS and for the health and safety of humans who may consume food

processed from these animals. Several studies have already investigated mycotoxin

contaminated meal through ethanol fermentation processes (Chu et al., 1975; Dam et al.,

1977; Lillehoj et al., 1979; Bothast et al., 1992). All agree that no mycotoxins were detected in the distilled alcohol. It is interesting to note however, that several studies were

at variance with one another. Two studies reported that aflatoxin is degraded during

fermentation (Dam et al., 1977; Okoye, 1986); one (Okoye, 1986) was based on

traditional brewing and the other (Dam et al., 1977) simulated industrial fermentation

procedures. Two additional studies based on industrial fermentation procedures reported

that aflatoxin did not degrade during the fermentation (Lillehoj and Lagoda, 1979;

Bothast et al., 1992).

Many studies have investigated into the detoxification of aflatoxin using heat,

gamma radiation, nixtamalization, and ammoniation (Southern and Clawson, 1980;

Bothast et al., 1992; Buser and Abbas, 2002; Méndez-Albores et al., 2004; Ghanem et

al., 2008; Herzallah et al., 2008) and have shown varying amounts of success in reducing aflatoxin in feeds and foods. However, some of these methods would not prove cost

effective for ethanol plants and would negatively impact the nutritional value of the

DDGS. Another alternative that is gaining attention is the option of incorporating binders

(sequestering agents) in with the contaminated feed meal that would bind aflatoxin, thereby making the meal non-toxic and safe for animal consumption (Klich, 2007).

Adding binders in with the DDGS would be easy and cost effective, requiring minimal modifications to the ethanol plant facilities. Currently no binder is approved by the FDA

45

at this time. Some recent studies have assessed a variety of binders for detoxification of aflatoxin and how they affect the physiology of animals that consume them (Piva et al.,

1995; Diaz et al., 2002; Peraica, 2002; Diaz et al., 2004; Tenorio Arvide et al., 2008).

This study was undertaken to determine the fate of aflatoxin during ethanol fermentation

of corn and to explore a strategy using binders to help salvage contaminated DDGs for

marketing.

Materials and Methods

Corn Sample Information

Corn meal (yellow 100% whole grain) purchased from the local food market was

used as a negative control (no aflatoxin was detected using the Enzyme-link

immunosorbent assay (ELISA) method described below). To ensure that aflatoxin levels

could be detected among the aliquots taken during ethanol fermentation of corn, four

samples of highly aflatoxin-contaminated ground corn were obtained from the USDA

ARS Corn Host Resistance Unit. The corn was planted in a randomized complete block

design on March 24, 2008 with four reps with approximately 20 plants per row. The corn

was infected by side needle injections with A. flavus (NRRL 3357) inoculum 14 days

after mid-silk (mid-silk is when 50% of the plants on a row have visible silks). Inoculum

injected was a 3.4 mL suspension containing 3 x 10^8 A. flavus conidia. The ears were

harvested by hand approximately 60 days after inoculation and dried at 38 °C for 5 to 7

days and then machined shelled. Samples were grounded using a Romer mill (Romer

46

Laboratories Inc., Union, MO, USA). The mean aflatoxin level across all reps was 10800

ppb.

Preparation and Fermentation of Corn

Four corn samples and their replicates were subject to a lab-scale ethanol

fermentation process. All samples were treated the same (Figure 2.1). Both subsamples of

a corn sample were carried through the fermentation process at the same time. The

ground corn sample was weighed (100 g) into a 2 L flask and filled to the 1 L mark with

water. The pH was then measured using litmus paper. All samples tested were within the

recommended pH range of 5.5-6.0. The corn meal slurry was brought up to a rapid boil

on a hot plate while mixing with a stir bar and then allowed to cool to 74-45 °C. Next, 0.1

g of α-amylase (Mile Hi Distilling; Lakewood, CO) was added. This converts starch to

maltose and oligosaccharides by randomly hydrolyzing α-1,4-glucosidic bonds. Ten mL

of 4000 mg/L Ca+2 solution (made up with calcium and distilled water) was added to the

slurry, stabilizing and activating the enzyme. The mixture was allowed to sit for 45

minutes to allow the enzymes to liquefy the mash. Afterwards, the pH was adjusted to 4.0

before adding 0.1 g of glucoamylase (Mile Hi Distilling; Lakewood, CO). This converts the long chain sugars produced by α-amylase into glucose sugars by cleaving α-1,4-

glucosidic and α-1,6-glucosidic bonds from the non-reducing ends. The efficiency of the

enzymatic conversion of starch to sugar was measured in the mash using a Brix

refractometer.

