The Evaluation of Adsorbents for the Removal of Aflatoxin M1 from Contaminated Milk

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Erika D. Womack

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The evaluation of adsorbents for the removal of aflatoxin M1 from contaminated milk

Erika D. Womack

A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Molecular Biology in the Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology

Mississippi State, Mississippi

December 2015

Erika D. Womack

2015

The evaluation of adsorbents for the removal of aflatoxin M1 from contaminated milk

Erika D. Womack

Approved:

______Darrell L. Sparks, Jr. (Major Professor)

______Ashli Brown-Johnson (Minor Professor)

______Janice DuBien (Minor Professor)

______Stephanie H. Ward (Committee Member)

______Xueyan Shan (Committee Member)

______Kenneth O. Willeford (Graduate Coordinator)

______George Hopper Dean College of Agriculture and Life Sciences

Name: Erika D. Womack

Date of Degree: December 11, 2015

Institution: Mississippi State University

Major Field: Molecular Biology

Major Professor: Darrell L. Sparks, Jr.

Title of Study: The evaluation of adsorbents for the removal of aflatoxin M1 from contaminated milk

Pages in Study: 210

Candidate for Degree of Doctor of Philosophy

Taking precautions to restrain aflatoxin M1 (AFM1) from milk is critical, particularly due to the health and economic impact AFM1 imposes. The predominant post-harvest means of reducing AFM1 in milk includes the addition of sequestering agents to feed to diminish the bioavailability of aflatoxin B1 (AFB1), the parent compound of AFM1 found in contaminated feed. Still, residual AFM1 has been found in the milk.

Using sequestering agents added to raw milk, we found that activated carbon was the most effective binder to reduce AFM1 contamination. The combination of 0.75% granular activated carbon (GAC) and a flow rate of 0.4 mL/min to pump contaminated milk through a glass column were chosen as optimum conditions for the removal of

AFM1. These conditions obtained a 98.4% reduction of 0.75 ng/mL AFM1 from raw milk. The treated milk was also analyzed to assess the effects of GAC on milk constituents. The results determined that GAC had no significant effect on major nutritive milk constituents: total protein, lactose, minerals, and fat. Additionally, we optimized an extraction method coupled to high performance liquid chromatography tandem mass

spectrometry (HPLC-MS/MS) that minimized matrix effects, lowered the levels of

detection, and reduced analysis costs. The optimized extraction method was based on

QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe). Results determined 5

mL milk (15°C) with 10 mL acetonitrile, 3200 g centrifugation, and 0.2 µm syringe filter were the optimum conditions for the extraction of 0.5 ng/mL AFM1 from raw milk. The

method was validated according to AOAC guidelines.

This study reports experimental results on AFM1 remediation from raw bovine

milk. The use of GAC for the removal of AFM1 in raw milk has reduced the amount of

AFM1 below the FDA action limit and European Union maximum regulatory level. This method could have a global health impact, particularly, for people in developing nations and for infants and children who are more susceptible to the adverse effects of AFM1.

DEDICATION

My graduate work is dedicated to my mom, Lenna B. Womack. Her strength and faith surpassed anything I could ever hope to accomplish. I really would not have made it this far without her encouragement and love. It has impacted who I am today and for that

I am eternally grateful. Rest in Heaven.

ACKNOWLEDGEMENTS

I would first like to thank my major professor, Dr. Darrell L. Sparks. Without his guidance and knowledge throughout my entire doctoral career, this dissertation would not be possible. Even through my “…so Dr. Sparks…” questions, he’d effortlessly guide me through any problem. I also would like to thank Dr. Ashli Brown who has guided me as well through this degree process. She has offered tremendous support and introduced me to a number of opportunities I may have never experienced. I would like to thank the rest of my committee members, Dr. Janice DuBien, Dr. Stephanie H. Ward, and Dr. Xueyan

Shan, for providing guidance and mentoring me through this process. I would especially like to thank Dr. DuBien for helping me through some difficult statistical analysis.

I would like to thank Kenneth Graves for supplying me with limitless fresh raw milk from MSU’s dairy farm. I would like to thank the Mississippi State Chemical

Laboratory for, not only analyzing samples for me but also teaching me to use very expensive laboratory equipment and instruments. Thank you for putting a little trust in me that I may not break your instruments. I would like to thank the Mississippi

Agricultural and Forestry Experiment Station (MAFES) for supporting my graduate assistantship.

Lastly, I would like to thank my family and friends for their love and support.

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TABLE OF CONTENTS

DEDICATION ...... ii

ACKNOWLEDGEMENTS ...... iii

LIST OF TABLES ...... vii

LIST OF FIGURES ...... x

CHAPTER

I. GENERAL INTRODUCTION ...... 1

Background of the Problem ...... 1 Statement of the Problem and Study Justification ...... 4 Significance, Objectives, and Goals of the Project ...... 5

II. LITERATURE REVIEW ...... 7

Mycotoxin-Producing Fungi ...... 7 Aspergillus ...... 11 Aflatoxin ...... 13 Background ...... 13 Economic and health implications of aflatoxin ...... 15 Factors influencing aflatoxin contamination ...... 18 Aflatoxin biosynthesis ...... 22 Pre-harvest Management of Aflatoxin ...... 28

III. A RECENT REVIEW OF NON-BIOLOGICAL REMEDIATION OF AFLATOXIN-CONTAMINATED CROPS ...... 31

Abstract ...... 31 Introduction ...... 31 Sorting of aflatoxin-contaminated grains ...... 34 Binding agents for the reduction of aflatoxins ...... 38 Irradiation for the reduction of aflatoxins ...... 41 Chemical methods for the detoxification of aflatoxins ...... 46 Conclusion ...... 50

IV. AFLATOXIN M1 IN MILK AND MILK PRODUCTS...... 52 iv

Abstract ...... 52 Introduction ...... 53 Toxicity ...... 55 Analytical methodology ...... 57 Sample preparation and clean-up ...... 57 Detection and quantitative techniques ...... 62 Occurrence ...... 66 Prevention and Control of AFM1 ...... 74 Conclusion ...... 78

V. THE EVALUATION OF ADSORBENTS FOR THE REMOVAL OF AFLATOXIN M1 FROM CONTAMINATED MILK ...... 79

Abstract ...... 79 Introduction ...... 80 Materials and Methods ...... 84 Milk preparation...... 84 Adsorbent materials ...... 84 Sample Preparation ...... 85 Analytical instrument conditions ...... 87 Linearity ...... 87 Statistical Analysis ...... 88 Results and Discussion ...... 88 Conclusion ...... 92

VI. THE VALIDATION OF QuEChERS AS AN EXTRACTION METHOD FOR AFLATOXIN M1 IN RAW MILK ...... 94

Abstract ...... 94 Introduction ...... 94 Materials and Methods ...... 98 Chemicals and reagents ...... 98 Milk samples ...... 99 Sample preparation ...... 99 Extraction optimization ...... 100 Analytical instrument conditions ...... 102 Method validation ...... 103 Results and Discussion ...... 105 Extraction optimization ...... 105 MS/MS Optimization ...... 108 Method validation ...... 109 Conclusion ...... 114

VII. THE EFFECTS OF GRANULAR ACTIVATED CARBON ON RAW MILK CONSTITUENTS ...... 116

Abstract ...... 116 Introduction ...... 117 Materials and Methods ...... 120 Milk preparation...... 120 Adsorbent materials ...... 120 Sample Preparation ...... 120 Experiment 1: Optimization of granular activated carbon ...... 121 Calibration curve for the peristaltic pump ...... 121 Experiment 2: Effects of granular activated carbon on milk constituents ...... 121 Statistical Analysis ...... 122 Results and Discussion ...... 122 Experiment 1: Optimization of granular activated carbon ...... 122 Experiment 2: Effects of granular activated carbon on milk constituents ...... 126 Conclusion ...... 129

VIII. GENERAL DISCUSSION AND SUMMARY ...... 130

REFERENCES ...... 134

APPENDIX

A. RAW DATA AND STATISTICAL ANALYSES FOR CHAPTER V ...... 164

B. RAW DATA AND STATISTICAL ANALYSES FOR CHAPTER VI ...... 182

C. RAW DATA AND STATISTICAL ANALYSES FOR CHAPTER VII ...... 201

LIST OF TABLES

3.1 Physical removal of contaminated nuts and grains through sorting and processing...... 37

3.2 Reduction of aflatoxins by the addition of adsorbents ...... 39

3.3 Detoxification of aflatoxin through irradiation...... 44

3.4 Detoxification of aflatoxin through chemical methods...... 48

4.1 Sample preparation involving extraction of AFM1 from milk ...... 58

4.2 Sample preparation involving extraction and clean-up of AFM1 from milk products ...... 60

4.3 Occurrence of AFM1 in milk between 2010 and 2015 in Africa...... 68

4.4 Occurrence of AFM1 in milk between 2010 and 2015 in the Americas...... 69

4.5 Occurrence of AFM1 in milk between 2010 and 2015 in Asia...... 71

4.6 Occurrence of AFM1 in milk between 2010 and 2015 in Europe...... 73

5.1 The analysis of variance (ANOVA) used to determine the significance of the main effects and interactions ...... 89

5.2 ANOVA to determine the significance of the main effects and nested interactions between binder type and binder concentration ...... 91

5.3 Least squares means for the significant main effect, binder type ...... 91

6.1 ANOVA analysis showing significant main effect of the QuEChERS extraction method ...... 101

6.2 A 23 Factorial design of varied factor combinations using QuEChERS ...... 102

6.3 ANOVA analysis of the 23 factorial design of varied factor combinations using QuEChERS ...... 106

6.4 Recovery and relative standard deviation (RSD) from spiked raw milk ...... 113

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6.5 HPLC optimization using peak response ...... 114

7.1 One sample t-test below the FDA AL of 0.5 ng/mL ...... 126

7.2 The effects of GAC on milk constituents when applied to blank milk and aflatoxin M1-contaminated milk...... 127

A.1 Raw data of the crossed, nested statistical design ...... 166

A.2 Summary statistics of the crossed, nested statistical design ...... 169

A.3 ANOVA analysis of the crossed, nested statistical design ...... 172

A.4 Least squares means of milk*binder concentration nested in binder type ...... 173

A.5 Least squares means of milk*binder type ...... 176

A.6 Raw data for nested design ...... 179

A.7 Summary statistics of the nested statistical design ...... 180

A.8 ANOVA analysis of the nested statistical design ...... 181

A.9 Least squares means of the nested statistical design ...... 181

B.1 Raw data of the 27-4 fractional factorial design ...... 184

B.2 Summary statistics of the 27-4 fractional factorial design ...... 185

B.3 ANOVA analysis of the 27-4 fractional factorial design ...... 187

B.4 Raw data of the 23 factorial design ...... 189

B.5 Summary statistics of the 23 factorial design ...... 190

B.6 ANOVA analysis of the 23 factorial design ...... 191

B.7 Least squares means of ACN volume*milk volume ...... 192

B.8 Least squares means of milk temperature*milk volume ...... 193

B.9 Raw data of the 23 factorial design ...... 196

B.10 Summary statistics of the 23 factorial design ...... 197

B.11 ANOVA analysis of the 23 factorial design ...... 198

B.12 Least squares means of ACN volume*milk volume ...... 199

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B.13 Least squares means of milk temperature*milk volume ...... 200

C.1 Raw data for 52 factorial design for contact study ...... 203

C.2 Summary statistics for 52 factorial design for contact study ...... 205

C.3 ANOVA for 52 factorial design for contact study ...... 206

C.4 Least squares for 52 factorial design for contact study ...... 206

C.5 One sample t-test below the FDA AL of 0.5 ng/mL ...... 209

LIST OF FIGURES

1.1 Robinson projection map highlighting subtropical and tropical regions where Aspergillus is most prominent...... 2

1.2 Chemical structure of aflatoxin M1...... 3

2.1 Chemical structures of mycotoxins found in food and feed...... 8

2.2 Chemical structures of aflatoxin B1, B2, G1, and G2...... 9

2.3 Aspergilli species grown on media...... 11

2.4 Metabolism of AFB1...... 15

2.5 Aflatoxin biosynthesis gene cluster in A. flavus and A. parasiticus...... 23

2.6 Proposed mechanism for the conversion of AFB1 to AFM1 by cytochrome P450 enzymes...... 26

3.1 Metabolism of AFB1 by cytochrome P450...... 33

3.2 Proposed pathway for degradation of AFB1 by UV...... 42

4.1 AFM1 is the hydroxylated derivative of AFB1...... 54

5.1 Burrell Wrist Action used for sample homogenization ...... 85

5.2 The extraction of a milk sample containing AFM1 and 0.4% powdered activated carbon (PAC) using QuEChERS extraction method...... 86

5.3 Maximum percent binding of PAC when exposed to 50 ng/mL AFM1 in skim milk...... 89

5.4 Reduction of AFM1 when sequestering binders are added to milk ...... 90

6.1 HPLC-MS/MS used for the quantification of AFM1 ...... 98

6.2 Geno-Grinder used to vigorously vortex milk samples...... 100

6.3 Linearity assessed using a matrix-matched 13-point calibration curve (replicated 3 times) ranging from 0.002 to 8 ng/mL...... 104 x

6.4 A comparison of milk matrices by milk volume using varied parameters of QuEChERS...... 107

6.5 Fragmentation pattern for AFM1 as proposed by Biancardi et al. (2013)...... 109

6.6 A comparison of chromatogram displaying a blank milk (top) and milk spiked with 0.5 ppb AFM1 (bottom)...... 110

6.7 The limit of detection (LOD) displayed (top) at a concentration of 0.002 ng/mL AFM1...... 112

7.1 Carbon structures containing pentagonal, hexagonal, and heptagonal rings ...... 119

7.2 Calibration curve for peristaltic pump setting ...... 123

7.3 Illustration of granular activated carbon added to the bed of a glass column ...... 124

7.4 The effects of varying concentrations of granular activated carbon for the removal of a mean 0.78 ng/mL AFM1 from milk at varying flow rates...... 125

7.5 Plot of distribution of AFM1 with 90% lower confidence ...... 126

B.1 Plot of ACN volume*milk volume ...... 194

B.2 Plot of milk temperature*milk volume ...... 194

C.1 Plot of distribution of AFM1 with 90% lower confidence ...... 210

GENERAL INTRODUCTION

Background of the Problem

Aflatoxins are a group of structurally related difuranocoumarins produced as

secondary metabolites mainly by fungal strains of Aspergillus flavus (A. flavus), which is found on aerial parts of plants (leaves and flowers), and parasiticus (A. parasiticus), which can be found in soils (Mclean and Dutton, 1995). Aflatoxins, the most extensively studied mycotoxin, exhibit mutagenic, immunosuppressive, teratogenic, and carcinogenic effects where the main target organ is the liver.

Aflatoxin exposure in humans is a global concern and difficult to avoid as

Aspergillus is ubiquitous in nature. These toxic compounds occur widely in temperate,

sub-tropical, and tropical areas (Klich, 2002) that lie between the latitudes of 40°N and

40°S (Figure 1.1) where the climate promotes the growth of Aspergillus on important

agricultural commodities including maize (Zea mays), cotton (Gossypium), peanuts

(Arachis), and tree nuts (Magan and Aldred, 2007; Paterson and Lima, 2011). These

regions include Sub-Saharan Africa and Southeast Asia where liver cancer is most

prominent, and where the prevalence of aflatoxin exposure is 16-32 times higher

compared to that of developed nations (Mitchell et al., 2014; Diaz and Sanchez, 2015).

Aflatoxins are produced in response to high temperatures, high humidity, and drought

stress in the soil during pre-harvest, post-harvest processing, or storage conditions. 1

Figure 1.1 Robinson projection map highlighting subtropical and tropical regions where Aspergillus is most prominent.

Adapted from Arizona Geographic Alliance.

Surveys reveal sufficiently high occurrences and concentrations of aflatoxins to suggest that they are a constant financial concern (Santos et al., 2014; Zheng et al., 2013;

Vardon, 2003). Economic injury due to aflatoxins include: (1) deficit due to diseases induced by toxigenic fungi; (2) deficit due to animal productivity (animal reproduction, performance, and health); (3) the cost of preventive measures; and (4) the reduced value and quality of crops (CAST, 2003). Furthermore, aflatoxin-contaminated commodities cannot be exported. These economic impacts are often felt amongst crop producers, animal producers, grain handlers and distributers, and consumers.

Aflatoxin contaminated crops result not only in economic losses, but also have a tremendous impact on human health. The evaluation of epidemiological and laboratory results carried out in 1993 by the World Health Organization’s International Agency of 2

Research for Cancer (IARC) found that there is sufficient evidence of the carcinogenic effect of aflatoxins to classify it as a Group 1, known carcinogen to humans (IARC,

1993). Aflatoxin B1 (AFB1), the most toxic aflatoxin, is also the most potent naturally

occurring liver carcinogen known. Consumption of contaminated foods, implicated as the

primary route of AFB1 exposure, results in an increased risk for the development of liver cancer (hepatocellular carcinoma, HCC) and hepatic failure resulting in death (Lewis et

al., 2005). AFM1 (Figure 1.2), reported as a Group 2B, possible human carcinogen

(IARC, 2002), is the major oxidized metabolite of AFB1 found primarily in the urine and

milk of lactating animals, including humans, that have ingested AFB1-contaminated crops. Although not as carcinogenic or mutagenic as AFB1, AFM1 is associated with

cytotoxicity, DNA damage, and gene mutation (Caloni et al., 2006; Prandini et al., 2009).

If consumed, AFM1 can have a significant impact on animal and human health including

immunosuppression, liver cancer, and death (Williams et al., 2004). Due to the toxic nature of AFM1 in milk, decontamination processes are desirable.

Figure 1.2 Chemical structure of aflatoxin M1.

Statement of the Problem and Study Justification

The addition of sequestering agents or binders to AFB1-contaminated feed has

been considered the most promising dietary approach to reduce AFM1 toxicity (Galvano

et al., 2001; Whitlow, 2006). The remediation of AFB1 includes the use of adsorbent

materials including activated carbon, silicates (phyllosilicate, clay, bentonite,

montmorillonite, zeolite, etc.), and complex indigestible carbohydrates (cellulose, polysaccharides in the cell walls of yeast and bacteria, etc.). The sequestering agents added to contaminated feed bind strongly enough to the toxin to reduce AFB1 intestinal

absorption in the digestive tract (Pasha et al., 2007; Kutz et al., 2009). Yet, not all AFB1

is bound and maybe metabolized to AFM1 in the liver and excreted in the milk.

Provisions to limit AFM1 in milk are necessary. The introduction of sequestering binders

directly to contaminated milk may be an effective method for the removal of AFM1

residues. Only a few studies have been conducted on the remediation of AFM1 directly in

milk using sequestering binders (Di Natale et al., 2009; Soha et al., 2006).