47

Control Aflatoxin Contaminated Corn

Corn Control Sample 1 Sample 2 Sample 3 Sample 4 Samples NDa (7600 ppb) (10800 ppb) (14800 ppb) (11200 ppb)

EtOH Fermentation Fermentation Sub-a Sub-b Sub-a Sub-b Sub-a Sub-b Sub-a Sub-b Sub-a Sub-b (Fermented Mash, EtOH,

48 and Stillage) DDG DDG DDG DDG DDG DDG DDG DDG DDG DDG DDG

LC/MS/MS R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 Immunoassays

Figure 2.1 Schematic showing experimental set-up of a lab scale ethanol fermentation for negative and aflatoxin- contaminated corn samples

a ND, no aflatoxin detected.

The digested mash was then allowed to cool and settle for 30 minutes. The supernatant

from the mash was then removed from the solids (course grains). Sugars that cling to the

solids are removed with the solids; therefore the supernatant was used to wash the solids

to help retrieve most of the sugars back into the supernatant. The course grain portion

was then set aside and allowed to completely dry forming dried distillers grain (DDG).

The supernatant was cooled to 37 °C using an ice bath before stirring in approximately

0.3 grams of yeast, Saccharomyces cerevisiae (purchased from the local market). The

mash was incubated at 29 °C for four days in a loosely sealed container.

Distillation of Ethanol

All of the fermented mash was distilled for ethanol on a micro-scale still (Figure

2.2) consisting of a Bunsen burner, fractionating column, thermometer, Graham condenser and graduated cylinder for collection of ethanol. Distilled ethanol fractions were collected between 78-85 °C. To ensure that no contamination of aflatoxin occurred between samples, a rinse solution of 50:50 ethanol:water was distilled through the fractionating column and Graham condenser three times and collected. The collected rinsed fractions were analyzed via LC-MS/MS.

49

Figure 2.2 Picture showing the micro-scale set-up for ethanol collection

Preparation, Extraction, and Analysis of Samples

Corn

The total aflatoxin in the four corn samples obtained for this experiment was

determined by two immunoassays: Immunoaffinity column (IAC) and ELISA.

The samples were extracted and analyzed as indicated by the test kit instructions

(outlined below).

50

DDGs

The total aflatoxn in the DDGs fractions collected from the experiment were

analyzed by IAC, ELISA, and LC-MS/MS. For the two immunoassays, the

samples were extracted and analyzed as indicated by the test kit instructions. For

LC-MS/MS analysis, the extraction of aflatoxin from DDGs followed the ELISA

extraction procedure using 5 g of grounded sample in 25 mL of methanol:water.

The filtrate was then diluted to fall within the standard curve. No additional clean-

up step was needed before LC-MS/MS analysis (outlined below).

Fermented Mash

The total aflatoxin in the fermented fractions collected from the experiment were

analyzed by LC-MS/MS. Aliquots taken from the fractions collected were

cleaned-up using a 0.45 µm nylon filter before injection with no dilution step

necessary. Fermented mash fractions were not a suitable matrix for the two

immunoassay analysis as indicated by the test kit instructions.

Distilled Ethanol

The total aflatoxin in the distilled ethanol fractions collected from the experiment

were analyzed by LC-MS/MS. Aliquots taken from the fractions collected were

cleaned-up using a 0.45 µm nylon filter before injection with no dilution step

necessary. Distilled ethanol fractions were not a suitable matrix for the two

immunoassay analysis as indicated by the test kit instructions.

51

Stillage

The total aflatoxin in the distilled ethanol fractions collected from the experiment

were analyzed by LC-MS/MS. Aliquots taken from the fractions collected were

cleaned-up using a 0.45 µm nylon filter before injection with no dilution step

necessary. Stillage fractions were not a suitable matrix for the two immunoassay

analysis as indicated by the test kit instructions.

Immunoassay Conditions

Immunoaffinity column assay

An IAC kit using monoclonal antibody-based affinity chromatography, which is

commercially available and is approved by AOAC International and Federal Grain

Inspection Service (FGIS), was purchased. This method was followed as directed by the

instruction manual. A brief outline describing the protocol is as follows: Aflatoxin is

extracted by mixing 50 grams of grounded sample and salt with 100 mL of

methanol/water mixture and then filtered. The filtered extract is then diluted and filtered

again before passing the filtrate over monoclonal antibody affinity column. The column

is then washed with water and the aflatoxins are eluted with methanol into a cuvette.