Special attention has been made to milk and restrictive levels of AFM1 have been

imposed. Thus, limits as low as 0.05 ng/mL, as enforced by the European Union, and 0.5

ng/mL, as set by the United States Food and Drug Administration (FDA), have been

established for the regulation of AFM1. With more stringent regulations of AFM1, the development of rapid, efficient, and accurate analytical methods for the detection and quantification of AFM1 is warranted to determine AFM1 at these very low levels. Hence,

the need for discriminating extraction techniques for the determination of AFM1 in milk

is desirable. Liquid-liquid extraction (LLE), the conventional method, and solid-phase

extraction (SPE) are commonly utilized for the separation of AFM1 from milk (Fallah,

2010a; Wang et al., 2012). Although effective, these methods are time-consuming and costly (Paya and Anastatassiades, 2007). The development of extraction techniques to achieve a simple, fast, and reliable method is ongoing and optimal conditions for the extraction of AFM1 from milk should be attained.

Significance, Objectives, and Goals of the Project

Milk is a good source of many nutrients, and used extensively throughout the world. In consideration of milk consumption, especially of children, exposure to AFM1 in

milk has gained particular attention as a matter of considerable concern. This toxin has

also been the subject of legislative regulations in many countries (Food and Agricultural

Organization, FAO, 2004). The sequestering agents offer an approach to the reduction of

AFB1 toxicity in feed (Phillips et al., 2008); and therefore, these binders might offer the potential reduction of AFM1 levels in milk (Soha et al., 2006). This research investigated the physical adsorption of AFM1 from artificially contaminated raw milk using sequestering agents. The hypothesis for this research stated that if a sequestering agent is added to milk, then the agent will bind strongly enough to AFM1 to prevent the toxicity

of raw milk. The main objectives of this research were:

1. To examine the proficiency of adsorbents (Biofix, Mycofix, MTB-100,

and powdered activated carbon) to detoxify AFM1 from raw milk

2. To develop and validate the QuEChERS (Quick, Easy, Cheap, Effective,

Rugged, and Safe) extraction method for the quantitation of AFM1 in

bovine, unpasteurized raw milk using high performance liquid

chromatography tandem mass spectrometry (HPLC-MS/MS)

3. To determine the effectiveness of the “best” adsorbent on raw milk

constituents: water, lactose, protein, fat, vitamins, and minerals

The overall goal is to offer an alternative means for the removal of AFM1 from milk which can be used to improve the economic and health effects AFM1 inflicts globally, and to gather a better understanding of the problem it imposes on the dairy industry.

LITERATURE REVIEW

Mycotoxin-Producing Fungi

The term mycotoxin is usually reserved for the toxic chemicals produced by various filamentous fungi that have adverse effects on humans, animals, and crops that result in illnesses and economic losses (Wood, 1992). Mycotoxins are produced as a result of high temperatures and humidity, as well as temperate zones, poor harvesting practices, and improper storage. Mycotoxin ingestion by humans and animals can lead to deterioration of liver and kidney function. Some mycotoxins are neurotoxic, while others act by interfering with protein synthesis, and producing effects ranging from skin sensitivity or necrosis to extreme immunodeficiency (Sweeney et al., 2000). Mycotoxins produce diseases known as mycotoxicoses causing various acute and chronic effects on humans and animals, especially monogastrics. Ruminants generally have been more resistant to the deleterious effects of mycotoxins due to their rumen microbiota that is capable of degrading mycotoxins (Zain, 2011). There are many such compounds, but only a few of them are regularly found in food and animal feedstuffs (Figure 2.1).

Figure 2.1 Chemical structures of mycotoxins found in food and feed.

Aflatoxin, the most abundant and toxic compound, is principally produced by fungal species A. flavus and A. parasiticus, which are the primary causal agents of food and feed contamination. There are over 20 aflatoxins identified with the most thoroughly studied mycotoxins being aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2)

(Figure 2.2), which are named based on their fluorescence under ultraviolet light (blue or green). AFM1 and aflatoxin M2 (AFM2), produced in milk and dairy products, are oxidative metabolic products of AFB1 and AFB2, respectively. AFB1 is the most potent

toxin and a known natural human carcinogen. It is usually the major mycotoxin produced

by toxigenic strains (Dohnal et al., 2014). Foods particularly susceptible to aflatoxin

contamination are those cultivated in the tropic and sub-tropic areas (Klich, 2002).

Figure 2.2 Chemical structures of aflatoxin B1, B2, G1, and G2.

Fumonisins are produced by Fusarium, a filamentous fungi widely distributed in

the soil. Because of the relative abundance of Fusarium, these mycotoxins can enter the food chain through commonly consumed cereals such as maize and wheat (Schaafsma and Hooker, 2007). There are over 15 fumonisins identified with the most thoroughly studied and toxic of the group being fumonisins B1 (FB1) and B2 (FB2). FB1 and FB2,

produced primarily by F. proliferatum and F. verticillioides, are cancer-causing

metabolites targeting the liver, kidneys, and esophagus (Stockmann-Juvalla and

Savolainen, 2008). The IARC has classified FB1 as a Group 2B carcinogen (IARC,

1993).

Deoxynivalenol (DON) is a type of mycotoxin produced by certain Fusarium species that frequently infect maize (Zea mays), wheat (Triticum), barley (Hordeum), oats

(Avena), rice (Oryza), and other grains in the field or during storage (Sobrova et al.,

2010). DON is capable of producing a wide range of toxic effects, affecting animal and

human health causing acute temporary nausea, vomiting, diarrhea, abdominal pain,

headache, dizziness, and fever (Sobrova et al., 2010). At the molecular level, DON

disrupts normal cell function by inhibiting protein synthesis through the binding of ribosomes and by activating critical cellular kinases involved in signal transduction related to proliferation, differentiation, and apoptosis (Pestka and Smolinski, 2005).

Zearalenone (ZEN), also produced by Fusarium, is found in maize, barley, oats,

and wheat (Suzuki et al., 2007). ZEN is a non-steroidal estrogenic mycotoxin that causes

alterations in the reproductive tract of animals (Belhassen et al., 2015). This mycotoxin

has been shown to competitively bind to estrogen receptors in the uterus, mammary

gland, liver, and hypothalamus of different species. Various estrogenic effects such as

decreased fertility, increased embryolethal resorptions, reduced litter size, changed

weight of adrenal, thyroid, and pituitary glands, and changes in serum levels of

progesterone and estradiol have been observed (Gajecki, 2002).

Ochratoxin is a ubiquitous mycotoxin produced mainly by fungal species,

Penicillum verrucosum and Aspergillus ochraceus. Ochratoxin A (OTA) is the most prevalent, occurring in barley, wheat, coffee beans, cocoa beans, fruit, wine, and milk

(Mateo et al., 2007). OTA is a nephrotoxin and is suspected of being the main etiological

agent responsible for human Balkan endemic nephropathy (BEN) and associated urinary

tract tumors (Pfohl-Leszkowics and Manderville, 2007). IARC classified OTA as a

Group 2B, possible human carcinogen (IARC, 1993).

Aspergillus

One of the oldest named genera of the fungus, Aspergillus, received its name from

an Italian priest and biologist named Pier Antonio Micheli in 1729. It was so named

because Micheli was reminded of the shape of a holy water sprinkler, called aspergillum

when viewing the spore-bearing organism under the microscope (Bennett, 2010). By the

early 1900s, Aspergillus had become one of the most studied fungal groups. Aspergillus

was found to be very prevalent in the environment and has become an important

scientific tool because of its ease of cultivation on laboratory media (Figure 2.3), positive

impact as fermentation agents, and negative impact as degraders of agricultural products

(Klich, 2002).

Figure 2.3 Aspergilli species grown on media.

Aspergillus colonies are usually fast growing, white, yellow, yellow-brown, brown to black, or green. They mostly consist of a dense felt of erect conidiophores. Picture adapted from Wikipedia.org. 11

Aspergillus, a soil-borne filamentous fungus, grows abundantly as a saprophyte on damaged and decaying crops (McMillian et al., 1985). Aspergillus spends most of its

development as a saprophyte in the soil where it accumulates nutrients from plants,

especially grains (corn, peanuts, wheat, cottonseed, and nuts) (Scheidegger and Payne,

2003). Aspergillus gains access to nutrients on damaged and decaying vegetation when the fungal hyphal tips grow into the food substrates. Aspergillus has asexual reproductive

properties and during most of the life cycle, it grows rapidly, sporulating a few days after

germination (Lee and Adams, 1994). The spores (conidia) produced are converted into

aerosols and are distributed into the environment. Aerosolized Aspergillus spores are

found nearly everywhere and therefore, we are constantly exposed to them (Bennett,

2010).

Aspergillus thrives in favorable environmental conditions such as warm

temperature, low soil moisture, and insect infestation (Scheidegger and Payne, 2003). It

has been shown that contamination requires a drought period of 30-50 days and a mean

soil temperature of 29-31°C for optimum Aspergillus growth (Cole et al., 1989; Dorner et

al., 1989). Most Aspergilli grow optimally at temperatures between 25°C and 40°C in an

acidic environment (pH 4.0-6.5) and have the least growth around 10°C (Klich, 1992). A.

flavus is most frequently found in warm temperate zones (subtropical latitudes) rather

than cooler temperate zones (Klich, 2002). The ability of Aspergillus to survive these

harsh conditions allows it to easily out-compete other organisms for nutrients in the soil or on its host (Bhatnagar et al., 2000). The optimal water potential for growth of

Aspergillus is -2 MPa (~0.8 aw; severely drought stressed soil) with a moisture percentage of 22-25% under field conditions (Kozakiewicz and Smith, 1994). Yet, Aspergillus can

grow at water potentials down to -3.5 MPa, which is lower than the minimum for many

other fungal species giving Aspergillus a competitive advantage (Klich 1992).

Aflatoxigenic fungal species produce fungal toxins that are deleterious to plants

and animals, including humans. Aspergillus frequently causes life-threatening infections called aspergillosis and produces allergens that may cause cancer, and can damage the immune and nervous systems (Segal, 2009). Aspergillus is a very common fungus and

people with weakened immune systems or lung diseases are at a greater risk of

developing health problems. Because of their airborne behavior, Aspergillus spores can

pass through the respiratory tract causing an increased risk for developing allergens,

asthma, and sinus problems (Zmeili and Soubani, 2007).

Aflatoxin

Background

The discovery of aflatoxins occurred in 1960, England when they were identified

as a causative agent resulting in the deaths of numerous turkey poults, ducklings, and

chicks (Blount, 1961). More than 100,000 young turkeys died over a course of a few

months. The outbreaks affected 2- to 20-week old turkeys, with the heaviest mortality in

4-week old turkeys. The outbreak was characterized by nervous system deterioration,

coma, and death. After the death of the turkeys, postmortem dissection exposed lesions

on the liver (Wannop, 1961). Subsequent studies revealed that the unknown causative

agents were capable of inducing acute liver disease and liver cancer (Sargeant et al.,

1961). The disease came to be called Turkey “X” because of its unknown etiology.

Eventually Turkey “X” was attributed to the contaminated feed, the Brazilian peanut

meal (Asao et al., 1965). Investigations revealed that the toxicity of the Brazilian peanut 13

meal was associated with the presence of A. flavus. The toxin this fungus produced was

named “aflatoxin” for Aspergillus flavus toxin (Wannop, 1961). Vigorous research

ensued following the discovery of the fungus, Aspergillus flavus that produced the

possibly deadly toxin. These extensive research efforts assessed potential health hazards

to the human food supply (Latha et al. 2008).

AFB1 is likely the best known and most extensively researched mycotoxin to date

due to its potent carcinogenic effect enacted on animals and humans. Following

absorption, AFB1 undergoes an initial oxidation in the liver by major cytochrome (CYP)

P450 enzymes, CYP1A1, 1A2, 2A4, 2A6, 3A3, 3A5, and 3A7, which are involved in

aflatoxin metabolism to yield aflatoxin-8,9-epoxide stereoisomers (Dohnal et al., 2014).

These isomers have been shown to cause carcinogenesis and the AFB1-N7-guanine adduct can be excreted in urine (Bennett et al., 1981). AFB1-N7-guanine can be used as a biomarker in urine to measure human exposure to the carcinogen. While levels of the

AFB1-albumin adduct in blood maybe better to evaluate repetitive or long-term human exposure, this method may not be economically feasible where repeated measurements over days, weeks, or months are required (Egner et al., 2006). AFB1 can undergo

metabolic processes in the liver to produce other secondary metabolites such as aflatoxins

P1, Q1, B2a, B3, G2a, D1 and M1 (Figure 2.4).

Figure 2.4 Metabolism of AFB1.

Adapted from Dohnal et al., 2014.

Economic and health implications of aflatoxin

The economic impact of mycotoxin, including aflatoxin, contamination has become a distress on the agricultural market (FAO, 2004). An estimated $0.5 million to over $1.5 billion are lost annually in the United States from mycotoxins (aflatoxin, fumonisin, and deoxynivalenol) through market rejection and animal health impacts

(Vardon, 2003). Annual aflatoxin-related crop losses are estimated to range from $160 million to $225 million (Wu et al., 2008; Vardon, 2003). Estimates of annual costs in the

United States for crops where aflatoxin contamination is prevalent include: $4.4 million and $7 million loss for cottonseed in Arizona and Texas respectively; $25 million for 15

peanuts in Georgia; $15 million and $2 million for corn in Texas and Mississippi, respectively; and California lost $38.7 million for walnuts and $47 million for almonds

(Robens and Cardwell, 2003). In the United States, areas with severe contamination may

yield crops with > 500 µg/kg total aflatoxins (Jaime-Garcia and Cotty, 2003). In

developing countries with limited regulations, aflatoxin contaminated crops result not

only in economic losses, but they also have a tremendous impact on human health.

Aflatoxins are toxic, immunosuppressive, mutagenic, carcinogenic, and

teratogenic compounds with the liver as the primary target organ (Richard, 2007; Bennett

and Klich, 2003). Acute aflatoxicosis in humans may manifest as abdominal pain and

vomiting, hemorrhaging, and mental impairment (Abdulrazzaq et al., 2004; Turner et al.,

2007). Clinical symptoms also include acute toxic liver injury and death (Abdulrazzaq et

al., 2004). Acute aflatoxicosis has been implicated in the pathogenesis of malnutrition

diseases, increased neonatal susceptibility to infections, and jaundice. Children are more

susceptible than adults to the effects of aflatoxins and aflatoxicosis leading to child

mortality and immunological deficiencies (Magoha et al., 2014).

There have been several outbreaks, mostly occurring in developing countries where poor levels of regulation, poor agricultural practices, malnutrition, and poor health statuses are prevalent. The largest known outbreak case reported of aflatoxicosis occurred

in India, 1974 with 397 cases and 106 deaths. Unseasonal rains resulted in aflatoxin

concentrations ranging from 6.3-15.6 µg/kg (Krishnamachari et al., 1975). In 2002, a

severe outbreak of aflatoxicosis was reported in Kenya. Locally grown maize was

contaminated with aflatoxin during storage under damp conditions. A total of 317 cases

were reported with 125 deaths. A total of 31 samples were tested and 15 samples had

AFB1 contamination ranging from 20-8,000 µg/kg (Centers for Disease Control, 2004).

There have been no human aflatoxicosis outbreaks reported in the United States to date.

Chronic exposure to aflatoxin in the diet can also be detrimental to the human health causing liver cancer (Williams et al., 2004). Liu and Wu (2010) determined that aflatoxin might play a causative role in 6-28% of all global HCC cases. Chronic dietary exposures to aflatoxin have also been linked to immunosuppression in humans causing a decrease in resistance to secondary infections by fungi, bacteria, and parasites (Marin et al., 2013).

Exposure of animals to aflatoxin contaminated feed may have a greater economic impact as a result of reduced feed efficiency, immunosuppression, and reduced reproducibility (Kaleibar and Helan, 2013). Acute aflatoxicosis in animals consists of reduced feed consumption, dramatic drops in milk and egg production, weight loss, and liver damage (Kaleibar and Helan, 2013). Aflatoxin consumption may manifest as severe hepatotoxicity that may lead to liver necrosis and eventually, death. The effect of aflatoxin contamination in cattle depends on the age, health, diet, dose, and length of exposure. Aflatoxin contamination has been shown to affect rumen function by decreasing cellulose digestion, volatile fatty acid formation, proteolysis, and rumen mobility (Smith, 1992). Yiannikouris and Jouany (2002) reported that the aflatoxin concentration ranging from 0.1 to 10 µg/g are poorly degraded in the rumen as a result of bacteria in the gut. However, ruminants generally have been more resistant to the adverse effects of aflatoxin than simple-stomached animals due to the nature of the ruminant’s

digestive system where aflatoxin is partially detoxified by the rumen microbiota (Fink-

Gremmels, 2008). Susceptible non-ruminant species such as rabbits and ducks have a low

median lethal dose (LD50) of 0.3 mg/kg, whereas chickens (18 mg/kg) and rats (18

mg/kg) have a greater tolerance (Williams et al., 2004). Newman et al. (2007) examined

an acute aflatoxicosis outbreak in the United States from 2005 to 2006 in nine dogs after

exposure to contaminated commercial dog food. Aflatoxin concentration in feed samples

was consistently greater than 598 µg/kg. For dogs, the toxic dose of aflatoxin is <60

µg/kg and the LD50 is 500-1000 µg/kg. Histopathologic findings included hepatic lipidosis, fibroplasia, and biliary hyperplasia consistent with subacute toxic hepatopathy in eight of the nine dogs. An outbreak of acute aflatoxicosis on a chinchilla farm occurred in Argentina where commercial feed was suspected of causing the death of 200 animals.

Acute aflatoxicosis in chinchillas was characterized by low feed intake, diarrhea, weight loss, fur discoloration, sudden death, and a predisposition to secondary infections

(González Pereyra et al., 2008). Evidence of chronic toxicity in the literature also exists

in cattle, poultry, and swine (Ortatali et al. 2005; Yunus et al., 2011). Chaytor et al.

(2011) studied the effects of chronic exposure of diets with reduced concentrations of

aflatoxin and deoxynivalenol (DON) on growth and immune status of pigs. Collectively,

this study showed that diets containing both aflatoxin and DON greater than 60 and 300

µg/kg, respectively, might reduce growth and decrease feed intake. Diets containing 120

µg/kg aflatoxin and 600 µg/kg DON, may result in altered immune health, systemic

inflammation, and partial liver damage, causing further reduction in growth of pigs.

Factors influencing aflatoxin contamination

Aflatoxin contamination is the consequence of the interaction between the host

and the environment. Aflatoxin contamination occurs in crops in more temperate climates

and in warm drought years. Aflatoxin producers, A. flavus and A. parasiticus, are favored

by warm conditions, particularly in temperate climates (Milani, 2013). Therefore, it is not 18

surprising that chronic aflatoxin problems occur in the southeastern United States which is associated with external environmental factors such as high temperatures, high humidity, and drought stress in the soil. Maize is often harvested with a moisture content of about 18-20% (0.9-0.93 aw) and subsequently dried. If not dried properly, sometimes this process can lead to rapid spoilage and aflatoxin production, as optimum temperature and water activity (aw) for aflatoxin production has been shown to be 33°C and 0.99 aw, respectively (Nesci et al., 2003). In a field study performed by Abbas et al. (2002), it was

found that high heat stress resulted in the highest levels of aflatoxin contamination in

corn in comparison to moderate heat stress. In 1998, corn losses in Mississippi were

extremely severe resulting in high aflatoxin contamination (20-150 µg/kg) of 50 million

bushel as a result of high heat stress and drought stress (Robens and Cardwell, 2003).

Cole et al. (1985) found that irrigation relieved drought stress, reduced soil temperature,

and reduced aflatoxin contamination in peanuts.

Insect infestation has been associated with an increase in aflatoxin contamination

in crops. Aspergillus spores have been isolated from corn earworm (Helicoverpa zea),

corn borer (Diatraea, Ostrinia) belonging to the order, Lepidoptera, and the maize weevil

(Sitophilus zeamais) (McMillian, 1987). In addition to disseminating A. flavus spores on

their bodies into close proximity of the plant, the insect has been reported to injure the

crop creating an entry for the fungus. The insect breaks the pericarp while feeding,

rendering the grain more vulnerable to invasion (Wicklow, 1988). This damage results in

increased humidity, allowing favorable conditions for fungal growth which can easily be

identified by the accumulation of fungal spores. Increased A. flavus infection and the

subsequent accumulation of aflatoxin, are frequently associated with southwestern corn

borer infestation of corn grown in the southern United States (Windham et al., 1999;

Williams et al., 2002a; Williams et al., 2002b). Schatzki and Ong (2000) conducted a

study that investigated the distribution of aflatoxin in almonds. The insects entered the

kernel during split hulls late in the growing season. It was found that the highest

concentration of aflatoxin contamination was distributed in a lot assigned to navel orange

worm (Amyelois transitella). They also found that at the point of entry made by the

insect, the Aspergillus fungus colonized the damaged crop, which lead to aflatoxin

contamination.

Carbon utilization by Aspergillus has been shown to have a dramatic effect on

aflatoxin biosynthesis. The presence of glucose, ribose, xylose or glycerol may induce

vegetative growth and aflatoxin production (Woloshuk et al., 1997; Woloshuk and Shim

2012). It was demonstrated that readily metabolized carbon sources from glucose and the

pentose phosphate pathway are the best inducers of aflatoxin production. Yu et al. (2003)

examined the effects on aflatoxin production by A. flavus and A. parasiticus in soybean

and peanut. It was reported that the addition of oil to a non-inductive medium resulted in

60% and 10% of the aflatoxin produced by A. flavus and A. parasiticus, respectively,

when they were grown in glucose-containing medium. Fakhoury and Woloshuk (1999)

suggested that the α-amylase of A. flavus promotes aflatoxin production in the endosperm

of infected maize kernels. When the amylase gene, Amy1, was disrupted in an

aflatoxigenic strain of A. flavus, the mutant failed to produce extracellular α-amylase and

grew at 45% the rate of the wild-type strain on starch medium. The mutant produced

aflatoxin in medium containing glucose but not in a medium containing starch.

Expression of α-amylase inhibitors from maize, or stronger inhibitors from other

organisms, may limit the production of aflatoxin by limiting the availability of simple sugars (Warburton and Williams, 2014).

Because fungi are aerobic organisms, the formation of reactive oxygen species

(ROS) is a natural byproduct of the metabolism of oxygen. Oxidative stress is an imbalanced state where excessive amounts of highly reactive oxygen species (oxygen ions, free radicals, and peroxides) overcome endogenous cell scavenging (antioxidant) capacity, leading to the oxidation of a wide array of macromolecules such as enzymes, proteins, nucleic acids and lipids (Dai and Mumper, 2010). It has been reported that oxidative stress is a stimulator of aflatoxin biosynthesis in Aspergillus (Scott and Eaton,

2008). During times of environmental stress, ROS levels can increase dramatically

resulting in significant oxidative stress inside the fungal cell that may lead to a plant

hypersensitivity response to the fungus (Reverberi et al., 2010). The production of

secondary metabolites occurs at a particular stage of growth in the fungi. Reverberi et al.

(2008) reported that in A. parasiticus, a burst of ROS was produced in the transition from

conidia to hyphal growth phases and from mycelial to conidiogenesis. During this

senescence process, an oxidative burst occurred in parallel with the beginning of

aflatoxin synthesis suggesting that ROS and aflatoxin biosynthesis are closely

interconnected. Narasaiah et al. (2006) showed that A. parasiticus mutants were blocked

at various stages in the synthesis of aflatoxin resulting in the generation of ROS. The

demand for oxygen for growth increased suggesting that aflatoxin synthesis may occur as

a compensatory response to ROS accumulation.

Under oxidative stress, the fungus may preserve its cells by activating antioxidant

enzymes. Maintaining an appropriate balance between oxidant and antioxidants species is

important for aflatoxin biosynthesis. Interestingly, when antioxidants (i.e. catalase and glutathione) were stimulated in the A. parasiticus cells, aflatoxin synthesis was inhibited

by the downregulation of aflatoxin genes (Reverberi et al. 2006). Nebert and Vasiliou

(2004) found that an increase in glutathione S-transferase in the fungus lowered aflatoxin

levels by protecting the fungus during respiration when large amounts of ROS were being

produced. Magbanua et al. (2007) evaluated H2O2 (peroxide) and salicylic acid levels in

maize embryos from two resistant and two susceptible maize lines. They observed a

significant reduction in H2O2, a ROS, in the resistant compared to susceptible maize

embryos along with a significant increase in salicylic acid. In addition, they observed an

increase in catalase activity in the resistant lines.

Toxigenic fungi on grain products have the potential of leading to significant

health hazards. The pre-/post-harvest production of Aspergillus, especially on grains, is

highly dependent on environmental factors such as temperature and moisture content.

Aspergillus accumulation is optimum at soil temperature of 29-31°C and water activity of

0.8 aw while aflatoxin production is optimum at a temperature 33°C and a water activity

of 0.99 aw. The production of aflatoxin is climate dependent, associated with ROS, affected by the bioavailability of micronutrients, and insect damage. However, climate is the key environmental factor that drives fungal colonization and aflatoxin production.

Aflatoxin biosynthesis

Aflatoxin biosynthetic pathway involves more than 20 enzymes. Most of the aflatoxin-related genes are clustered within a 75 kb region of the genome (Yu et al.,

2006). AFLR is a Zn2Cys6-type sequence-specific DNA-binding protein that is thought to

be necessary for the expression of most of the genes in the aflatoxin pathway gene 22

cluster. RThe afl genes within A. flavus and A. parasiticus regulate these clustered genes.

The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing

a zinc-finger DNA-binding motif that transcriptionally activates most of the structural

pathway genes (Yu et al., 2006). An in-depth understanding of the molecular biology of

the aflatoxin biosynthetic pathway will be reviewed.

Enzymes and regulatory proteins are encoded by approximately 25 clustered

genes in a 70-75 kb DNA region (Figure 2.5) for the synthesis of aflatoxin in Aspergillus

(Cleveland and Bhatnagar, 1991). Each gene codes for an enzyme. For example, pksA

(old gene name) or aflC codes for the enzyme polyketide synthase. This enzyme is used

to convert polyketide to norsolorinic acid in the polyketide pathway. The 3’ end of this

gene contains a well-defined sugar utilization gene cluster. The 82 kb DNA sequence in

A. parasiticus contains the aflatoxin pathway gene cluster and the sugar utilization gene

cluster.

Figure 2.5 Aflatoxin biosynthesis gene cluster in A. flavus and A. parasiticus.

Adapted from Yu, et al., 2006. The new gene names are at the bottom and the old gene names are at the top. The direction of each transcription is represented by the arrow and is unique in each gene.

The polyketide pathway is the most characteristic route of the fungi. Aflatoxins

are difuranocoumarin derivatives produced by a polyketide pathway according to the 23

following scheme (Cleveland and Bhatnagar, 1991): malonyl-CoA  polyketide 

norsolorinic acid  averantin  hydroxyaverantin   versiconal

hemiacetal acetate     demethysterigmatocystin  aflatoxin. Initially, a head-to-tail condensation of a linear

polyacetate chain occurs. The aflA, aflB, and aflC genes that code for the enzymes, fatty

acid synthase α, fatty acid synthase β, and polyketide synthase, respectively, are

responsible for the conversion of malonyl-CoA to polyketide to norsolorinic acid (NOR).

There is a direct analogy between the biosynthesis of fatty acids and the polyketides.

However, the polyketide pathway is different from fatty acid synthesis in that it lacks the

dehydration and reduction reactions (Yu et al., 2002).

Polyketides are formed by linkages of multiple acetate units, followed by

cyclization. NOR has been shown to be the most stable and earliest precursor in the

aflatoxin biosynthetic pathway (Bennett et al., 1971). This discovery has led to the

identification of other key aflatoxin intermediates. Yu et al. (2012) described the

aflatoxin biosynthesis pathway summarized here. The aflD, aflE, and aflF genes are

involved in the conversion of NOR to averantin (AVN). The genes encode enzymes

reductase, NOR-reductase, and a dehydrogenase, respectively. The aflG gene encodes a

cytochrome P450 monooxygenase that converts AVN to 5’-hydroxyaverantin (HAVN).

The aflH encodes the enzyme alcohol dehydrogenase and is involved in the conversion of

HAVN to averufanin (AVF). The aflI gene encodes the enzyme oxidase for the

conversion of AVF to versiconal hemiacetal acetate (VHA). The aflJ gene encodes for

esterase and is involved in the conversion VHA to versiconal (VAL). The aflK gene

encodes for versiconal cyclase for the conversion of VAL to versicolorin B (VERB).

VERB is converted to VERA with the aflL gene that encodes for a desaturase. This

conversion has been proposed to serve as a branching point separating biosynthesis of

AFB1 and AFG1 from AFB2 and AFG2. The aflM and aflN genes are involved in the

conversion of VERA to demethysterigmatocystin (DMST). These genes encode for the

enzymes dehydrogenase and a monooxygenase, respectively. The aflO gene encodes the

o-methyltransferase B that coverts DMST to sterigmatocystin (ST) for the AFB1 and

AFG1 pathway or demethyldihydrosterigmatocystin (DMDHST) to

dihydrosterigmatocystin (DHST) for the AFB2 and AFG2 pathway. As an intermediate of

aflatoxin biosynthesis, ST has a chemical structure very similar to that of AFB1. ST is a mycotoxin that is also a liver carcinogen and forms DNA adducts after metabolic activation to an epoxide at the furofuran ring (Pfeiffer et al., 2014). The aflP gene

encodes for the enzyme o-methyltransferase A for the conversion of ST to o-

methylsterigmatocystin (OMST) or the conversion of DMST to dihydro-O-

methylsterigmatoccystin (DHOMST). The aflQ gene encodes for the cytochrome P450

monooxygenase for the conversion of OMST to AFB1 and AFG1 or DHOMST to AFB2

and AFG2 (Yu et al., 2006). Experiments with A. flavus, A. parasiticus, and A. nidulans,

showed that aflR is the transcriptional regulator for the biosynthesis of aflatoxin. Because

the aflR region is highly conserved, the alteration or elevated transcription of aflR leads

to changes in the expression of the pathway genes and high levels of aflatoxin production

(Yu et al., 1996).

After consumption of contaminated food and feed, AFB1 enters the hepatocyte

and is metabolized via monooxygenases to form AFM1. AFM1 is a hydroxylated

metabolite of AFB1 found in milk. Cytochrome P450 monooxygenases are implicated as

key enzymes that are heme-thiolate proteins that introduce an oxygen atom into an organic substrate (RH) while the other oxygen atom is reduced to water [Equation 1.1].

+ + O2 + H + NAD(P)H + RH → H2O + NAD(P) + ROH (2.1)

A theory of the conversion of AFB1 to AFM1 has been made using the literature

of Meunier et al. (2004) which describes the mechanism of oxidation reactions catalyzed

by cytochrome P450 enzymes (Figure 2.6).

Figure 2.6 Proposed mechanism for the conversion of AFB1 to AFM1 by cytochrome P450 enzymes.

The active site of cytochrome P450 contains a prosthetic group, heme linked with a cysteine residue via a thiolate (sulfur, S, containing organic compound). The reversible binding of the substrate AFB1 to the active site of the monooxygenase enzyme occurs.

The first transfer of an electron ensues when water is displaced. This causes the reductase

to reduce the iron(III) center to the ferrous state from NAD(P)H to NAD(P)+ via the

electron transport center (iron-sulfur center) producing the reductant. Arginine has an

important role in the binding of dioxygen to the ferrous state of the heme-enzyme

complex occurs. Oxygen binds to the Fe-S center creating a heterolytic cleavage of the

O-O bond. The iron-oxygen complex can dissociate to an iron(III) and superoxide anion.

The dissociation of the superoxide can lead to harmful radicals and the generation of

nd H2O2. The transfer of the 2 electron to P450 and the second reduction step occurs. The

unprotonated iron(III)-peroxo complex forms a nucleophilic oxidant intermediate

III creating an AFB1-P450-Fe -OOH complex (not shown). Two proton transfers via

threonine and asparagine occur and hydroxylation occurs converting AFB1 to AFM1. It

should be noted that the replacement of threonine with alanine results in the over

production of H2O2 and water. AFM1 favors a polar environment causing the entrance of

bulk water molecules into the active site displacing AFM1.

Biosynthesis of aflatoxin is a highly conserved and complex process governed by

genes maintained in 25 clustered genes in a 70-75 kb DNA region. The regulatory gene,

aflR, encodes a protein that controls transcription regulation. The clustering of these

genes implies that the cluster plays an important role in aflatoxin production. The

conversion of AFB1 to AFM1 was also discussed. The mechanism of the binding of AFB1 in the active site of cytochrome P450, which contains a prosthetic group, heme linked

with a cysteine residue via a thiolate, showed the role this enzyme plays in the formation of AFM1 furthering our understanding of the regulation of gene expression of aflatoxin

production.

Pre-harvest Management of Aflatoxin

Aflatoxins are potent metabolites that contaminate crops in warm regions

worldwide and reduce health and economic welfare. The aflatoxin that has caused the

most concern is AFB1 due to its widespread occurrence, its prevalence among the four

naturally occurring aflatoxins, and its acute toxicity and carcinogenicity. While the acute

toxicity of the aflatoxins is noteworthy, it is the carcinogenic potential of AFB1 that has

been the focus of considerable research for the management of aflatoxin contamination.

The best management of aflatoxin contamination is prevention prior to harvest.

Pre-harvest bio-control, using atoxigenic strains to competitively exclude toxigenic strains, has been utilized for the management of aflatoxin contamination (Abbas et al.,

2011; Atehnkeng, et al., 2008; Dorner et al., 1999; Dorner et al., 2003; Dorner, 2009;

Kabak and Dobson, 2009; Reddy et al., 2009). The use of bio-competitive agents as a biological control for the pre-harvest management of aflatoxin contamination offer a number of advantages over other biological control methods such as fungicide and insect management. Non-toxigenic strains of A. flavus and A. parasiticus may be used to

competitively exclude toxigenic strains by occupying the same ecological niche.

Conditions that favor the production of toxigenic strains will also favor production of

atoxigenic strains. Drought and high temperatures that may be harmful to other bio-

competitive agents, will be the same for toxigenic and atoxigenic strains. In other words,

the non-toxigenic strains have the same ability to survive in the natural environment and possibly out-compete toxin-producing fungal strains.

Cotty and Bhatnagar (1994) studied five atoxigenic strains of A. flavus (AF36,

NRRL-5918, NRRL-5565, NRRL-5917, and NRRL-1957) for the ability to prevent a toxigenic strain (AF13) from contaminating developing cottonseeds. It was suggested that atoxigenic strains that did not produce certain enzymes in the aflatoxin biosynthesis pathway did not reduce contamination of toxigenic strains. Instead, a strain that produced many of the enzymes in the pathway and did not produce aflatoxins (AF36) was the most effective for the reduction of toxigenic aflatoxin in cottonseed. It was found that AF36 reduced aflatoxin contamination in cottonseed by 94%. Dorner and Horn (2007) conducted a study to determine the effect of applying non-toxigenic strains of A. flavus and A. parasiticus to aflatoxin-contaminated peanuts. Treatments included a control, soil

inoculated with non-toxigenic strain of A. flavus only (NRRL-21882), and A. parasiticus

(NRRL-21369) only, and soil inoculated with the 2 non-toxigenic strains. Results showed significant displacement of toxigenic A. flavus (70%) in year one. By year two there was a significant 91.6% reduction in aflatoxin. It was concluded that treatment with the non- toxigenic strain of A. flavus alone was more effective than the A. parasiticus strain alone

and in combination with A. flavus.

Despite improved biological methods, handling, processing, storage, and

regulatory guidelines, aflatoxin contamination still remains a serious problem and poses a

unique challenge to food safety. Therefore, post-harvest management to remediate

contaminated products are needed to limit economic and health impacts and add value to

the agricultural industry. Post-harvest strategies include: the separation of aflatoxin from

contaminated grains through sorting, the addition of adsorbents to contaminated diets, radiation, ozonation, and ammoniation of aflatoxin.

A RECENT REVIEW OF NON-BIOLOGICAL REMEDIATION OF AFLATOXIN-

CONTAMINATED CROPS

Abstract

Aflatoxins are highly toxic, mutagenic, teratogenic, and carcinogenic compounds

produced predominantly as secondary metabolites by fungi belonging to certain

Aspergillus species. Due to the significant health risks and economic impacts associated

with the presence of aflatoxins in agricultural commodities, a considerable amount of

research has been directed at finding methods to prevent toxicity. This review compiles

the recent literature of methods for the detoxification and management of aflatoxin in

post-harvest agricultural crops using non-biological remediation.

Introduction

Aflatoxins are a group of highly toxic secondary metabolites produced mainly by strains of A. flavus and A. parasiticus. Aflatoxins have been shown to be very stable under most conditions during growth, harvest, processing, and storage. Toxin levels may accumulate to dangerously high levels under suitable environmental conditions including heat stress, moisture deficit, and insect infestation (Pitt et al., 2013). The main aflatoxin

groups to be considered are aflatoxins B1 (AFB1), B2 (AFB2) and G1 (AFG1), G2 (AFG2),

which appear blue and green under UV light, respectively (Asao et al., 1965). AFB1 is

usually the major aflatoxin produced by toxigenic strains, the most potent aflatoxin, and also the most toxic naturally occurring liver carcinogen known (Asao et al., 1965).

Aflatoxin M1 (AFM1) is found as a metabolite of AFB1 in milk as mammals consume

aflatoxin-contaminated feeds (Patterson et al., 1980).

The economic impact of aflatoxin contamination has become a worldwide

concern on the agricultural market affecting approximately 25% of the world’s food

supply as estimated by the Food and Agriculture Organization (FAO) of the United

Nations (FAO 1997). Economic costs of aflatoxins include the cost of preventative and

mitigation measures, the reduced value of contaminated feeds, and the reduction in

animal reproduction, performance, and health. An estimated 500 million dollars in the

United States, and as much as a billion dollars globally, are lost annually as a result of

aflatoxin toxicity (Vardon 2003; Wu et al., 2008).