Developer is then added to the elute before reading the sample with a calibrated Series-

4EX Fluorometer (VICAM, Watertown, MA) to determine aflatoxin concentration. The

IAC kit detection limits range from 1-300 ppb for grains.

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Enzyme-link immunosorbent assay

A direct competitive ELISA kit using polyclonal antibodies, which is

commercially available and is approved by Grain Inspection, Packers and Stockyards

Administration (GIPSA) and FGIS was purchased. The method was followed as directed

by the instruction manual. A brief outline describing the protocol is as follows: Aflatoxin

is extracted by taking 5 g of grounded sample (50 g of sample was stated for

USDA/GIPSA standards but using 5 g of sample was specified in the manual as also

being acceptable) and blending it with 25 mL of a methanol/water solution before

filtering. The filtrate is then diluted if necessary before being sampled and mixed with

enzyme-labeled toxin (conjugate). The mixed solution is transferred to antibody-coated

wells, where free toxin and conjugate compete for antibody binding sites. The unbound

conjugate and other soluble phase substances are then rinsed away and a substrate is

added. Color develops as a result of the presence of bound conjugate. Red stopping

reagent is added and the color of the resulting solution is observed. Samples were

analyzed on Neogen stat fax microwell strip reader (Neogen, Lansing, MI). Absorbance

(OD650) readings of samples are compared with OD650 readings of controls, and the concentration of total aflatoxin in the samples are calculated in ppb. The ELISA kit has a lower detection limit of 2 ppb with a range of quantitation from 5-50 ppb.

LC-MS/MS conditions

For sample separation, a Varian ProStar duel solvent delivery module (Palo Alto,

CA) was used and coupled to with a Bruker Esquire Mass Spectrometer (Billerica, MA)

capable of tandem MS for positive identification. Sample volumes of 50 µL were injected

53

at 25 °C with a Varian ProStar autosampler onto a 5µm particle size C18 column (150 x

2.1 mm). A gradient elution method (Table 2.1) was used for the mobile phase consisting

of water with 0.1% formic acid plus 1.25 mM of ammonium acetate and methanol. The

total Retention times of aflatoxins B1, B2, G1, and G2 are as follows based on elution: 16

min. for G2, 16.6 min. for G1, 17.1 min. for B2, and 17.6 min for B1. A secondary UV scan at a wavelength of 356 nm was done using a Waters 2587 module (Waters

Corporation, Milford, MA). For the MS method, electrospray ionization was used in the

+ positive mode yielding [M+H] parent ions 313, 315, 329, 331 m/z of the aflatoxins B1,

B2, G1, and G2, respectively. The most intense daughter ions, resulting from collision-

induced dissociation were used for identification for B1 (285.1 + 298.1), B2 (297.1 +

287.1 +259.1), G1 (311.1 + 301.1 + 283.1 + 243.1), and G2 (313.0 + 285.1 + 303.1).

Table 2.1 Gradient method used for the mobile phase in separation of samples for the LC-MS/MS method

Time (min) Water% MeOH% Flow (mL/min)

0 100 0 0.3 16 30 70 0.3 40 30 70 0.4 45 0 100 0.4 50 0 100 0.4

54

Standards used for LC-MS/MS Analysis

The standard curve for LC-MS/MS analysis was made by combining separate stock solutions of aflatoxins: 2.0 µg/mL B1, 0.5 µg/mL B2, 2.0 µg/mL G1, 0.5 µg/mL G2

(SUPELCO; Bellefonte, PA) into a working solution of 800 ng/mL total aflatoxin, each aflatoxin equaling 200 ng/mL. A seven point standard curve plotting 8, 40, 80, 200, 400,

600, 800 ng/mL total aflatoxin (2, 10, 20, 50, 100 ng/mL for each of the four individual aflatoxins) was injected on the LC-MS/MS using the same method as described above with an overall, correlation coefficient, R2, ranging from 0.9979 to 0.9990 of all four

aflatoxins.