The health implication of aflatoxins is another global concern exhibiting acute

and chronic toxicity including mutagenic, carcinogenic, and teratogenic effects in a wide

range of organisms. The International Agency for Research on Cancer (IARC) of the

World Health Organization (WHO) has classified aflatoxins as Group 1, established

carcinogens to humans (WHO IARC 1993). Aflatoxin contamination has also been linked

to other diseases such as hepatitis B and C, and other kidney, lung and immune system

diseases (Wild and Gong, 2010; Wild and Montesano, 2009). Hsu et al. (1991) described

the mechanism by which AFB1 is metabolized to a carcinogen. The conversion of AFB1

to AFB1-8,9 epoxide through cytochrome P450-mediated oxidation as the result of the

formation of a reactive epoxide at the 8,9 position of the terminal furan ring was

described (Figure 3.1). The epoxide then intercalates between DNA strands and binds at

the N7 position of guanine to form AFB1-N7-guanine adduct. AFB1-8,9-epoxide reacts with guanine residues of specific sites of DNA and protein. The missense mutation from

AGGarg to AGTser of codon 249 of the p53 tumor suppressor gene causes carcinogenesis.

Figure 3.1 Metabolism of AFB1 by cytochrome P450.

Adapted from Hsu et al. (1991).

Management of aflatoxins includes prevention, regulation, and detoxification.

Because aflatoxins have been shown to be potent carcinogens, regulatory guidelines have

been established in more than 100 countries to reduce risk of exposure (FAO, 2004).

Regulation of aflatoxin in foods, dairy products, and feeds has significantly evolved over

the last decades. The United States began regulating mycotoxins in food and feed in

1968, following incidents of animal and human health. The United States Food and Drug

Administration (FDA) uses guidelines to maintain a safe level of contaminants in human food and animal feed. The safety range for corn, peanut, and cottonseed intended for feed and breeding animals is 100-200 µg/kg; smaller animals’ action level is 20 µg/kg. For human consumption, all foods have a safety maximum of 20 µg/kg, except milk (AFM1),

which has a level of 0.5 ng/mL (FDA, 1994). The impact that aflatoxin contamination has

on the economy and on the health of humans and animals highlight the importance of

establishing food safety management programs. Recently, food safety management

practices such as the Food Safety Objective (FSO) has been applied to understand

increases and decreases in mycotoxin levels in foods internationally, on the basis that the

maximum permissible levels are equivalent (Pitt et al., 2013; Garcia-Cela et al., 2012).

FSO focuses on good agricultural practices and proper storage and handling for the

reduction of aflatoxin levels.

Aflatoxin contamination has become a global concern and considerable research

has been directed at finding methods to prevent toxicity. There is excellent potential for

non-biological means to help manage the aflatoxin problem. Physical strategies such as

the separation of aflatoxin from contaminated grains through sorting, the addition of

adsorbents to contaminated diets, and radiation of aflatoxin have been examined to

reduce aflatoxin contamination post-harvest. Furthermore, chemical methods, ozone, and

ammonia have also been employed for the reduction of aflatoxins. Here, recent non-

biological methods are reviewed for the management and detoxification of aflatoxins.

Sorting of aflatoxin-contaminated grains

Physical removal or separation of aflatoxin-contaminated crops is an important

strategy for reducing aflatoxin levels and can be achieved based on differing physical 34

properties such as size, shape, color, and visible fungal growth on the affected commodity (Doko and Park, 2008; DeMello and Scussel, 2007; Sinha, 2009). Instead of discarding an entire lot, a reasonable approach for reducing aflatoxin contamination would be manual or, preferably, mechanical selection for the physical removal of only a percentage of discolored, small, or irregularly shaped contaminated nuts or grains.

A recent advancement in sorting incorporates infrared and UV coupled with color detection technology to enable the inspection of aflatoxin-contaminated agricultural products. There have also been advancements in optical and high-speed sorters improving the detection and separation of aflatoxins from contaminated nuts or grains allowing recovery of quality products. De Mello and Scussel (2009) used physical characteristics

(color, size, density) and sorting by near infrared reflectance (NIR) for mechanical in-

shell Brazil nut to assess nut quality and reduce aflatoxin contamination. After sorting,

there was no aflatoxin detected in the final batch of Brazil nuts. Contamination was

detected only in the pool of rejected nuts at a level of 16.4 µg/kg AFB1 corresponding to

5% of the initial nut batch (7135 nuts).

The viability of bright, greenish, and yellow fluorescence (BGYF) as an aflatoxin

screening method for the removal of contaminated agricultural crops has been evaluated.

The fluorescence is produced by the oxidative action of heat-labile enzymes

(peroxidases) in living plant tissue on kojic acid which is produced by A. flavus (Hadavi,

2005; Pearson et al., 2010; Lundadei et al., 2013; Yao et al., 2010). Color and form

changes detected visually are preceded by chemical changes in the grains caused by the

fungus (Hadavi, 2005). Yao et al. (2010) examined the relationship between fluorescence

emissions of corn kernels inoculated with A. flavus and aflatoxin-contamination levels

within the kernels. Emission data was taken with a fluorescence hyperspectral image data when the corn kernels were excited with UV light. Of the total 504 single corn kernels,

180 kernels were found to have aflatoxin levels < 1 µg/kg, 130 kernels (25.8% between 1 and 20 µg/kg, 31 kernels between 20 and 100 µg/kg, and 163 > 100 µg/kg). The results indicated that fluorescence hyperspectral imaging was applicable in the estimation of aflatoxin concentration in individual corn kernels.

Pearson et al. (2004) used high-speed dual-wavelength sorting for the reduction of

aflatoxin and fumonisin contamination in yellow corn and found that absorbances at 750

and 1200 nm could correctly identify > 100 µg/kg of aflatoxin-contaminated corn. The

high-speed sorter could also reduce aflatoxin levels by 81% from an initial 53 µg/kg.

Pearson et al. (2010) tested several sorting schemes for the removal of mycotoxin-

contaminated kernels using visible and NIR spectra, optical sorting, and sorting based on

size. The asymptomatic kernels were found to have an average AFB1 concentration of 6 ±

4 µg/kg (5.2% total AFB1 of 10 kg sample). White corn kernels with BGYF discolorations over at least ≥ 25% of the kernel surface comprised over 57% of the aflatoxin found in the sample collected. At wavelengths of 500 nm, approximately 87% of the kernels with high levels of aflatoxin were identified. However, it was found that kernels with minor discolorations that compose a substantial portion of the total aflatoxin were difficult to sort. Other studies have also found discriminant methods used to detect

BGYF discolorations as a means of physically reducing aflatoxin (Lundadei et al., 2013).

Despite advances in the technology of sorting, sorting by hand may be more feasible in less industrialized countries as these developing countries may not have access to such machinery. Fandohan et al. (2005) studied the fate of two mycotoxins, fumonisin

and aflatoxin, through traditional processing (sorting, winnowing, and washing) in naturally contaminated maize products, mawe, ogi, and owo. The total aflatoxin level for

mawe before processing was 15.28 ± 0.32 µg/kg; the total aflatoxin levels for both ogi

and owo before processing was equal to or greater than 22 ± 0.26 µg/kg. There was a

91% aflatoxin reduction in mawe and an 80% reduction of total aflatoxin level from raw

maize to ogi (from ≥ 22 to 4.5 µg/kg) after processing. A significant reduction of total

aflatoxin level from raw maize to owo was also observed (from ≥ 22 to 12.62 µg/kg).

This study and others (Filbert and Brown 2012) have reported that the systematic

removal of moldy and damaged agricultural products by hand-sorting and washing before

processing can significantly reduce overall aflatoxin levels (Table 3.1).

Table 3.1 Physical removal of contaminated nuts and grains through sorting and processing.

Agricultural % Processing Aflatoxin Reference Product Reduction Hand-sorting Pistachios 15 µg/kg 40-80% Garcia-Cela et al., 2012 Hand-sorting Brazil nuts - ND DeMello and Scussel, 2007 NIR and Brazil nuts - ND DeMello and Scussel, 2009 hand-sorting BGYF Pistachio - 99.2% Hadavi, 2005 High-speed dual Yellow corn, 53 µg/kg 81% Pearson et al., 2010 wavelength sorting Sorghum Pistachio 92% BGYF - Lundadei et al., 2013 Cashews 82% BGYF White corn - 67% Yao et al., 2010 Hand-sorting, Maize 22 µg/kg 93% Fandohan et al. 2005 washing, winnowing Hand-sorting Peanuts 7.9-799.8 µg/kg 97.7% Filbert and Brown 2012 in the shell Abbreviations: Non-detectable (ND), near infrared reflectance (NIR), and blue green yellow fluorescence (BGYF)

Binding agents for the reduction of aflatoxins

The practice of adding binders or sequestering agents to feed has been studied to reduce toxicity by acting as an enterosorbent, binding aflatoxins, and reducing bioavailability in intestinal absorption (Phillips 1999; Phillips et al., 2008). These dietary additives (inorganic silica-based compound, organic charcoal-based polymer, or an indigestible carbohydrate) offer one of the greatest potentials for preventing toxicity in a stable digestive tract where the bound aflatoxin can be excreted via urine or feces.

Binders are sold as anti-caking agents and their use in detoxifying mycotoxins has not yet been approved by the FDA.

Clay additives such as bentonite (montmorillonite), hydrated aluminum silicate

(HSCAS), zeolite, and sepiolite (Table 3.2) have been utilized to improve the physical properties of animal feeds and offer the absorption of aflatoxin (Diaz et al., 2004;

Pimpukdee et al., 2004; Bailey et al., 2006; Magnoli et al., 2011; Nuryon et al., 2012;

Rao and Chopra 2007; Gallo et al., 2010; Pasha et al., 2007). It is suggested that these specific silicate minerals can bind with aflatoxin by chelating the β-dicarbonyl moiety in aflatoxin with uncoordinated metal ions in the clay materials (Phillips 1999). Pasha et al.

(2007) studied the efficiency of sodium bentonite (NaB; 0.5% and 1%), 0.5% Sorbatox

(HSCAS), or 0.2% Klinofeed (HSCAS) to bind 100 µg/kg aflatoxins in broiler chick feed; body weight was also assessed. During the starter phase, the highest weight gain was observed in the negative control (1029.4 g) and the least amount in the positive control (663.7 g), Sorbatox (649.4 g) and Klinofeed (638.6 g). The NaB at 0.5% (856.8 g) was shown to increase weight gain the most when compared to the other treatments.

Between the two levels of NaB, 0.5% level gave better results than 1%. The findings of

this study indicated that the addition of NaB was an effective method to reduce aflatoxin toxicity to improve the weight of broiler chickens when added to feed.

Table 3.2 Reduction of aflatoxins by the addition of adsorbents

Initial Adsorbent Target Product Aflatoxin Measured Improved Ref. (%) (µg/kg) 1.2% NaB 50 - 65% Corn, % Red. 1.2% CaB 31% Diaz et al., Cows alfalfa 55 AFB1 (AFB1 to 0.25% AC 5.4% 2004 silage AFM1) 0.05% YCW 58.5% 1% - 2% NaB 15.33 63 -76% Gallo et al., Cow Corn meal % Rec. 1% - 2% AC AFB1 52 -37% 2010 0.56% HSCAS corn, % Red. 47% Kutz et al., 0.56% HSCAS Cows alfalfa 112 AFB1 (AFB1 to 44% 2009 0.56% YCW cottonseed AFM1) 5% + control 32.4 MNC 100% HSCAS 211.88 Genotoxic 8.4 [S] Shebl et al., Chicks Feed 98% YCW Total effect 7.2 [S] 2010 HSCAS + YCW 6.2 [S] + control 42.4% DNAF 100% HSCAS 211.88 31.6% [S] Shebl et al., Chicks Feed DNAF 98% YCW Total 28.8% [S] 2010 HSCAS + YCW 23.8%[ S] Control 57.2 g Egg 300 AFB1 56.46 g Manafi et al., 0.1% Bentonite Chicks Rice Weight 400 AFB1 55.74 g 2012

500 AFB1 55.61 g [S] Control 57.2 g Egg 300 AFB1 57.76 g Manafi et al., 0.2% GMA Chicks Rice Weight 400 AFB1 56.68 g 2012

500 AFB1 55.96 g [S] Maize, % Red. 1% AC 76% Rao and Goats mustard, 100 AFB1 (AFB1 to 1% NaB 67% Chopra 2001 wheat AFM1) Abbreviations: Sodium bentonite (NaB), Calcium bentonite (CaB), hydrated aluminum silicate (HSCAS), activated carbon (AC), Yeast cell wall (YCW), glucomannan (GMA), DNA fragmentation (DNAF), micronucleated cells (MNC), recoveries (rec.), reduction (red.), statistically significant [S] from control (p-value < 0.05)

The HSCAS is characterized as an aflatoxin-selective clay and may not be as effective for other mycotoxins (Phillips 1999). Kutz et al. (2009) conducted a study that evaluated the binding efficacy of 3 adsorbents: NovasilPlus (HSCAS), Solis (HSCAS), and MTB-100 (yeast cell wall). The final dietary concentration of AFB1 in the feed and 39

the dietary concentration of the adsorbents were 112.2 µg/kg and 0.56%, respectively.

After consumption of the AFB1-contaminated feed and/or binders, the carry-over

reduction of AFB1 to AFM1 concentration was determined. The AFM1 concentration

absent of a binder resulted in a carry-over of 1.92 µg/kg. With the addition of adsorbents,

Solis, NovasilPlus, and MTB-100, the carry-over reduction of AFB1 to AFM1 were

determined to be 1.06 (47%), 1.00 (44%), and 1.84 µg/kg (5%), respectively. Carry-over

from AFB1 to AFM1 concentrations in milk were significantly reduced with the addition

of the HSCAS while the addition of yeast cell wall adsorbent was not effective. Other

studies suggest that the use of yeast cell wall and HSCAS either alone or in combination

were efficient in the prevention of the toxic effect of aflatoxin (Shebl et al., 2010;

Masoero et al., 2007; Stroud 2006; Li et al., 2010).

The effectiveness of some binders are debatable due to the complex process involving a combination of porosity characteristics, surface acidity, temperature, pH, and distribution of exchangeable cations (Johnston et al., 2012; Yener et al., 2012; Liu et al.,

Manafi et al., 2012). For instance, Johnston et al. (2012) conducted a study that

investigated the glucomannan (MTB-100) as a possible agent for sequestering aflatoxin

in contaminated dried distillers grains (DDGs) resulting in an 80% reduction in total

aflatoxin levels. Additionally, it was revealed that environmental factors such as pH and

temperature affect the binding abilities of MTB-100 to sequester the aflatoxin in DDGs.

It was concluded that temperature near 0°C (pH 6) resulted in near negligible binding and

maximum binding percentage was 19.7% (pH 8). However, an increase in temperature to

40°C resulted in binding of 36%, 47%, and 45% at pH 6, 7, and 8, respectively.

The effectiveness of activated carbon (AC) to bind aflatoxin is variable (Diaz et

al., 2004; Galvano et al., 1998; Edrington et al., 1996; Jaynes and Zartman 2011). Diaz et

al.(2004) evaluated three NaB (Astra-Ben 20, Flow Guard, and Mycrosorb), calcium

bentonite (CaB; Red Crown), AC (SA-20), and an esterified glucomannan along with other binders as sequestering agents to bind dietary AFB1 and reduce absorption from an

animal’s gastrointestinal tract and consequently reduce the transfer of AFB1 to AFM1 in

milk. Contaminated corn of 55 µg/kg of AFB1 and NaB (1.2%) were given to 16 lactating

cows for 20 days. AFM1 residues were determined to be 1.24 µg/kg, 0.52 µg/kg, 0.43

µg/kg, and 0.62, respectively. The carry-over reduction of AFB1 from the feed to milk

was 64.6% (Astra-Ben 20), 31.4% (Red Crown), 58.5% (MTB-100), and 5.4% (AC). The

NaB, CaB, and esterified glucomannan were found to diminish AFB1 to AFM1

significantly. A concentration of 0.25% of AC had no significant effect on the reduction

of AFB1 to AFM1 in cow’s milk. However, other findings found that AC was comparable to other clay binders (Rao and Chopra 2001). Other studies also suggest that AC may be more suitable for other mycotoxins such as zearalenone and deoxynivalenol (Jard et al.,

2011; Sabater-Vilar et al., 2007) and in aqueous solutions.

Irradiation for the reduction of aflatoxins

The use of UV radiation as a detoxification agent for aflatoxin has been studied

and it is believed that irradiation at 362 nm activates AFB1 and increases its susceptibility

to degradation (Lillard and Lantin, 1970). However, longer UV exposures may have

some safety issues leading to the formation of less toxic photodegradation products. Liu

et al. (2010) revealed the appearance of new photodegradation products and a suggested

photodegradation pathway has also been proposed (Figure 3.2). 41

Figure 3.2 Proposed pathway for degradation of AFB1 by UV.

Adapted from Liu et al. (2010).

Tripathi et al. (2010) describes the efficiency of peroxidase (2-16 U) isolated

from garlic bulbs coupled with short duration UV exposure in degrading AFB1 (3.12-31.2

µg/kg) from contaminated red chili powder. The enzymatically detoxified sample was

exposed to long wave 365 nm UV light and the exposure was given for 15-60 min. The

optimum detoxification (66.02%) occurred with 12 U of enzyme, 60 nmol AFB1 per 100

g chili powder at 24 h of incubation. Time-dependent AFB1 degradation upon exposure

was observed with maximum detoxification (87.8%) recorded after 60 min. A recent

study concluded that when dried figs were subjected to UV irradiation for 90 minutes at a

wavelength of 365 nm, the levels of aflatoxin decreased 25% (Isman and Biyik, 2009).

Atalla, et al. (2004) investigated the effect of fluorescent and UV light on

aflatoxins B1, B2, G1, and G2 under various humidity levels in wheat grains (13%

moisture). The wheat grains were inoculated with A. parasiticus and three other fungal isolates that were known to produce toxins at a concentration of 1 mL spore suspension

100/g. The wheat grains were exposed to short wave 254 nm and long wave 362 nm light and fluorescent light at a distance of 5 cm and 25 cm from the UV lamp and fluorescent light, respectively for 30, 60, and 120 min. It was found that aflatoxins B1 and G1 were

completely eliminated when exposed to UV short, long-wave, and fluorescent light for 30

and 60 min and AFB2 decreased after treatment of 120 min at different humidity levels

(50, 74, and 80%).

It has been reported that 5 to 60 kGy of gamma ray dosage significantly reduced a

percentage of aflatoxin (Table 3.3). Nonetheless, the maximum allowable dosage, as

permitted by the FDA, is 30 kGy. Ghanem et al. (2008) irradiated (60Co) samples of rice,

pistachios, peanuts, corn, wheat, and bran with dosage of 4, 6, and 10 kGy at a rate of

1.449 kGy/hour. In all tested commodities, a positive correlation was found between the

increase in doses of gamma radiation applied to the commodity and the level of aflatoxin

breakdown.

Table 3.3 Detoxification of aflatoxin through irradiation.

Radiation Product Dosage Measured Aflatoxin Improved Ref. Tripathi Chili 365 nm and UV % Red. 60 nmol 100/g AFB1 66.02% powder (24 h) Mishra, 2010 Dried 365 nm Isman and UV % Red. 3.18 μg/kg total 25% figs (90 min) Biyik, 2009 60 Co Black B1 47%, 51%

gamma and 60 µg/kg (B1, G1) B2 39%, 35% Jalili et al., 30 kGy % Red. (18% white 18 µg/kg (B2, G2) G1 47%, 48% 2012

moisture) pepper G2 40%, 43% B1 43% 60 Co Black B2 24% Jalili et al., 60 kGy % Red. 55 µg/kg gamma pepper G1 40% 2012 G2 36% 60Co Corn- 5, 10 Weight NS in weight Simas et 200 µg/kg gamma Soybean kGy Gain gain al., 2010

22.46 µg/kg B1 1650W 36% AFB1 Perez- 69.62 µg/kg B2 Microwave Maize 2450 % Red. 58% AFB2 Flores et 141.47 µg/kg MHz 82% B1 + B2 al., 2011 B1+B2 51%-ND 92°C 5-183.2 µg/kg AFB1 Mobeen et Microwave Peanut % Red. AFB1 (5 min) 7- 46.6 µg/kg AFB2 al., 2011 ND AFB2 5, 10, 15, 210-965 µg/kg 60Co Chicken 34-40% Herzallah 20, 25 % Red. total gamma feed 33-43% et al., 2008 kGy 192-894 µg/kg AFB1 965, 421, 210 µg/kg 1450W 21-33% total Chicken total Herzallah Microwave 2450 % Red. 23-32% feed 894, 395, 192.1 et al., 2008 MHz AFB1 µg/kg AFB1 Abbreviations: Non-detectable (ND), no significant improvement (NS)

Jalili et al. (2012) evaluated the efficacy of gamma radiation (60Co) ranging from

5 to 30 kGy for the degradation of aflatoxin in black and white pepper. The moisture content of the pepper samples was 12 or 18%. It was found at 18% moisture level and 30 kGy proved to reduce AFB1, AFB2, AFG1, AFG2 by 50.6%, 39.2%, 47.7%, and 42.9%, respectively. Jalili et al. (2010) studied dosage of gamma radiation ranging from 0 to 60 kGy for the complete degradation of aflatoxin in black pepper (50 g). It was found that at

60 kGy AFB1 and AFG1 reached maximum reduction of 43% and 40%, respectively. It

was determined that AFB2 (24% reduction) and AFG2 (36%) were more “radio-resistant”

than AFB1 and AFG1.

Simas et al. (2010) studied the influence of gamma radiation on the productivity

parameters of chicken fed mycotoxin-contaminated corn. Of the mycotoxins examined,

aflatoxin-contaminated corn with a concentration 200 µg/kg was irradiated with 5 kGy or

10 kGy using 60Co irradiator. There was no statistical difference in mean body weight in aflatoxin-fed chicks at 0 (2.5 kg), 5 (2.52 kg), and 10 kGy (2.57 kg). An increase in the irradiation dose did not improve the body weight of chickens fed any level of gamma radiation (absent from mycotoxin).

Porez-Flores et al. (2011) studied the effect of microwave heating during alkaline- cooking of aflatoxin contaminated maize (10.8%) moisture using a modified tortilla-

making process. The initial concentrations of aflatoxin in 20 kg maize grain were 22.46,

69.2, and 141.48 µg/kg for AFB1, AFB2, and AFB1 + AFB2 respectively. The maize was

cooked in a microwave resistant plastic container, and the cooking stage had an average

cooking power of 100% during 5.5 min. The power output was 1650 W and the operating

frequency was 2450 MHz. Microwaving the maize resulted in a 36% reduction of AFB1

(14.41 µg/kg), 58% reduction of AFB2 (29.44 µg/kg), and 82% reduction of AFB1 +

AFB2 (24.95 µg/kg). Further processing to the tortilla resulted in a 68% reduction in

AFB1 (7.22 µg/kg), 80% reduction in AFB2 (13.58 µg/kg), and 84% reduction in AFB1 +

AFB2 (23.18 µg/kg). Mobeen et al. (2011) also evaluated the effectiveness of microwave

heating for the detoxification of aflatoxin in peanut and peanut products. Contaminated

peanut samples contained 5-183.2 µg/kg AFB1 and 7-46.6 µg/kg AFB2. The samples

were exposed to microwave heating up to 92°C for 5 min which resulted in a 51.1% -

100% reduction of AFB1 while AFB2 was not found within detectable limits.

Herzallah et al. (2008) studied the efficiency of total-aflatoxin (965, 421, and 210

µg/kg) and AFB1 (894, 395, 192.1 µg/kg) decontamination in poultry feeds through solar

(0 – 30 h), gamma (0-25 kGy), and microwave radiation (2 - 10 min). Sunlight exposure

of 30 hours resulted in maximum reduction of 65.9% (329 µg/kg), 74.5% (106.1 µg/kg),

and 60% (83.0 µg/kg) in total aflatoxin; and 75.5% (219 µg/kg), 74.6% (100.5 µg/kg),

and 60.4% (76.0 µg/kg) reductions of AFB1. Gamma radiation dosage of 25 kGy resulted

in 34.7% (630 µg/kg), 33.5% (280 µg/kg), and 40% (125 µg/kg) reductions of total

aflatoxin; and 32.9% (600 µg/kg), 34.2% (260 µg/kg), and 42.7% (110 µg/kg) reductions

of AFB1. Samples heated in a microwave oven at 100% power with an output of 2450

MHz and 1.45kW resulted in a maximum reduction of 21.2% (760 µg/kg), 21.6% (330

µg/kg), and 33.3% (140 µg/kg) of total aflatoxin at a heating time of 10 min; and 22.5%

(690 µg/kg), 32.3% (310 µg/kg), and 32.3% (130 µg/kg) reductions of AFB1. The results

of this study conclude that solar radiation was significantly more effective for the

reduction of aflatoxin.

Chemical methods for the detoxification of aflatoxins

Extensive research has been performed to consider ozonation as a practical

method for the decontamination of aflatoxins. Under various concentrations,

temperatures and time of exposure, ozonation has been shown to reduce aflatoxin levels.

McKenzie et al. (1997) conducted a study to investigate the degradation and

detoxification of mycotoxins from aflatoxin-contaminated rice in the presence of ozone.

This experiment involved electrochemical ozone generation, where water was the source 46

for the formation of ozone. Mycotoxins, including aflatoxins B1, B2, G1, and G2 (10

µg/g), were treated with 2, 10, and 20% ozone over a period of 5 min. The results showed

that AFB1 and AFG1 achieved total degradation by 15 sec at a 2% ozone concentration

(rate of 0.0603 and 0.0583 µg/sec), respectively. AFB2 and AFG2 required 20% ozone for

rapid degradation at a constant rate equaling 0.1014 and 0.0831 µg/sec, respectively.

Despite using an increased rate to achieve a faster rate of degradation, AFB2 and AFG2

were also shown to have a significantly reduced aflatoxin concentration within 15

seconds.

Proctor et al. (2004) examined the effects of gaseous ozonation and mild heat

treatment on the degradation of aflatoxins in peanut kernels and flour. Batches of 25 g of

either peanut samples were spiked with aflatoxins at a contamination level of 20 µg/kg

AFB1, AFB2, AFG1, and AFG2. Ozonation was carried out using ozone gas at 4.2% per

weight at 15 psi generated by an ozone generator. Ozone gas and heat (25, 50, 75°C)

treatments were applied to the samples at 5, 10, or 15 min. It was found that AFB1 was

reduced by 77% for 10 min at 75°C, while AFG1 showed a max degradation of 80%

under the same conditions in peanuts. Regardless of treatment combinations, AFB1 and

AFG1 showed the highest degradation levels while AFB2 maximum degradation was

obtained at 75°C despite reaction time (56%). AFG2 exposure at 75°C, for at least 10

min, resulted in degradation of 30%. It was shown that ozonation efficiency increased

with higher temperatures and longer treatment times. Also, higher levels of toxin

degradation were achieved in peanut kernels than in flour (Table 3.4). Other research has

verified the reduction of aflatoxin-contaminated peanuts by ozone under various

conditions (Table 3.4) (Diao, et al., 2013; Chen, et al., 2013; Akbas and Ozdemir, 2006).

Table 3.4 Detoxification of aflatoxin through chemical methods.

Dosage/ Initial Method Method Product % Reduction Ref. Aflatoxin Parameter 77% AFB1 (10 min, 75°C) 80% AFG1 4.2% per wt 25 g Peanut (75°C, any time) (15 psi) 20 µg/kg 51% AFB2, AFG2

AFB1 (75°C, any time)

AFB2 56% AFB1 Proctor et O3 AFG1 (10 min, 50°C) al., 2004

AFG2 61% AFG1

(10 min, 50°C) 5, 10, 15 min 25 g Flour 20% AFB2 25, 50, 75°C (5 min, 50°C) 30% AFG1 (5 min, 50°C) 55% at 16 mg/L 16, 33 mg L-1 75 g Red Pepper 20 µg/kg (60 min), Inan et al., O3 7.5, 15, 30, and (12.6% moisture) AFB1 80% at 33 mg/L 2007 60 min at 25°C (30 min) -1 50 mg L -1 Peanut 189.5 µg/kg 89.4% Diao et al., O3 (5 L min ) for (8% moisture) AFB1 (20 µg/kg) 2013 60 h 7.5 mg L-1 for Peanut 200 µg/kg Chen et al., O3 30 min at room 65.0% (5% moisture) AFB1 2013 temperature 9 mg L-1 10 µg/kg Akbas et al., O3 420 min at Pistachios 23% AFB1 2006 20°C Ammonium 178 µg/kg Allameh et 0.25% - 2.0% 50 g Corn 53% - 87% persulfate AFB1 al., 2002 650 µg/kg 1% AFB1 ND Allameh et Ammonia 40 - 50°C Maize 1280 µg/kg 10 µg/kg AFB1 al., 2005 for 48 h AFB1

Abbreviations: Non-detectable (ND), Ozone (O3)

Inan et al. (2007) conducted a study to determine the influence of gaseous ozone treatment on AFB1 in red pepper. A total of 75 g of red pepper (12.6% moisture) with an initial concentration of 20 µg/kg was exposed to various ozone concentrations of 16, 33, and 66 mg/L and exposure times of 7.5, 15, 30, and 60 min at 25°C. After 60 min exposure at 16 mg/L, AFB1 levels decreased to 11 µg/kg (55% reduction). An increase in 48

ozone to 33 mg/L after 30 min of exposure significantly decreased AFB1 concentration to

4 µg/kg (80% reduction).

Under various conditions, ammoniation has been shown to decontaminate

produce containing aflatoxin. Aflatoxin reduction by ammoniation is more effective

when subjected to long exposure time, high temperature and high pressure (Inan et al.,

2007). However, these conditions are not cost effective in developing countries and such

conditions may nutritionally degrade the product and safety options must be considered.

Ammonia has been shown to alleviate toxicity of AFB1 by converting it to a non-toxic

product, AFD1, through hydroxylation and decarboxylation (King and Prudente, 2005;

Banu and Muthumary, 2010).

Burgos-Hernandez et al. (2002) studied the decontamination of AFB1-

contaminated corn by ammoniation-fermentation integrated process. Contaminated corn

was exposed to 0, 0.5, 1.0, 1.5, or 2.0% (w/w) ammonium persulfate during the

fermentation process to ethanol. The corn had an initial AFB1 concentration of 178 ± 15

µg/kg. The fermentation process alone significantly reduced AFB1 concentration by

55.6% (79 ± 10 µg/kg). The statistically significant reduction of AFB1 concentration

ranged from 53 to 87% at 0.25 to 2% of ammonium persulfate, respectively. It was also concluded that the incorporation of ammonium persulfate in the fermentation of corn did not affect the production of ethanol.

Allameh et al. (2005) evaluated mortality and productivity in broiler chickens

when fed aflatoxin-contaminated maize. Dietary treatments included (A) uncontaminated

maize, (B) ammonia treated uncontaminated maize, (C) AFB1-contaminated maize (1000

µg/kg), (D) ammonia treated AFB1-contaminated maize (1000 µg/kg), (E) AFB1-

contaminated maize (2000 µg/kg), and (F) ammonia treated AFB1-contaminated maize

(2000 µg/kg). There was 650 µg/kg AFB1 in the feed from the initial 1000 µg/kg in the

maize in treatment C. This concentration was reduced to non-detectable when 1%

aqueous ammonia was applied (treatment D). For treatment E, AFB1 in the feed was

found to be 1280 µg/kg and was reduced to approximately 10 µg/kg in the feed when

subjected to 1% ammonia (treatment F). Additionally, there was no significant change in

dietary intake, and body weight gain in chickens fed ammonia treated aflatoxin- contaminated maize.

Conclusion

Aflatoxins occur naturally in many agricultural crops causing health hazards and economic losses. Despite improved biological methods, handling, processing, and

storage, aflatoxin contamination still remains a serious problem and poses a unique

challenge to food safety. Therefore, new ways to remediate contaminated products are

needed to limit economic and health impacts and add value to the agricultural industry.

Non-biological strategies such as the separation of aflatoxin from contaminated grains

through sorting, the addition of adsorbents to contaminated diets, radiation, ozonation,

and ammoniation of aflatoxin have been reviewed for the reduction of aflatoxin

contamination post-harvest.

Non-biological methods are being actively studied and can be a highly

encouraging choice to reduce or eliminate the possible contaminations of aflatoxins in

foods and feeds. However, no one method currently fulfills the necessary efficacy, safety,

and cost requirements needed for the removal of aflatoxin from contaminated agricultural

products. For example, adsorbents have been shown to bind aflatoxins when added to 50

feed, reduce aflatoxin uptake by animals, prevent acute aflatoxicosis, and decrease aflatoxin residues in milk. However, the addition of binders to feed does not meet safety requirements.

Discrepancies in the reviewed literature of the reduction of aflatoxin might be due to differences in experimental conditions, the varied aflatoxin concentrations, and varied agricultural product or animal model. Despite these variable findings, most of the data indicated that hand and mechanical sorting might be the most effective non-biological method for the detoxification of aflatoxin in agricultural products and animals post- harvest. Additionally, application of advanced mechanical sorting may produce a better quality, safer, and more consistent product. Ultimately, the best reduction of aflatoxin contamination is one that is approved by regulatory agencies, cost-effective, and one that is effective at the reduction of aflatoxin from contaminated agricultural products.

AFLATOXIN M1 IN MILK AND MILK PRODUCTS

Abstract

Aflatoxin M1 (AFM1) is associated with carcinogenicity, genotoxicity,

mutagenicity, and teratogenicity and as a result, represents a human health problem

worldwide. This review will detail the toxicity, analytical methodology, occurrence, and

prevention and control of AFM1 in milk and milk products. The probable daily intakes

(PDI) per bodyweight (b.w.) worldwide ranged from 0.002 to 0.26 ng/kg b.w./day for

AFM1. Nevertheless, the high occurrence of AFM1 demonstrated in this review

establishes the need for monitoring to reduce the risk of toxicity to humans. The

recommended extraction method of AFM1 from milk is liquid-liquid with acetonitrile because of the acceptable recoveries (85-97%), compatibility with the environment, and cleanest extracts. The recommended analytical technique for the determination of AFM1 in milk is the high performance-liquid chromatography-fluorescence detector (HPLC-

FLD), achieving a 0.001 µg/kg detection limit. The HPLC-FLD is the most common internationally recognized official method for the analysis of AFM1 in milk. The

suggested extraction and analytical method for cheese is dichloromethane (81-108%

recoveries) and ELISA, respectively. This review reports the projected worldwide

occurrence of AFM1 in milk for 2010-2015. Of the 7,841 samples, 5,873 (75%) were

positive for AFM1, 26% (2,042) exceeded the maximum residue levels (MRL) of 0.05 52

µg/kg defined by the European Union and 1.53% (120) exceeded the MRL of 0.5 µg/kg defined by the United States Food and Drug Administration. The most effective way of preventing AFM1 occurrences is to reduce contamination of AFB1 in animal feed using

biological control with atoxigenic strains of Aspergillus flavus, proper storage of crops,

and the addition of binders to AFB1-contaminated feed. Control measures include the

addition of binders and use of biological transforming agents such as lactic acid bacteria

applied directly to milk. Though the one accepted method for the control of AFM1 in

milk and milk products is the enforcement of governmental MRL.

Introduction

Aflatoxins are toxic metabolites produced mainly by Aspergillus flavus (A. flavus) and parasiticus (A. parasiticus) during pre-harvest, post-harvest, and during storage of

cereal grains particularly maize, rice, wheat, barley, and oats (Pitt et al., 2013). Aflatoxin

B1 (AFB1), the most potent mycotoxin, is also the most toxic naturally occurring liver

carcinogen known (McKean et al., 2006). Concurrently, AFB1 has been categorized by

the International Agency for Research on Cancer (IARC) as a Group 1 carcinogen, a

known human carcinogen (IARC, 1993).

AFB1, metabolized in the liver, produces a number of metabolites including

hydroxylated derivative, aflatoxin M1 (AFM1) shown in Figure 4.1. AFM1 appears in the

milk 2-3 days after the ingestion of AFB1 (Prandini et al., 2009). The carry-over from

feed to milk in dairy cows is influenced by various nutritional and physiological factors,

including feeding regimens, rate of ingestion, rate of digestion, health of the animal,

hepatic biotransformation capacity, and actual milk production (Duarte et al., 2013).

Consequently, the percent conversion of AFB1 in animal feed to AFM1 in milk can vary 53

from 0.3% to, as high as, 6.2% (Creppy, 2002). Cows in early lactation (2 to 4 weeks

after calving) show highest milk yields and a higher carry-over rate than cows in late

lactation (34 to 36 weeks after calving), when milk yield naturally declines (Veldman et

al., 1992). Once AFM1 is found in milk, it takes about 2-3 days for the milk to be AFM1 free (Prandini et al., 2009).

Figure 4.1 AFM1 is the hydroxylated derivative of AFB1.

AFM1, like AFB1 but less toxic, is associated with cytotoxicity, carcinogenicity,

genotoxicity, mutagenicity, and teratogenicity (IARC, 2002). AFM1, a Group 2B

carcinogen (possible carcinogen), is found in the milk of lactating animals that have consumed AFB1-contaminated feed (IARC, 2002). JECFA (Joint FAO/WHO Expert

Committee on Food Additives) noted that carcinogenic potency of AFM1 was

approximately one-tenth less than that of AFB1 and the genotoxicity of AFM1 ranged

from similar to one-sixth potent of AFB1 (World Health Organization, 2002).