Preparation and Analysis of DDGs mixed with Binders

Evaluation for decontamination of aflatoxin in DDGs using binders, MTB-100®, was carried out on DDGs made from contaminated corn sample 3. Triplet positive control samples were analyzed, each sample containing 5 g of contaminated DDGs

(approximately 14000 ppb). Duplicate samples containing binder and DDGs made from contaminated corn sample 3 were analyzed, each sample prepared by mixing 1 g of

MTB-100® (Alltech; Lexington, KY) with 5 g of contaminated DDGs. All five samples

were stored at 4oC for seven days to ensure binding before having the aflatoxin extracted

as outlined in the ELISA extraction procedure described above. The filtrate of all the

samples were then passed through a 0.45 µm nylon filter as a clean-up step before

analysis by the LC-MS/MS method described previously. Quantification was done using

peak areas. All solvents were HPLC grade (purchased from Sigma Aldrich, St. Louis,

MO).

55

Results

The total aflatoxin in the four aflatoxin-contaminated corn samples obtained for this experiment were measured by the IAC method and are as follows: Sample 1: 7600 ppb, Sample 2: 10800 ppb, Sample 3: 14800 ppb, and Sample 4: 11200 ppb. ELISA gave comparable values of the contaminated corn samples (shown in Table 2.2). Compilation of the results for the total aflatoxin of four duplicate samples of aflatoxin-contaminated corn plus a duplicate negative control (no aflatoxin detected) are found in Table 2.2. The

DDG samples were analyzed for total aflatoxin by LC-MS/MS, ELISA, and IAC. The fermented mash, distilled ethanol, and stillage fractions were analyzed for total aflatoxin via LC-MS/MS only because the matrices were unsuitable for the two immunoassay methods. No aflatoxin was detected in any of the fractions collected from the negative control corn sample. For the aflatoxin-contaminated corn samples, no aflatoxin was detected in the distilled ethanol fractions. Also, no aflatoxin was detected in the rinse fractions, which were distilled through the fractionating column and Graham condenser after each contaminated sample was distilled for ethanol. Ethanol yields averaged 2.1%

(v/v). Aflatoxin was detected in all fractions collected from the fermented mash with total aflatoxin levels ranging from approximately 130-160 ppb. Also, aflatoxin was detected in all stillage fractions collected with total aflatoxin levels ranging from approximately 130-

195 ppb. The highest aflatoxin levels were measured in the DDGs fractions. The mean total aflatoxin levels for the four DDG

56

Table 2.2 Distribution of aflatoxin B1 in products from ethanol fermentations

Total Aflatoxin (ppb)a

Control Corn Sample Corn Sample 1 Corn Sample 2

Mass Mass Mass Sample Immunoassays Immunoassays Immunoassays spectrometry spectrometry spectrometry

IAC ELISA LC-MS/MS IAC ELISA LC-MS/MS IAC ELISA LC-MS/MS

Corn NAb NDc NA 7600 8200 NA 10800 10000 NA 58 9300 ± 16300 ± 9867 ± 11300 ± 14000 ± 9756 ± DDG NA ND ND 3300 1600 3000 4900 1500 3000

Fermented NA NA ND NA NA 161 ± 7 NA NA 127 ± 13 Mash

Distilled EtOH NA NA ND NA NA ND NA NA ND

Stillage NA NA ND NA NA 133 ± 13 NA NA 166 ± 15

a ppb, nanograms per milliliter (mean of duplicate assays). b NA, not available. c ND, none detected.

Table 2.2 continued

Total Aflatoxin (ppb)

Corn Sample 3 Corn Sample 4

Mass Mass Sample Immunoassays Immunoassays spectrometry spectrometry

IAC ELISA LC-MS/MS IAC ELISA LC-MS/MS

Corn 14800 16000 NA 11200 10944 NA

14300 ± 20700 ± 14208 ± 14600 ± 22400 ± 14149 ± DDG 1300 2000 4000 700 0 3000 59 Fermented NA NA 196 ± 7 NA NA 161 ± 31 Mash

Distilled EtOH NA NA ND NA NA ND

Stillage NA NA 195 ± 29 NA NA 152 ± 6

samples and their replicates are as follows: 9900 ± 3000 ppb, 9800 ± 3000 ppb, 14200 ±

4000 ppb, 14100 ± 3000 ppb. The percentage of aflatoxin recovered was 31-53% in the

DDGs, 13% in the fermented mash and 13% in the stillage.