AFM1 poses a worldwide concern, particularly for infants and children who

consume large quantities of milk and who are more susceptible to the adverse effects of

AFM1 (Boudra et al., 2007). Additionally, there is evidence of hazardous human

exposure to AFM1 in milk and dairy products shown throughout literature (Ali et al.,

2014; Elzupir and Elhussein, 2010; Fallah, 2010a; Atasever et al., 2010b). Because 54

AFM1 is hazardous to human health, taking precautions to restrain AFM1 from milk and

milk products is necessary. Evaluating the toxic effects, choosing a normalized analytical

method for detection, determining the incidence, and understanding prevention and

control of AFM1 is vital for the reduction of AFM1 in milk and milk products. Therefore,

the toxicity, analytical methodology, occurrence, and the prevention and control of AFM1 will be reviewed.

Toxicity

Aflatoxicosis, distinguished between acute and chronic toxicity, is a disease that occurs from the ingestion of aflatoxins, primarily through the consumption of aflatoxin- contaminated food or feed. Though, Oluwafemi et al. (2012) has reported occupational aflatoxicosis through airborne inhalation of Aspergillus fungal spores. AFM1 and AFB1

cause almost identical effects of acute toxicity in several species (Caloni et al., 2012;

World Health Organization, 2002). Acute aflatoxicosis caused by AFM1 has been

implicated in the pathogenesis of malnutrition diseases, increased neonatal susceptibility

to infections, and jaundice (Abdulrazzaq et al., 2004; Turner et al., 2007). The largest risk of aflatoxin to humans, however, is usually the result of chronic dietary exposure.

Chronic aflatoxicosis results from ingestion of low to moderate levels of aflatoxin and have nutritional, immunological, and carcinogenic consequences (Williams et al., 2004).

Magoha et al. (2014) observed the effects of AFM1 in breast-milk on the growth of 143

infants (<6 months of age) in Northern Tanzania. The breast-milk samples contained

AFM1 concentrations ranging from 0.02 to 0.55 µg/kg. A significant (p<0.05) inverse

relationship was observed between AFM1 exposure and the weight and height in the

infants. 55

Exposure to toxic compounds such as AFM1 is problematic to human health.

Thus, it is important to determine the degree of human exposure. The degree of human

exposure is estimated based on consumption of contaminated food and the average

occurrence of the toxin and is measured in probable daily intake (PDI) per unit of

bodyweight (b.w.) in kilograms (WHO, 2002). The daily AFM1 intake, as estimated by

JECFA was 0.002, 0.1, 0.058, 0.11 and 0.20 ng/kg b.w./day for Africa, Middle East,

Latin America, Europe, and Far East, respectively (WHO, 2002). Contrary to the PDI

reported by JECFA, Jager et al. (2013) and Shundo et al. (2009) reported a PDI of 0.1

and 0.118 ng/kg b.w./day, respectively for Brazil, doubling the PDI as first reported in

2001. Ruangwises et al. (2011) estimated PDIs in five cohorts based on age for the intake of AFM1 from milk: (1) 0-3 years of age (PDI 0.16 ng/kg b.w./day); (2) 3-9 years of age

(PDI 0.26 ng/kg b.w./day); (3) 9-12 years of age (PDI 0.12 ng/kg b.w./day ); (4) 19-65

years of age (PDI 0.04 ng/kg b.w./day ); (5) >65 years of age (PDI 0.03 ng/kg b.w./day).

Leblanc et al. (2005) estimated the intake of AFM1 in the French population. For the

adults aged 15 years and older (n=1474), the PDI was 0.06 ng/kg b.w./day and the

children aged 3-14 (n=1018) PDI was 0.13 ng/kg b.w./day. In risk assessments, the PDI

is compared to tolerable daily intake (TDI) determined in toxicological studies to indicate

the degree of concern (Jager et al., 2013). The TDI for AFM1 was proposed to be 0.5

ng/kg b.w./day by Kuiper-Goodman (Silva et al., 2015). It should be noted that 0.5 ng/kg

b.w./day is not an official value, but a recommendation. In the current review, all PDIs

(ranging from 0.002 to 0.26 ng/kg b.w./day) estimated for AFM1 in the population were

below the proposed TDI for AFM1 (0.5 ng/kg b.w./day). However, the high occurrence of

AFM1, as shown later in this review, demonstrates the need for monitoring in milk and milk products to reduce the risk of toxicity to humans.

Analytical methodology

Sample preparation and clean-up

The methods described in literature for the extraction of AFM1 from milk utilized

different extraction techniques, such as liquid-liquid extraction (LLE), solid-phase

extraction (SPE), or LLE followed by a SPE or immunoaffinity (IA) clean-up step (Chen

et al., 2005; Fallah, 2010a; Sørensen and Elbaek 2005). SPE with IA sorbents has also

been used in AFM1 extraction and for clean-up purposes (Bascarán et al., 2007).

LLE is the conventional method for the separation of AFM1 from milk or milk

products (Pittet, 2005), using suitable solvents such as chloroform (CHCl3), acetonitrile

(ACN), methanol (MeOH), or dichloromethane (DCM). The first effective extraction of

AFM1 from milk (75 mL) utilized 400 mL of CHCl3 and 4% sodium chloride (NaCl).

Samples were analyzed using thin-layer chromatography (TLC) and the recovery of 0.5

ng/mL AFM1 was satisfactory (Jacobson et al., 1971). Kamkar (2005) extracted AFM1 from 50 mL milk using 125 mL CHCl3. The extract was purified by silica gel column chromatography and quantified using TLC. The recovery of AFM1 spiked in raw milk at

2 and 4 µg/L was 90%. Other authors (Table 4.1) have also seen acceptable extraction

recoveries of AFM1 from milk using CHCl3 (Herzallah, 2009; Fallah, 2010a) and other

solvents including MeOH and ACN (Navas et al., 2005; Temamogullari and Kanici,

2014; Wang et al., 2010). Studies have shown acceptable extraction recoveries of AFM1

from milk using CHCl3, ranging from 77 to 99% recoveries (Herzallah, 2009; Fallah,

2010a; Kamkar, 2005). However, the disadvantage of using CHCl3 for the extraction of 57

AFM1 from milk is the use of large volumes of solvents (125-400 mL) that have to be

disposed of creating environmental problems (Hussain, 2011). MeOH and ACN, on the

other hand, have become increasingly more popular to use for the extraction of aflatoxins

because of their better compatibility with the environment and the antibodies involved in

subsequent clean-up with IA columns (Manetta, 2011). Recoveries of AFM1 from milk

using MeOH and ACN ranged from 77 to 99% and from 85 to 97%, respectively (Navas

et al., 2005; Temamogullari and Kanici, 2014; Wang et al., 2010; Wang et al., 2011).

Table 4.1 Sample preparation involving extraction of AFM1 from milk

Extraction AFM1 Spike Recovery Clean-up Samples References Method (ng/mL) (%) Method centrifuged/ 0.05 114 Diaz and Espitia, milk IAC defatted 0.2 91 2006 125 mL 50 mL CHCl3 1-2 89.5 filtration Fallah, 2010a raw milk (NaCl) 150 mL 50 mL CHCl3 2-20 95-99 SPE Herzallah, 2009 raw milk (5% NaCl) 50 mL 125 mL 2-4 90 silica gel Kamkar, 2005 raw milk CHCl3 5 g QuEChERS NA 57.5-59.3 SPE Karaseva et al., 2013 raw milk and LLME 0.01 94 25 mL 35 mL 0.03 77 IAC Navas et al., 2005 breast milk MeOH 0.05 82 nylon 10 mL 50 69 QuEChERS syringe Rubert et al., 2014 breast milk 250 69 filter centrifuged/ 0.01 86 Shundo and Sabino, milk IAC defatted 0.5 74 2006 2 g Temamogullari and 5 mL MeOH 0.05 99 centrifuge raw milk Kanici, 2014 20 mL 0.02 85 30 mL ACN PTFE Wang et al., 2010 raw milk 1.0 86 Abbreviations: CHCl3 (chloroform); NaCl (sodium chloride); MeOH (methanol); QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe); LLME (liquid-liquid micro-extraction); ACN (acetonitrile); IAC (immunoaffinity column); PTFE (polytetrafluoroethylene) syringe filter; SPE (solid-phase extraction)

In addition to conventional extraction methods, there was a need to develop new techniques due to the cost of solvents, time-consumption, and the labor-intensiveness of

LLE (Karaseva et al., 2013). Relatively new approaches, such as QuEChERS (Quick,

Easy, Cheap, Effective, Rugged, and Safe), have been developed to remediate those problems. QuEChERS involves an initial extraction with ACN followed by a partitioning step with the addition of a salt mixture. Rubert et al. (2014) used QuEChERS to evaluate

AFM1 in human breast milk. Percent recovery of AFM1 from breast milk was 69% with

relative standard deviations (RSDs) of 12 and 13% at 50 and 250 ng/mL, respectively.

The extraction of AFM1 from milk and cheese using QuEChERS is a novel approach and

optimization of this method is necessary to improve percent recovery of AFM1.

The extraction of AFM1 from cheese mainly involves the use of the extraction

solvent, DCM, the evaporation of the solvent, and partitioning of the reconstituted

residue with hexane (Anfossi et al., 2008). DCM is commonly used for the extraction of

AFM1 from cheese. Recoveries of AFM1 from spiked cheese samples using DCM ranged

from 81 to 108% with relative standard deviations (RSDs) ranging from 1.0 to 15%

(Cavaliere et al., 2006; Kav et al., 2011; Kocasari et al., 2012; Martins et al., 2007).

Concurrently, extracting AFM1 from cheese using MeOH achieved lower mean

recoveries of 60-86% (RSDs 2-10%) compared to samples extracted with DCM (Iha et

al., 2011; Temamogullari and Kanici, 2014). Cavaliere et al. (2006) suggest that DCM is

a better extraction solvent than MeOH for the extraction of M1 from cheese (Table 4.2).

Concurrently, Iha et al. (2011) extracted AFM1 from cheese using MeOH achieving a

lower mean recovery of 61-86% (RSDs 2-10%) compared to samples extracted with

DCM. Temamogullari and Kanici (2014) also experienced relatively low mean extraction

recovery (60%) using MeOH. Karaseva et al. (2014) extracted AFM1 from cheese using

QuEChERS extraction method achieving a 72% recovery.

Table 4.2 Sample preparation involving extraction and clean-up of AFM1 from milk products

Extraction AFM1 Recovery Clean-up Samples Reference Method Spike (ng/g) (%) Method 200 g hard cheese DCM and 0.25-0.45 81-92 SPE Cavaliere et al., 2006 50 g med. cheese acetone 200 g hard cheese 10% MeOH 0.25-0.45 79-84 SPE Cavaliere et al., 2006 50 g med. cheese @ 150°C 22 mL water/ 8 g cheese 0.10-0.52 71 IAC Iha et al., 2011 13 mL MEOH QuEChERS 5 g cheese NR 72 SPE Karaseva et al., 2014 with LLME 2 g white cheese 40 mL DCM 0.01-0.08 102 centrifuge Kav et al, 2011 2 g white cheese 40 mL DCM NR NA NR Kocasari et al., 2012 80 mL DCM 10 g cheese 0.02-0.05 92-108 IAC Martins et al., 2007 7 g DME Temamogullari and 1 g cheese 5 mL MeOH 0.05 60 centrifuge Kanici, 2014 2 g yogurt 40 mL DCM NR NR centrifuge El Khoury et al., 2011 22 mL water/ 8 g yogurt 0.07-0.26 76 IAC Iha et al., 2011 13 mL MEOH 80°C/ yogurt NR NR NA Kocasari et al., 2012 pasteurization 80°C/ yogurt NR NR None Akkaya et al., 2006 pasteurization 80°C/ yogurt NR 100-120 None Gürbay et al., 2006 pasteurization 80 mL DCM 10 g yogurt 0.025-0.2 75-96 IAC Iqbal and Asi, 2013 10 g DME 80 mL DCM 10 g butter 0.025-0.2 76-91 IAC Iqbal and Asi, 2013 10 g DME 5 g butter 70% MeOH NR NR IAC Kocasari et al., 2012 centrifuge/ 2 g ice cream NR NR None Kocasari et al., 2012 defatted centrifuge/ ice cream NR NR None Khoshnevis et al., 2012 defatted Abbreviations: med. (medium); DCM (dichloromethane); DME (diatomaceous earth); MeOH (methanol); NR (information not reported); IAC (immunoaffinity column); SPE (solid-phase extraction)

Many other milk products such as yogurt, butter, and ice cream may contain

AFM1. Akkaya et al. (2006) analyzed 177 yogurt samples for the presence of AFM1 from

Turkey markets. Yogurt samples were heated to 80°C for 3 min to inactivate live yogurt bacteria, cooled, diluted, homogenized, and analyzed. Gürbay et al. (2006) used the same method as Akkaya et al. (2006) achieving recoveries of AFM1 in spiked yogurt samples

of 100 to 120%. Iqbal and Asi (2013) investigated the incidences and occurrences of

AFM1 in yogurt and butter. Samples were extracted with 10 g diatomaceous earth in

80 mL of DCM and passed through an IA column. Precision and recovery assays were

carried out on yogurt and butter samples fortified with AFM1 at concentrations of 0.025-

0.2 µg/L. The recoveries of AFM1 were 75-96% in yogurt and 76-91% in butter samples

with RSDs of 12-26%, and 11-22%, respectively. Authors (Table 3.2) have used other

methods for the extraction of AFM1 from dairy products (El Khoury et al., 2011; Iha et al., 2011; Khoshnevis et al., 2012; Kocasari et al., 2012).

AFM1 can be extracted from milk with SPE or IA. Extractions with LLE followed

by SPE or IA columns as a clean-up procedure have also been utilized. Manetta et al.

(2005) extracted AFM1 from milk samples using SPE obtaining average recoveries of spiked samples (0.025-0.075 µg/kg AFM1) of 89-90% (RSDs 1.5-2.5%). Wang et al.

(2012) compared the extraction efficiencies of SPE (OASIS HLB) and IA columns. The average recoveries of 0.05 µg/kg AFM1 in raw milk with OASIS HLB and IA were

96.9% (RSD 2.1%) and 92.6% (RSD 4.7%), respectively. Sørensen and Elbaek (2005)

developed a chromatographic method for the determination of mycotoxins, including

AFM1, in bovine milk. The milk samples were extracted with ACN followed by clean-up

by SPE using Oasis HLB. Average recoveries of 0.02, 0.1, and 1.0 µg/kg of AFM1 in

milk were 98, 87, and 80%, respectively. Biancardi et al. (2013) compared LLE method

to the IA chromatography column based on the Official International Organization for

Standardization, ISO14501 (ISO 2007). They concluded that LLE was comparable to IA chromatography.

Although comparable to other methods, IA extraction is an expensive technique.

Therefore, LLE technique is recommended for the extraction of AFM1 from milk. ACN

or MeOH should be considered the best organic solvent for the extraction of AFM1 from

milk because of the acceptable recoveries and compatibility with the environment.

Additionally, ACN was found to have given the cleanest extracts due to milk proteins

precipitating in it (Wang et al., 2011). The best organic solvent to be considered for the

extraction of AFM1 from cheese is DCM.

Detection and quantitative techniques

A number of quantitative methods, extensively reviewed annually by Shephard

(2008 and 2009), Shephard et al. (2010, 2011, 2012, and 2013), and Berthiller et al.

(2014 and 2015), for the analysis of mycotoxins, including AFM1, have been previously

reported. A suitable analytical method for the analysis of AFM1 in milk and milk

products can be found in these previously published articles. For this reason, this section

will give a brief overview of parameters used in analytical techniques for the quantitation

of AFM1. Present methods may be classified into two main groups according to where

analysis is needed: quantitative and qualitative. One point that should be clear from this

short review of a very broad topic is that there are many methods available for the

analysis of AFM1 in milk and milk products.

The quantitative analytical methods for AFM1 include thin-layer chromatography

(TLC) and LC (liquid chromatography). TLC has been widely accepted because of its

simplicity of operation, ability to repeat detection and quantification, and cost 62

effectiveness of analysis as many samples can be analyzed on a single plate with low solvent usage (Fuchs et al., 2011). Some authors have analyzed AFM1-contaminated milk and milk products according to TLC official methods (method 980.21 and method

2000.08) approved by AOAC International (AOAC, 2005) achieving a limit of detection

(LOD) as low as 0.01 μg/kg (Fallah, 2010b; Kafle et al., 2014). Yet with the development

of LC, most laboratories abandoned the use of TLC for the analysis of AFM1. Biancardi

et al. (2013) used HPLC-MS/MS (high performance liquid chromatography-tandem mass

spectrometry) equipped with an electrospray ionization (ESI) following LLE for the

analysis of AFM1 in milk. The limit of quantitation (LOQ) was 0.015 µg/kg. The major

advantages of the HPLC-MS/MS are the separation capability of the HPLC coupled to

the sensitivity (improved detection limits, ng to fg) and specificity (high resolution of

compounds) of the MS. The limitations include the complexity for novices, the expense

of the instrument, and large quantities of organic solvents. Therefore, HPLC-MS/MS may

not be practical for most routine analyses (Yao, et al., 2015). The most commonly found detection coupled to HPLC for the quantitation of AFM1 is fluorescence detectors

(HPLC-FLD), which rely on the presence of a chromophore in the molecules. AFM1 has

a natural fluorescence and can be detected directly by HPLC-FLD. HPLC-FLD following

IA column clean-up was adopted as a standard method by ISO, IDF (International Dairy

Federation) and AOAC International (ISO, 2007; IDF, 1995; AOAC, 2005). With FLD,

both excitation and emission fluorescence spectra help characterize compounds. Lee and

Lee (2015) identified AFM1 and aflatoxin M2 (AFM2) in dairy products (milk, yogurt,

milk powder, ice cream, and sherbet) utilizing wavelengths for excitation (360 nm) and

emission (450 nm). Trombete et al. (2014) analyzed 30 samples of grated Parmesan

cheese from Brazilian markets. Samples were extracted, passed through IA column for clean-up, and analyzed using HPLC-FLD. The excitation and emission were 360 and 430 nm, respectively. The LOD and LOQ were 0.02 and 0.05 µg/kg, respectively. Manetta et

al. (2005) determined AFM1 in milk and cheese using HPLC-FLD. Excitation and

emission wavelengths were 353 and 423 nm, respectively. The LOD was 0.001 ng/g, 40-

fold lower than the maximum acceptable level for AFM1 for EU, and 400-fold lower than

the limit set by the FDA. Other researchers have also found success using HPLC-FLD for

determining AFM1 in milk (Andrade, et al., 2013, Elgerbi et al., 2004; Navas et al.,

2005).

Recent developments have been made in the field of mass spectrometry for

quantifying AFM1. Busman et al. (2015) described the application of DART-MS (direct

analysis in real time-mass spectrometry) for the quantification of AFM1 in milk which

enables utilization of ambient desorption ionization techniques. The DART technique

relies upon excited-state helium atoms to produce reactive species leading to analyte

ionization. The lowest calibration level for this method was 0.1 µg/kg.

ELISA is the most used analytical method to measure AFM1 in milk and dairy products (Shephard et al., 2013). The use of ELISA immunoassay can be justified by the fact that it is affordable, simple and easy to use, and does not need expensive equipment such as liquid chromatography. There are only a few chromatographic methods that have been developed for the determination of AFM1 in cheese due to the complexity of the

food matrix (Monaci et al., 2007). The predominant method for the analysis of AFM1 in

cheese is ELISA (Arast et al., 2012; Iha et al., 2011; Kav et al., 2011; Rubio et al., 2011).

ELISA has also been used for the analysis of AFM1 in other dairy products (Atasever et

al., 2010a; Jafarian-Dehkordi and Pourradi, 2013; Temamogullari and Kanici, 2014).

Still, a major disadvantage of ELISA includes potential cross-reactivity and dependence

on a specific matrix (Pittet, 2005). Nevertheless, Guan et al. (2011) developed an ultrasensitive and specific monoclonal antibody-based competitive ELISA for the high affinity of AFM1 and no cross-reactivity to aflatoxin B1, B2, G1, and G2. The LOD was

3 ng/L for milk, the RSD was less than 10%, and the recovery ranged from 91% to 110%.

Li et al., (2015) developed an indirect competitive chemiluminescent enzyme

immunoassay for determining AFM1 in milk products. A luminol-hydrogen peroxide chemiluminescence system catalyzed by horseradish peroxidase was used as the signal detecting system. The LOD was 0.01 ng/mL and the linear range was 0.018 to 0.13 ng/mL. The recoveries ranged from 71.9 to 109%. The authors found a good correlation with the commercial available ELISA kit for AFM1 (r = 0.9978), indicating that the

developed chemiluminescence system can be used to determine AFM1 in real samples.

Vdovenko et al. (2014) developed an ultra-sensitive direct chemiluminescent enzyme

immunoassay for the determination of AFM1 in milk. To improve the sensitivity of the

assay, a mixture of 3-(10’-phenothiazinyl)-propane-1-sulfonate and 4- morpholinopyridine (MORPH) was used to enhance peroxidase-induced chemiluminescence. The LOD and dynamic working range were 0.001 ng/mL and 0.002–

0.0075 ng/mL, respectively. Values of recovery were 81.5 to 117.6%.

Portable and disposable tools, dipstick and lateral flow immunoassays, can provide qualitative determination of the presence or absence of AFM1 in large sample

sizes (Anfossi et al., 2013; Liu et al., 2015; Reybroeck et al., 2014). Advantages of the

dipstick and lateral flow immunoassays are their compactness, portability, and ease of

use. Disadvantages include further analysis of qualitative testing, a subjective interpretation, and a higher cost per test compared to ELISA (Pittet, 2005).

The quantitative analytical methods reviewed here can generally provide results with a high level of accuracy based on recovery for extracted AFM1 samples. The FLD is

the most common detector coupled to the HPLC for AFM1 analysis. It has been shown to

be highly accurate reaching detection limits as low as 0.001 µg/kg (Manetta et al., 2005).

Additionally, the determination of AFM1 in milk is internationally recognized as an

official method. Therefore, the most sensitive and recommended method for the

quantification of AFM1 in milk is the HPLC-FLD. The suggested method for the analysis of AFM1 in cheese is ELISA. While qualitative analysis can provide potential for

screening samples contaminated with AFM1, a major disadvantage for qualitative analysis is the inability of achieving quantifiable results. Yao et al. (2015) suggested an integrated approach which incorporates rapid sample screening techniques with analytical methods for the detection of aflatoxins in consumables.

Occurrence

This review reports the published occurrence of AFM1 in milk and milk products

from 2010-2015 (Tables 4.3-4.6). Between 2010 and 2015, an estimated total of 7,841

milk samples including raw, pasteurized, powdered, UHT (ultra-high temperature whole

milk), and breast milk were analyzed for AFM1 contamination in different countries. Of the 7,841 samples, 5,873 (75%) were positive for AFM1 contamination, 26% (2,042

samples) exceeded the maximum residue level (MRL) of 0.05 µg/kg for AFM1 as defined

by the European Union (EU) and 1.53% (120 samples) exceed the MRL of 0.5 µg/kg for

AFM1 as defined by the United States Food and Drug Administration (European 66

Commission, 2010; FDA, 2005). In the current review, the total milk products including cheese, yogurt, butter, and ice cream was 3,100 with 59% (1,828) of the milk products positive for AFM1 (Amr Amer and Ibrahim, 2010; Ashraf, 2012; Barjesteh et al., 2010;

Khoshnevis et al., 2012; Öztürk et al., 2014; Tavakoli et al., 2012; Trombete et al.,

2014). Flores-Flores et al. (2015) reviewed the presence of AFM1 in animal milk from

2001 to 2014. The authors reported 9.8% (2,190 samples) of the 22,189 milk samples

were contaminated above 0.05 µg/kg AFM1 and suggests that percentage of samples

could be higher as many surveys reported only the number of samples exceeding the

United States Food and Drug Administration (FDA) regulations for 0.5 µg/kg AFM1

(FDA, 2005).

Between 2010 and 2015, the occurrence of AFM1 in Africa was scarcely reported

(Table 4.3) with surveys only from Egypt, Morocco, Sudan, and Tanzania (Ali et al.,

2014; Amr Amer and Ibrahim, 2010; El Marnissi et al., 2012; El-Tras et al., 2011;

Elzupir and Elhussein, 2010; Magoha et al., 2014). The total number of milk samples was

582 with 405 (70%) of the total positive for AFM1. There were 270 (46%) samples that

exceeded the EU regulatory limit and 68 (11.7%) samples that exceeded the FDA

regulatory limit. The highest AFM1 concentration was found in raw milk collected from

dairy farms in Sudan at a concentration of 6.9 µg/kg (Elzupir and Elhussein, 2010). The

high levels were attributed to the alarmingly high AFB1-contaminated feed as a result of

poor manufacturing and storage practices. The authors suggest that government

authorities must educate stakeholders on the control of aflatoxins and the potential health

consequences they may cause. It should also be noted that Sudan does not impose

regulatory limits on milk or milk products.

Table 4.3 Occurrence of AFM1 in milk between 2010 and 2015 in Africa.

EU FDA Total Positive Range >0.05 >0.5 Origin Sample Analysis Reference (n) n (%) (μg/kg) μg/kg μg/kg n (%) n (%) RM 35 35 (100) 0.10-2.52 35 (100) 27 (77) Ali et al., Sudan1 ELISA POWM 12 12 (100) 0.01-0.85 5 (42) 4 (33) 2014 Amr Amer Egypt1 RM 50 19 (38) 0.02-0.07 10 (20) None ELISA and Ibrahim, 2010 HPLC- El Marnissi et Morocco* RM 48 13 (27) 0.01-0.10 4 (8.3) None FLD al., 2012 IPM 125 54 (43) 0.00-0.02 0 (0) El-Tras et al., Egypt1 None ELISA BM 125 87 (70) 0.01-0.33 65 (52) 2011 Elzupir and HPLC- Sudan1 RM 44 42 (96) 0.22-6.90 42 (100) 35 (83) Elhussein, FLD 2010 143 Magoha et Tanzania1 BM 143 0.01-0.55 109 (76) 2 (1) HPLC (100) al., 2014 Abbreviations: RM (raw milk); POWM (powdered milk); IPM (infant powdered milk); BM (breast milk); ELISA (enzyme-linked immunosorbent assay); HPLC (high performance liquid chromatography); FLD fluorescence detector 1 * No regulation established for that country; Limit of 0.05 µg/kg AFM1 Note: A value of 0.00 µg/kg AFM1 indicates less than 0.005 µg/kg AFM1, but does not suggest non-detectable levels.

Between 2010 and 2015, the occurrence of AFM1 in the Americas has also been

scarcely reported (Table 4.4). The review has only captured data from Argentina,

Ecuador, Mexico, and Brazil (Alonso et al., 2010; Bahamón, 2014; Gutiérrez et al., 2013;

Iha et al., 2013; Picinin et al., 2013; Santos et al., 2014; Silva et al., 2015). Canada the number 7 exporter of maize and United States, the main exporter of maize (Wu, 2012), do not have AFM1 surveys reported in literature between 2010 and 2015. There was a

total of 641 milk samples evaluated for AFM1 in the Americas, of which 412 (64.3%)

were contaminated with AFM1. There were 72 (11.2%) samples that exceeded the EU

regulatory limit and 14 (2.2%) samples that exceeded the FDA regulatory limit. The

highest AFM1 contamination of 7.7 µg/kg in organic raw milk originated from Mexico in

October 2008 during the rainy season (Gutiérrez et al., 2013). Concurrently, it has been 68

previously reported that AFM1 contamination is the highest during the rainy season in the

fall (Ruangwises and Ruangwises, 2009). Since harvesting is mainly in the rainy season,

maize drying is difficult during this period resulting in an increase in aflatoxin levels

(Prawat Tanboon-ek, 1991).

Table 4.4 Occurrence of AFM1 in milk between 2010 and 2015 in the Americas.

EU FDA Total Positive Range >0.05 >0.5 Origin Sample Analysis Reference (n) n (%) (μg/kg) μg/kg μg/kg n (%) n (%) HPLC- Alonso et al., Argentina** RM 94 60 (64) nd-0.07 10 (11) None MS/MS 2010 HPLC- Bahamón, Ecuador1 BM 78 9 (11.5) 0.01-0.04 None None FLD 2014 RM 12 10 (83) 0.03-3.81 8 (67) 6 (50) HPLC- Gutiérrez et Mexico** ORM 11 7 (64) 0.23-7.66 7 (64) 6 (54) FLD al., 2013 UHT 17 13 (76) 0.01-0.22 6 (35) 0 (0) PM 30 26 (87) 0.01-0.44 18 (60) 0 (0) HPLC- Iha et al., Brazil** POWM 12 12 (100) 0.02-0.76 NR 2 (17)+ FLD 2013 RM+ 17 13 (76) 0.01-0.06 1 (6) 0 (0) IPM 7 None None None 0 (0) 129 Picinin et al., Brazil** RM 129 0.05-0.11 18 (14) None ELISA (100) 2013 Santos et al., Brazil** PM 82 None None None None ELISA 2014 133 HPLC- Silva et al., Brazil** UHT 152 0.00-0.12 4 (2.6) None (87.5) FLD 2015 Abbreviations: RM (raw milk); BM (breast milk); ORM (organic raw milk); UHT (ultra- high temperature pasteurized whole milk); PM (pasteurized milk); POWM (powdered milk); RM+ (raw milk + additives); IPM (infant powdered milk); HPLC (high performance liquid chromatography); MS/MS (tandem mass spectrometry); FLD fluorescence detector; ELISA (enzyme-linked immunosorbent assay); ND (non- detectable) 1 ** No regulation established for that country; Limit of 0.5 µg/kg AFM1 established for + that country; Limit of 5 µg/kg AFM1 established for that country Note: A value of 0.00 µg/kg AFM1 indicates less than 0.005 µg/kg AFM1, but does not suggest non-detectable levels.

In this review of Asian countries between 2010 and 2015 (Table 4.5), there was a

total of 5,577 milk samples. Positive AFM1 milk samples were found in 4,227 (76%).

There were 1,575 (28.2%) samples that exceeded the EU regulatory limit (20.1% of the 69

total) and 27 (0.5%) samples that exceeded the FDA regulatory limit (0.34% of the total).

China reported 402 out of 890 positive samples (45%) with 162 (18.2%) exceeding the

EU limit and 2 (0.2%) exceeding the FDA limit. (Guan et al., 2011; Guo et al., 2013;

Han et al., 2013; Huang et al., 2014; Wang, et al., 2010; Wang, et al., 2011; Wang, et al.,

2012; Xiong et al., 2013; Zhang et al., 2012; Zheng et al., 2013). Of the 3,140 milk

samples obtained from Iran, 2,806 (89.3%) samples were contaminated and 966 (31%)

exceeded the EU limit and 5 (0.16%) exceeded the FDA limit (Arast et al., 2012; Fallah,

2010a; Fallah, 2010b; Garmakhany et al., 2011; Jafarian-Dehkordi and Pourradi, 2013;

Khosravi et al., 2013; Nemati et al., 2010; Rafiei et al., 2014; Sani et al., 2012; Sefidgar

et al., 2011; Vagef and Mahmoudi, 2013). Turkey reported 495 samples of which 272

(55%) were contaminated and 58 (11.7%) exceeded the EU regulatory limit and only 1

sample (0.2%) exceeded the FDA limit (Aksoy et al., 2010; Atasever et al., 2010a;

Atasever et al., 2010b; Kara and Ince, 2014; Kocasari et al., 2012; Temamogullari and

Kanici, 2014). Other reports were obtained from Jordan (Omar, 2012), Lebanon (Hassan

and Kassaify, 2014), Pakistan (Iqbal and Asi, 2013), and Thailand (Ruangwises and

Ruangwises, 2010; Suriyasathaporn and Nakprasert, 2012). The maximum AFM1 contaminated milk (3.8 µg/kg) in Asia was found in India in both raw and pasteurized milk (Siddappa et al., 2012).

Table 4.5 Occurrence of AFM1 in milk between 2010 and 2015 in Asia.

EU FDA Total Positive Range >0.05 >0.5 Origin Sample Analysis Reference (n) n (%) (μg/kg) μg/kg μg/kg n (%) n (%) RM 15 14 (93.3) 0.00-0.28 7 (47) China** None ELISA Guan et al., 2011 POWM 9 7 (77.7) 0.01-0.22 1 (11) China** RM 233 112 (48) NR-0.095 48 (21) None ELISA Guo et al., 2013 China** RM 200 65 (32.5) 0.01-0.06 3 (1.5) None ELISA Han et al., 2013 RM 30 24 (80) NR-0.237 NR UHPLC- China** LM 12 4 (33.3) NR-0.046 None None Huang et al., 2014 MS/MS POWM 8 2 (25) NR-0.022 None PM HPLC- China** 50 15 (30) 0.01-0.25 NR None Wang, et al., 2010 UHT MS/MS PM HPLC- China** 50 3 (6) 0.01-0.25 NR None Wang, et al., 2011 UHT MS/MS RM 4 1 (25) 0.07 1 (25) PM 5 2 (40) 0.05-0.09 1 (20) HPLC- China** None Wang, et al., 2012 UHT 6 1 (16.7) 0.13 1(16.7) FLD POWM 16 4 (25) 0.09-0.21 4 (25) China** RM 72 43 (59.7) 0.01-0.42 34 (47) None ELISA Xiong et al., 2013 lateral China** PM 27 21 (77.8) NR NR 2 (7.4) Zhang et al., 2012 flow China** UHT 153 84 (54.9) 0.01-0.20 62 (41) None ELISA Zheng et al., 2013 Iran* PM 75 75 (100) 0.01-0.06 3 (4) None ELISA Arast et al., 2012 PM 116 83 (71.5) 0.01-0.53 31 (27) 2 (1.7) Iran* ELISA Fallah, 2010a UHT 109 68 (62.3) 0.01-0.52 19 (17) 3 (2.8) Iran* PM 91 66 (72.5) 0.01-0.25 33 (36) None TLC Fallah, 2010b PM 37 32 (86.5) 0.00-0.11 6 (16) HPLC- Garmakhany et al., Iran* None RM 37 31 (83.8) 0.00-0.17 2 (5.4) FLD 2011 Jafarian-Dehkordi Iran* BM 80 1 (1.25) 0.01 0 (0) 0 (0) ELISA and Pourradi, 2013 2160 722 Khosravi et al., Iran* RM 2160 0.00-0.15 None ELISA (100) (33) 2013 RM Iran* SM 90 90 (100) 0.00-0.09 30 (33) None ELISA Nemati et al., 2010 PM 0.00- Iran* BM 87 24 (27.6) None None ELISA Rafiei et al., 2014 0.005 Iran* PM 42 41 (97.6) 0.01-0.07 3 (1.6) None ELISA Sani et al., 2012 72 Iran* PM 72 72 (100) NR NR ELISA Sefidgar et al., 2011 (100) RMF 54 34 (63) 0.06-0.09 31 (57) Vagef and Iran* RMI 48 19 (39.6) 0.05-0.06 8 (17) None ELISA Mahmoudi, 2013 PM 42 10 (23.8) 0.05-0.06 6 (14) RM 45 45 (100) 0.10-3.80 22 (49) 6 (13) HPLC- Siddappa et al., India** UHT 45 29 (64.4) 0.06-2.10 29 (64) 10 (22) FLD 2012 PM 7 3 (42.9) 1.8-3.80 3 (43) 3 (43) 100 70 Jordan1 RM 100 0.07-2.03 NR ELISA Omar, 2012 (100) (100) 103 Hassan and Lebanon1 DP 388 254 (65) NR NR ELISA (26) Kassaify, 2014

Table 4.5 (Continued)

HPLC- Pakistan1 LM 107 76 (71) 0.00-0.85 44 (41) NR Iqbal and Asi, 2013 FLD 240 HPLC- Ruangwises and Thailand1 RM 240 0.01-0.20 85 (35) None (100) FLD Ruangwises, 2010 HPLC- Suriyasathaporn and Thailand1 PM 120 NR 0.00-0.29 33 (28) None FLD Nakprasert, 2012 HPLC- Turkey* RM 36 22 (61) NR None None Aksoy et al., 2010 MS/MS Atasever et al., Turkey* UHT 150 89 (59) 0.01-0.19 16 (11) None ELISA 2010b HPLC- Turkey* RM 124 None 0.01-0.03 None None Kara and Ince, 2014 MS/MS RM 45 41 (91) 0.02-0.08 16 (36) Turkey* None ELISA Kocasari et al., 2012 PM 45 30 (67) 0.01-0.06 2 (4.4) Turkey** POWM 45 42 (93) 0.06-0.80 NR 1 (2.2) ELISA Kocasari et al., 2012 RM 38 36 (95) 0.08-0.13 21 (55) None Temamogullari and Turkey* ELISA UHT 12 12 (100) 0.02-0.09 3 (25) None Kanici, 2014 Abbreviations: RM (raw milk); POWM (powdered milk); LM (liquid milk); PM (pasteurized milk); UHT (ultra-high temperature pasteurized milk); BM (breast milk); SM (sterilized milk); RMF (raw milk farm); RMI (raw milk industrialized); DP (dairy products); ); HPLC (high performance liquid chromatography); MS/MS (tandem mass spectrometry); FLD fluorescence detector; ELISA (enzyme-linked immunosorbent assay); TLC (thin-layer chromatography); NR (not reported); ND (non-detectable) 1 * No regulation established for that country; Limit of 0.05 µg/kg AFM1 established for ** that country; Limit of 0.5 µg/kg AFM1 established for that country Note: A value of 0.00 µg/kg AFM1 indicates less than 0.005 µg/kg AFM1, but does not suggest non-detectable levels.