Figure 2.3 shows the chromatogram obtained from LC-MS/MS analysis of

aflatoxin-contaminated DDGs (14000 ppb) collected from DDGs fractions from corn

sample 3 in the ethanol fermentation experiment previously described. The

chromatogram shows decrease levels of detected aflatoxin in DDGs mixed with binders

(2 replicates) when compared the positive controls (3 replicates) containing only DDGs.

The binders reduced aflatoxin levels by 80%.

60

DDG Replicate 1 Controls DDG Replicate 2 DDG Replicate 3

DDG + Binder Replicate 1 Samples DDG + Binder Replicate 2

Figure 2.3 Chromatogram obtained from LC-MS/MS analysis showing positive controls using aflatoxin-contaminated DDGs (14000 ppb total aflatoxin) and samples containing sorbents mixed in with contaminated DDGs

61

Discussion

During the experiment of ethanol fermentation of corn, the efficiency of the enzymatic conversion of starch to sugar was measured in the mash using a Brix refractometer after removal of DDGs but before fermentation. Controls averaged 8%

Brix, meaning 8 grams of sugar per 100 g of corn was measured. The contaminated corn mash measured an average of 4% Brix. The low Brix of the contaminated mash would account for the low ethanol yields (2.1% v/v). The variation seen here is likely from difference in corn composition and also the amount of starch saccharified.

For analysis of samples results based on the ELISA method were 1.4 to 1.6 times higher than the LC-MS/MS method. The results from the IAC method gave comparable results to LC-MS/MS. Likely cross reactivity with other metabolites is the cause, for

ELISA measures the response from each cross-reacting analogue into a single response, therefore increasing the sensitivity of the ELISA when other analogues are present. In comparison, LC-MS/MS only quantitates the analyte of interest. These findings are in agreement with multiple other studies giving reports that higher recovery values were observed in ELISA assays when comparing them with HPLC and LC/MS methods

(Samdal et al., 2005; Zheng et al., 2005; Colak et al., 2006; Faupel-Badger et al., 2010), but in all cases it was stated that the correlation between the compared methods were good. In one particular study (Faupel-Badger et al., 2010) when LC-MS/MS, RIA, and

ELISA methods were compared for the measurement of urinary estrogens, it was reported that absolute concentrations of estrogen metabolites for RIA and ELISA methods were 1.6 to 2.9 and 1.4 to 11.8 times higher in premenopausal and

62 postmenopausal women, respectively. Their results suggest that LC-MS/MS is a preferable method for comparing absolute or relative amounts of metabolites of interest.

In another study (Zheng et al., 2005) when an ELISA test kit was compared to HPLC for detecting ochratoxin A in food commodities, it was observed that the results from the

ELISA test kit were 1.7 to 1.8 higher than HPLC. Also, in a study conducted by Samdal et al. (2005), blue mussels were analyzed for yessotoxins by ELISA and LC-MS. They observed ELISA responses were 3-13 times higher than LC-MS, probably due to antibodies binding to other yessotoxins analogues not included in the LC-MS analysis.

However, the correlation between ELISA and LC-MS was good, with r2 values ≥ 0.8, indicating that that the ELISA is a reliable method for estimating the total level of yessotoxins in mussels. The variation between methods makes it difficult to compare results across studies that use different detection methods and emphasizes the need for standardization across assays to ensure maximal quality control.

The negative control corn sample showed no aflatoxin in the ground corn sample,

DDGs, fermented mash, distilled ethanol, and stillage. For all aflatoxin-contaminated corn samples, no aflatoxin was detected in the distilled ethanol, which agrees with many reported studies also investigating of mycotoxins during ethanol fermentation (Dam et al., 1977; Lillehoj and Lagoda, 1979; Okoye, 1986; Bothast et al., 1992). Also, no aflatoxin was detected in the rinse fractions, which were distilled through the fractionating column and Graham condenser after each contaminated sample was distilled for ethanol, to make sure no aflatoxin was carried over, if possible, between samples.

63

Aflatoxin was detected in the fermented mash, stillage, and DDGs in each of the contaminate samples.

Liquid chromatography coupled to tandem mass spectrometry analysis of the fermented mash (containing no DDGs) collected before distillation of ethanol measured total aflatoxin levels ranging from approximately 130-160 ppb, an average of 13% total aflatoxin recovered from the starting material. Since no aflatoxin was detected in the distilled ethanol, similar values obtained from the stillage fractions (containing no DDGs) collected after distillation of ethanol measuring 130-195 ppb total aflatoxin is expected.

The greatest concentration of aflatoxin was recovered in the DDGs. From the original

starting weight (100 g) of the contaminated corn samples (1-4), 37, 34, 47, and 42 g,

respectively, were recovered as DDGs and consequently, accounts for 37, 34, 47, and

42% of the weight of the original corn samples. From these DDG samples, 9900, 9800,

14200, 14100 ppb of total aflatoxin were detected via LC-MS/MS accounting for 48, 31,

45, and 53% recovery of aflatoxin from the starting material. The average of a 44%

recovery is comparable to 35% recovery of total aflatoxin reported by Bothast et al.

(1992). The unrecovered aflatoxin may have ended up in the solubles, which were not

analyzed in this study.

MTB-100®, an esterified glucomannan processed from yeast cell walls, was

mixed in with contaminated DDGs (14000 ppb), making up 16.7% of the total weight.

After allowing the mixed DDGs and binders sit for seven days, arbitrarily decided to

allow for binder/DDGs interaction, analysis via LC-MS/MS showed a remarkable

decrease in aflatoxin concentration. The binders were able to sequester large amounts of

64 aflatoxin as demonstrated by the chromatogram in figure 2.3, reducing aflatoxin concentration by 80% according to the ratio in peak’s heights when comparing the positive control with the contaminated samples containing the sorbent. These results agree with two thorough studies (Diaz et al., 2002; Diaz et al., 2004) investigating MTB-

100® and several other possible sequestering agents on the reduction of aflatoxin in vitro

® and in vivo. Results showed that MTB-100 bound over 95% of aflatoxin B1 in vitro and reduced aflatoxin M1 contamination 59% in lactating Holstein cows when MTB-

100®consisted 0.05% of the diet. For the present experiment, further testing would need to be done to determine how aflatoxin is associating with the binders, if it is covalently bounded or tightly interacted and how the binder-aflatoxin interaction affects the physiology of the animals.

Conclusion

Corn is a major crop affected by aspergillus mold and hence aflatoxin contamination. With the growing need for alternative fuels sources, the demand for corn among ethanol production plants is increasing, therefore diverting corn from the consumer’s market and feed for cattle. The overwhelming competition for corn, makes aflatoxin contamination a serious problem in the U.S. Therefore, determining where aflatoxin is concentrated during ethanol fermentation process of contaminated corn is important in the effort to keep corn prices low, help in the sustainability of ethanol plants and for the health of animals consuming DDGs from the co-products of ethanol plants. In this study aflatoxin was not detected in the distilled ethanol. Most aflatoxin was

65 recovered in the DDGs, and was also detected in the fermented mash and stillage but at much lower levels. Binders may be a very easy and cost effective means for treating contaminated DDGs collected after fermentation. This current study investigated MTB-

100® as a possible agent for sequestering aflatoxin in contaminated DDGs with results showing 80% reduction in total aflatoxin levels. Currently, no binders are FDA approved for treating contaminated DDGs (Whitlow, 2006). Numerous studies have already investigated many of the concerns associated with using binders, but more studies are needed to address how aflatoxin is interacting with the sorbents, how the pH may affect aflatoxins binding to sorbents after ingestion by animals, and how this physiologically affects the animals.

66

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71

CHAPTER III

DETOXIFICATION OF AFLATOXIN USING CLOROX® ANALYZED BY LC-

MS/MS

Abstract

An experiment was conducted to investigate the effectiveness of commercial

® bleach (Clorox : active ingredient NaOCl) in detoxifying aflatoxin B1 upon contact. It

® was shown that a 0.1% solution of Clorox reduced 500 ng/mL of aflatoxin B1 by 99%

when analyzed by LC-MS/MS two minutes later. Aflatoxin B1 reduction was inversely

proportional to the logarithm of bleach concentration.

Introduction

It was not until 1960 when over a 100,000 turkey poults died in Europe from

aflatoxin-contaminated feed meal that the era of mycotoxin research began in earnest

(Blount, 1960). Aflatoxin’s toxicity has classified it as a Group I carcinogen by the

International Agency for Research on Cancer (IARC, 1985) where the liver is the primary

target (Cullen and Newberne, 1994; Roebuck and Maxuitenko, 1994). Aflatoxins are

produced by several fungal species, most commonly Aspergillus flavus and A. parasiticus

(Bennett and Klich, 2003; Yu et al., 2004). The four main aflatoxins studied are B1, B2,

G1, and G2 (Wei and Jong, 1986; Klich and Pitt, 1988;

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Klich, 2007) with B1 being the most toxic and predominately expressed among the four

(Wogan, 1966; Maggon et al., 1970; Squire, 1981; Bennett and Klich 2003). Aflatoxin is

found worldwide contaminating agricultural crops consisting of corn, wheat, and grain

and other food commodities such as figs, nuts, tobacco, and fruits (Payne, 1983; Klich et

al., 1986; Lillard et al., 1970; Price et al., 1993; Martins et al., 2003; Bircan et al., 2008;

Delage et al., 2008; Rahimi et al., 2008). Aflatoxin may also indirectly contaminate dairy

products (milk, cheese, etc.) when dairy animals consume contaminated feeds (Van

Egmond, 1989; Chun Pei et al., 2009). A major problem in dealing with aflatoxin

contamination is that it is impossible to completely eliminate its source of production since Aspergillus is a natural contaminant of food crops (Wood, 1992) and contamination

may occur during pre and post harvest conditions. Consequently, aflatoxin is one of the

few mycotoxins regulated by the U.S. Food and Drug Administration to help reduce

potential health risks to animals and humans through the consumption of aflatoxin-

contaminated food commodities.

Aflatoxins are destroyed by strong oxidizing reagents (Fischbach and Campbell,

1965; Stoloff and Trager, 1965; Maggon et al. 1970; Yang, 1972; Natarajan et al., 1975).

To date, it is common among laboratories conducting aflatoxin research to use

commercial bleach solution containing NaOCl as a convenient and simple method for

decontaminating laboratory utensils and work stations. This method was originally

proposed by Fischbach and Campbell (1965) and Stoloff and Trager (1965),

recommending a 10% bleach solution and is upheld by Official Methods of Analysis of

AOAC International (2002) as a suitable practice for aflatoxin-decontamination. The

73

purpose of the present study was to investigate the effectiveness of commercial

household bleach on standard grade aflatoxin B1, and what levels of bleach concentration

were no longer effective on aflatoxin B1.

Materials and Methods

Standards Information

Aflatoxin B1 standard was purchased from Sigma-Aldrich, St Louis, MO.

Commercial bleach, Clorox®, (active ingredient 6% NAOCl) was purchased locally.

Sample Preparation

Various concentrations of commercial bleach (Clorox®, active ingredient 6%

NaOCl) were made using bleach diluted with distilled water and kept at room

temperature. A working solution of 1000 ng/mL of aflatoxin B1 in methanol was

prepared.

® A negative control sample of 500 ng/mL aflatoxin B1 (no Clorox ) in water was

prepared from aflatoxin B1 working solution. Negative control samples of aflatoxin B1 were tested before and after samples solutions of Clorox®/aflatoxin. The samples

containing Clorox® and aflatoxin were prepared by making a 1:1 solution of known

® concentration of Clorox and aflatoxin B1 working solution (1000 ng/mL). The sample

was briefly vortexed and diluted 1:5 before analyzed by LC-MS/MS. Approximately two

minutes elapsed from the preparation of the sample to injection onto LC-MS/MS.

74

LC-MS/MS Conditions

For sample separation, a Varian ProStar duel solvent delivery module (Palo Alto,

CA) was used and coupled to with a Bruker Esquire Mass Spectrometer (Billerica, MA)

capable of tandem MS for positive identification. Sample volumes of 50 µL were injected

at 25 °C with a Varian ProStar autosampler onto a 5µm particle size C18 column (150 x

2.1 mm). A gradient elution method (Table 3.1) was used for the mobile phase consisting

of water with 0.1% formic acid plus 1.25 mM of ammonium acetate and methanol. The

total Retention time of aflatoxin B1 by this method was 17.6 min. A secondary UV scan at a wavelength of 356 nm was done using a Waters 2587 module (Waters Corporation,

Milford, MA). For the MS method, electrospray ionization was used in the positive mode

+ yielding [M+H] parent ions 313, 315, 329, 331 m/z of the aflatoxins B1, B2, G1, and G2,

respectively. The most intense daughter ions, resulting from collision-induced

dissociation were used for identification for B1 (285.1 + 298.1).

Table 3.1 Gradient method used for the mobile phase in separation of samples used for the LC-MS/MS method

Time (min) Water% MeOH% Flow (mL/min)

0 100 0 0.3 16 30 70 0.3 40 30 70 0.4 45 0 100 0.4 50 0 100 0.4

75

Results

Figure 3.1 depicts how various concentrations of Clorox® (ranging from 0.001 to

0.1%) effect aflatoxin B1 at 500 ng/mL. The results show a logarithm relationship

between aflatoxin and various concentrations of Clorox® with a correlation coefficient, R2,

® of 0.9953. A 0.1% solution of Clorox reduced 500 ng/mL of aflatoxin B1 by 99%. A

0.5% Clorox® solution showed no detection of aflatoxin (results not shown). A replicate

sample of 0.1% Clorox® solution was measured (Figure 3.1) with standard deviation of

2.76.

Aflatoxin B1 (ng/mL)

600

500

400 (ng/mL) 1 300

200 Aflatoxin B Aflatoxin 100

0 0 0.02 0.04 0.06 0.08 0.1 0.12 % Clorox in water solution

Figure 3.1 Graph showing seven different Clorox®: water solutions ranging from ® 0.001 to 0.1% Clorox , each mixed with (500 ng/mL) of aflatoxin B1. The data follows a logarithmic equation (y = -120.71Ln(x) - 261.56 with a correlation coefficient, R2, of 0.9953, showing that the more concentrated the Clorox® solution the lower the aflatoxin concentration. Error bars for a 0.1% Clorox® replicate sample is shown

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Discussion

Several studies have investigated the effect of Clorox® on aflatoxins (Fischbach

and Campbell, 1965; Stoloff and Trager, 1965; Yang, 1972) using fluorescence, UV

spectrophotometry, and thin layer chromatography methods. The use of LC-MS/MS

methods, could better extrapolate at what concentrations of Clorox® is not longer effective on eliminating aflatoxin. Since Clorox® is a corrosive chemical, after mixing the

® working solution of aflatoxin B1 with one of the Clorox concentrations, the aflatoxin-

Clorox® solution was diluted 1:5 so as to help minimize corrosion of the LC column. The

reproducibility of the negative control samples tested before and after samples

(containing Clorox®/aflatoxin) analysis indicates that Clorox® did not cause any negative effects on the LC column. Quantification of aflatoxin levels were based on peak areas compared with the negative control sample. Samples were made so that the final concentration measured by LC-MS/MS was 500 ng/mL of aflaotxin B1 and

concentrations ranging from 0.001 to 0.1% of Clorox®. Figure 3.1 shows LC-MS/MS

® analysis of six samples containing aflatoxin B1 and Clorox . A surprising find is the

logarithmic trend Clorox® has on aflatoxin (correlation coefficient, R2, of 0.9953). From the

® data, 0.1% solution of Clorox reduced 500 ng/mL of aflatoxin B1 by 99% when

analyzed by LC-MS/MS two minutes later. A 0.5% Clorox® solution showed no

detection of aflatoxin B1 (results not shown). Based on the trend line calculated a 0.12%

Clorox® solution is predicted to completely destroy aflatoxin B1 at 500 ng/mL. A replicate

sample of 0.1% Clorox® solution was measured (Figure 3.1) with standard deviation of

2.76.

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These results are in agreement with a thorough study conducted by Yang (1972)

on detoxification of aflatoins by sodium hypochlorite and commercial bleaches. Yang

investigated how effective diluted bleach is in destroying aflatoxins produced by

toxigenic fungi based on UV spectrophotometry and thin-layer chromatographic analyses

and concluded that the recommended 10% bleach solution (containing 5-6% active

ingredient NaOCl) proposed by Fischbach and Campbell (1965) and Stoloff and Trager

(1965) is sufficient. Results obtained from this experiment confirm that concentrations of

5 to 6% Clorox® is sufficient for complete destruction of aflatoxin within minutes.

Conclusion

Clorox® at a 10% solution is an AOAC international approved method for decontamination of aflaxin-contaminated glassware and work bench areas in laboratories.

The use of Clorox® as an adequate method of clean-up was first published by Fischbach

and Campbell (1965) and Stoloff and Trager (1965). The results of this experiment

confirmed that Clorox® is an effective means for aflatoxin decontamination and indicates

that the proposed 10% Clorox® solution is more than adequate for laboratories handling

aflatoxin-contaminated substances.

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