Between 2010 and 2015, the occurrence of AFM1 in Europe has also been

reported (Table 4.6). The review collected data from Croatia, Spain, Serbia, Italy, and

Greece (Bilandžić et al., 2010; Cano-Sancho et al., 2010; Kos et al., 2014; Rubio et al.,

2011; Santini et al., 2013; Tsakiris et al., 2013). There was a total of 1,041 milk samples, of which 829 (79.6%) were contaminated with AFM1. There were 125 (12.0%) samples that exceeded the EU regulatory limit and 11 (1.1%) samples that exceeded the FDA regulatory limit. The highest AFM1 contamination of 1.2 µg/kg in pasteurized milk came

from a farm in Serbia (Kos et al., 2014). The authors suggest that hot and dry weather

conditions during the growing season could be the possible reason for high contamination

frequency of AFM1-contaminated milk in Serbia. There was a prolonged drought period during the maize growing season in 2012, promising for Aspergillus growth and aflatoxin

production. Consequently, maize was the majority of the animal feed and the authors

proposed that there was a great possibility of AFM1 appearance in milk and milk products. It should be noted that there is no published data from Europe regarding occurrence of AFM1 in milk from 2012 to 2013 period, which was characterized with

advantageous weather conditions for Aspergillus species growth and aflatoxin

production, especially in Southeast Europe (Kos et al., 2014).

Table 4.6 Occurrence of AFM1 in milk between 2010 and 2015 in Europe.

EU FDA Total Positive Range >0.05 >0.5 Origin Sample Analysis Reference (n) n (%) (μg/kg) μg/kg μg/kg n (%) n (%) Bilandžić et Croatia** RM 61 NR 0.01-0.06 1 (1.6) None ELISA al., 2010 Cano-Sancho Spain* UHT 72 68 (94.4) NR None None ELISA et al., 2010 PMI 22 22 (100) 0.06-0.70 21 (95.5) 1 (4.5) UHT 69 69 (100) 0.02-0.41 63 (91.3) None Kos et al., Serbia** OMI 6 6 (100) 0.01-0.08 1 (16.7) None ELISA 2014 PMF 13 13 (100) 0.08-1.20 8 (61.5) 5 (38.5) RM 40 40 (100) 0.005-0.9 25 (62.5) 5 (12.5) RM 407 404 (99) 0.00-0.08 3 (0.73) None Rubio et al., Spain* ELISA SM 82 81 (99) 0.00-0.13 1 (1.2) None 2011 HPLC- Santini et al., Italy* RM 73 35 (48) NR-0.02 None None FLD 2013 Tsakiris et al., Greece* RM 196 91 (46.5) NR 2 (1) None ELISA 2013 Abbreviations: RM (raw milk); (UHT) ultra-high temperature pasteurized milk; PMI (pasteurized milk industrialize); PMF (pasteurized milk farm); OMI (organic milk industrialized); SM (silo milk) ; ELISA (enzyme-linked immunosorbent assay); HPLC (high performance liquid chromatography); FLD fluorescence detector; NR (not reported) * ** Limit of 0.05 µg/kg AFM1 established for that country; Limit of 0.5 µg/kg AFM1 established for that country Note: A value of 0.00 µg/kg AFM1 indicates less than 0.005 µg/kg AFM1, but does not suggest non-detectable levels.

Weather conditions (temperature, humidity, etc.) and agricultural practices have been proven to have a drastic effect on the influence of AFM1 in milk. Therefore, future

studies that follow agricultural practices such as post-harvest storage conditions and

climate trends could help alleviate food security concerns particularly in developing

countries where regulation is limited.

Prevention and Control of AFM1

The most effective way of preventing AFM1 in the food supply is to reduce

contamination of AFB1 in animal feed. Preventive measures must be applied to reduce fungal growth and subsequently, AFB1 production in agricultural commodities intended

for dairy feed. A very attractive area for the prevention of aflatoxin in maize, and thus

AFM1, is the biological control method involving field inoculations with atoxigenic

strains of A. flavus. A number of relatively recent articles focus on the strategic

suppression of toxigenic strains and the decrease production of aflatoxin in maize

(Atehnkeng et al., 2014; Shan and Williams, 2014). Hruska et al. (2014) tracked

colonization of aflatoxigenic A. flavus strain AF70 with green fluorescent protein (GFP) in the presence of atoxigenic AF36 to better understand the competitive interaction between the two strains. The AF70-GFP inside the kernel was suppressed 82% when co-

inoculated with AF36. The growth suppression of AF70-GFP corresponded to 73%

suppression of aflatoxin production. Scarpari et al. (2014) studied the oxidative stress pathway and oxylipin (produced by the host during defense reaction) as it relates to the process of host-recognition and the pathogenic phase in A. flavus. The oxylipin profile of

the wild type strain and three mutants of A. flavus were deleted at the Aflox1 gene level

during maize kernel invasion. The results showed the difference between the oxylipin 74

profiles of the wild type and mutants highlighting the importance of maize oxylipin in driving secondary metabolism in A. flavus.

The most critical environmental factors determining whether a substrate will support fungal growth during storage are moisture content, temperature, and storage time.

Thus, drying, proper storage, and suitable transportation are of prime importance in prevention (Boutrif, 1998). Villers (2014) discussed the use of safe storage of agricultural crops using ultra-hermetic airtight containers used in over 100 countries. Williams et al.

(2014) determined if storage of maize in Purdue Improved Crop Storage (PICS) bags prevented fungal growth and aflatoxin accumulation. PICS bags are a three-layer, hermitic bag-system that forms a barrier against the influx of oxygen and the escape of carbon dioxide. A. flavus growth and aflatoxin accumulation were not observed in maize

stored in any PICS bags after 1 and 2 months incubation. There was no AFB1 detected in

woven bags containing low-moisture maize (12 and 15%), but detectable levels of

aflatoxin were observed in high moisture maize (18 and 21%). Villers suggested that

when airtight, this unit drastically inhibited fungal growth since fungi need oxygen and

high humidity. However, much scientific work is needed to invest long-term storage in

this container.

Another preventative measure to reduce AFM1 from milk involves the addition of

sequestering binders to AFB1-contaminated feed. Some sequestering agents include

indigestible adsorbent materials such as silicates, activated carbons, and complex

carbohydrates among others. These binders adsorb AFB1 and the bound aflatoxin can be

expelled via fecal material. If the binder adsorbs AFB1, then it theoretically cannot be

metabolized into AFM1 (Kutz et al., 2009). Although the FDA has not, yet, been

approved the use of binding agents to remove AFM1 from milk, this method does offer

some insight for the reduction of AFM1 in the future.

Regulation of AFM1 by strict governmental control is the only accepted method

for the control of AFM1 in milk and milk products. As of 2003, there were over 60

countries that had established regulatory limits for AFM1 ranging from not detectable to

15 µg/kg (FAO, 2003). The main limits enforced are 0.05 and 0.5 µg/kg adopted in 34

and 22 countries, respectively. The ten-fold discrepancies between the limits have

become the subject of debates within Codex Alimentarius, leading to the request to re-

evaluate the human health risk of AFM1 (Van Egmond and Jonker, 2000). The

establishment of a maximum allowable concentration of AFM1 may help to create and

enforce uniform regulatory guidelines for AFM1 reduction worldwide. The EU has imposed a limit of 5 µg/kg AFB1 for dairy feed. This level has, in turn, resulted in further

stringent regulation of AFM1 in milk at 0.05 µg/kg (European Commission, 2010).

Because quantitative carryover of AFB1 to AFM1 has been reported up to 6.2%, lactating cows that consume AFB1-contaminated feed containing 20 µg/kg (maximum allowable limit in feed) or greater may produce AFM1-contaminated milk that exceeds the FDA

maximum allowable limit of 0.5 µg/kg AFM1 (Diaz et al., 2004; Vardon, 2003). Other

countries have also set AFM1 limits at this action level including Asian and Latin

America countries (Alonso et al., 2010; Han et al., 2013; Huang et al., 2014). Still, there are many countries that lack regulatory limits for the control of AFM1 in milk and milk

products. Despite the regulatory control measures taken by some countries, the

production of aflatoxin-free milk is almost impossible to achieve (Nachtmann et al.,

2007) and other control methods may be beneficial.

Studies have shown that there have been no significant changes in AFM1

concentration after heat processing such as with pasteurization and sterilization (Jasutiene

et al., 2007). Still, little research has been done on the addition of sequestering binders to

milk for the removal of AFM1. Soha et al. (2006) studied the reduction of AFM1 in

naturally contaminated milk exceeding 0.05 µg/kg by the addition of hydrated sodium

calcium aluminosilicate (HSCAS) and bentonite at 0.5 and 2%. Results indicated that

bentonite reduced AFM1 levels by 90% at both the 0.5 and 2% levels and HSCAS obtained 47.7 and 77.8% reductions at 0.5 and 2%, respectively. This study showed that utilization of chemisorption compounds directly in milk had a high efficiency for the removal of AFM1 from milk. An alternative approach to removing aflatoxin is the use of

biological transforming agents (Bovo, et al., 2013; Elsanhoty et al., 2014; Montaseri et

al., 2014). El Khoury et al. (2011) investigated the binding ability of AFM1 by lactic acid

bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus) from contaminated

milk. The AFM1-contaminated milk (0.05 µg/kg) was inoculated with pure culture of the

Lactobacillus bulgaricus and Streptococcus thermophiles. AFM1 binding by

Lactobacillus bulgaricus for 2 and 6 hours after inoculation was 46.1 and 58.5%,

respectively. AFM1 binding by Streptococcus thermophiles for 2 and 6 hours after

inoculation was 22.6 and 37.7%, respectively. Dehydroacetic acid (DHA), a toxic

antimicrobial agent, has been evaluated as a means to reduce AFM1 from raw milk.

Duraković et al. (2012) investigated the reduction of AFM1 in raw milk using a new

synthesized analogue of DHA, 3-/2-aminophenylimino-(p-toluoyl)/-4-hydroxy-6-(p- tolyl)-2H-pyrane-2-one, a Schiff base. The toxicity of the Schiff base was evaluated by using brine shrimp (Artemia salina) larvae as a screening system for the determination of

its sensitivity to some chemical agents. At pH 5.5, 0.1 µmol/L of the Schiff base resulted in the 55% reduction AFM1 over a period of 35 days. However, at pH 6.5 the most

effective concentration for the reduction AFM1 was 0.5 µmol/L. The Schiff base was not

effective at pH value of 7 or higher. The authors concluded that the ability of Schiff base

to act as anti-aflatoxigenic agent provides new perspective for the control of AFM1

contamination in milk.

Conclusion

AFM1 in milk and milk products is hazardous to human health. The high

occurrence of AFM1, detected primarily by HPLC-FLD and ELISA, in the large majority

of milk and milk products discussed in this review, confirms the potential risk associated

with the presence of this toxic compound in food products. For this reason, it is extremely

important to maintain low levels of AFM1. The deterrence of AFB1-contaminated feed

consumed by dairy cattle has proven to be the most successful system of the prevention

of AFM1 in human food supply. Additionally, several strategies have been suggested in

order to decontaminate AFM1 in milk. Though the one accepted method for the control of

AFM1 is governmental regulation.

THE EVALUATION OF ADSORBENTS FOR THE REMOVAL OF AFLATOXIN M1

FROM CONTAMINATED MILK

Abstract

There is an increasing awareness of the hazards aflatoxin M1 (AFM1) pose by its presence in milk and milk products. A number of approaches, such as physical removal procedures, have been used to counteract this toxin including the adsorption of dietary sequestering agents of dairy feed contaminated with aflatoxin B1 (AFB1), the

carcinogenic parent compound of AFM1. This method has seen variable successes but,

still AFM1 residues may remain in milk after this remediation process. Another approach

to thwart AFM1 contamination is the direct addition of a sequestering agent to contaminated milk. This method is scant in the literature; and therefore, it is the subject

of this chapter.

A crossed, nested design of 3 factors, factor A-milk type (raw, skim, or whole

milk); factor B-sequestering binder (powdered activated carbon (PAC), Biofix, MTB-

100, and Mycofix); and factor C-binder concentration (0.1, 0.25, and 0.4% for PAC and

2.0, 3.0, and 4.0% for Biofix, MTB-100, and Mycofix), was performed for the removal of spiked 0.5 ng/mL AFM1. It was found that 0.25% and 0.4% PAC applied to skim milk

had the greatest reduction of 0.46 ng/mL AFM1. The mean AFM1 concentrations were

0.061 ng/mL (87% reduction) and 0.046 ng/mL AFM1 (90% reduction), respectively. The 79

highest percent reduction of all industrial sorbents was obtained with 4.0% Mycofix applied to whole milk with a reduction of 34% (0.358 ng/mL) from 0.54 ng/mL AFM1.

There was no statistical difference with the reduction of 0.56 ng/mL AFM1 in raw milk of

0.1 and 0.4% PAC obtaining percent reductions of 56.9% (0.24 ng/mL) and 57.4% (0.24

ng/mL), respectively. Governing bodies have not officially approved the use of such

binders for the removal of AFM1 from milk. However, the possibility of PAC to make

AFM1-contaminated milk safe for human consumption is an encouraging avenue to

explore. This method should be considered for the removal of AFM1 from milk.

Introduction

Discovered over 50 years ago, aflatoxins have become recognized as ubiquitous

contaminants of animal feed and human food supply throughout the world. The acute and

chronic toxicity these compounds inflict include hepatocellular carcinoma,

immunosuppression, and even death. People chronically infected with hepatitis B virus

are at increased risk of liver cancer when exposed to aflatoxin, demonstrating that even

low levels of this toxin in the diet can pose serious health effects (Kensler et al., 2011).

Aflatoxin B1 (AFB1) is the most toxic compound to humans and animals. AFB1 can be

metabolized to aflatoxin M1 (AFM1), a hydroxylated derivative found in the milk of

lactating animals that also demonstrates toxic consequences. Current effective removal

methods for the reduction of AFM1 and one of the most hopeful tactics to minimize the

adverse effect of aflatoxin in food and feed include the use of sequestering agents to

exploit the binding affinity of AFB1 to the dietary binder (Phillips et al., 2008).

The highly variable metabolic conversion of AFB1 to AFM1 ranging from 0.5-5%

in humans and up to 6% in dairy cows, is usually considered to be a removal process as 80

the in vivo carcinogenicity of AFM1 is approximately 2-10% of AFB1 (Creppy, 2002;

Neal et al., 1998). Furthermore, in the presence of metabolic activation in vitro, AFM1

has also been shown to be 10% as mutagenic as AFB1 (Neal et al., 1998). Moreover,

AFM1 is heat stable and no reduction of toxin levels has been observed after

pasteurization or sterilization processes (Jasutiene et al., 2007). AFM1 has been identified

in raw and ultra-high temperature (UHT) whole milk and milk products including yogurt,

butter, cheese, and dried and cultured milk. Because AFM1 has been linked to casein

fraction in milk, AFM1 levels can be concentrated 3-5 folds higher in dairy products as a

result of processing (Barbiroli et al., 2007; Manetta et al., 2009). Due to the stability of

AFM1, various methods, including physical, have been developed for the remediation of

AFM1 (Mishra and Das, 2003; Piva et al., 1995).

Adsorbents such as silicates, activated carbons, and complex carbohydrates have

been studied for the reduction of AFM1 by reducing AFB1 availability in dietary feed.

Silicates are divided into subclasses according to their structure. One group belonging to

the silicate family is phyllosilicate. Hydrated sodium calcium aluminosilicate (HSCAS), a

type of phyllosilicate, is the most extensively studied of these materials. Research has

shown a 32-80% reduction of AFB1 when added to feed (Kutz et al., 2009; Phillips et al.,

1988; Shebl et al., 2010). HSCAS is thought to absorb aflatoxin selectively during the digestive process, rendering much of the aflatoxin unavailable for absorption from the gastrointestinal tract. Evidence suggests that aflatoxins may react at multiple sites on

HSCAS particles especially within the interlayers (Phillips et al., 1999). The

chemisorption of aflatoxin to HSCAS involves the formation of a complex by the β-keto-

lactone of aflatoxin with uncoordinated metal ions in HSCAS (Sarr et al., 1990). Other

silicates used for the removal of AFB1 in feed include bentonite, composed of

montmorillonite clay, and zeolite. Activated carbon (AC), also called activated charcoal,

is a common porous carbon material with a large surface area in excess of 1500 m2 that

enhances its adsorption properties. It can be powdered or granular in nature. The

preparation of AC can be achieved by using a physical or chemical activation process. In

physical activation, the generation of porosity usually takes place via selective

elimination of the more reactive carbon of the structure and further gasification. In the

chemical activation process, the precursor is mixed with a chemical such as ZnCl2 or

H3PO4, carbonized, and washed to produce the porous AC (Abdullah et al., 2001).

Because of the porosity AC exhibits, it has been shown to adsorb aflatoxins in aqueous

solutions (Jard et al., 2011). However AC, for the removal of AFB1 in feed, has been

shown to have variable results ranging from 5.4-76% reduction (Diaz et al., 2004; Rao and Chopra, 2001). Therefore, AC may not be as effective in binding aflatoxin from feed as silicates-based binders and more studies of decontamination of AC in contaminated feed are warranted. Complex indigestible carbohydrate polymers derived from yeast cell

walls have also been shown to be effective in binding aflatoxin and restoring

performance to animals consuming multiple mycotoxins (Whitlow, 2006). The United

States Food and Drug Administration (FDA) have not approved any adsorbent material

for the removal of aflatoxins. However, several of these adsorbent materials have been

recognized as safe feed additives under the GRAS Act and are used in diets for purposes

such as flow agents and pellet binders (Whitlow, 2006).

Physical removal or separation of aflatoxin-contaminated crops is an important

strategy for reducing aflatoxin levels. The practice of adding binders or sequestering

agents to feed has been studied to reduce toxicity by acting as an enterosorbent and reducing the bioavailability in intestinal absorption (Gallo et al., 2010; Phillips et al.,

2008). These dietary additives (inorganic silica-based compound, organic charcoal-based

polymer, or an indigestible carbohydrate) offer one of the greatest potential solutions for

preventing toxicity in a stable digestive tract where the bound aflatoxin can be excreted.

Research studies have demonstrated the utilization of activated carbon, glucomannan,

bentonite clay, and hydrated sodium calcium aluminosilicate (HSCAS) that effectively

reduced AFB1 toxicity in feed (Li et al., 2010; Kutz et al., 2009; Masoero et al., 2007;

Pasha et al., 2007; Shebl et al., 2010). However, not all AFB1 is bound, and some maybe

metabolized to AFM1 and excreted in the milk.

The blending of a food or feed containing a substance exceeding the FDA AL

with another food or feed is unlawful. As so, legal action may be taken to remove the

contaminated products from the market (FDA, 2000). To use AFM1-contaminated milk

(≥ 0.5 ng/mL) for the production of cheese (FDA AL 20 ng/mL AFM1) is also problematic as AFM1 has been linked to milk casein fraction. This could result in dairy

products contaminated at even higher AFM1 concentration than the original milk

(Barbiroli et al., 2007). Therefore, once the AL has been exceeded, the AFM1-

contaminated milk should be discarded (FDA, 2000). Instead of discarding an entire

batch of milk that has exceeded the FDA AL of AFM1, resulting in economic loss, a

reasonable method for reducing aflatoxin contamination would be the physical removal

of AFM1 from milk.

Although an attractive method, studies have shown residual AFM1 levels in milk

after application of adsorbents to aflatoxin-contaminated feed. Yet, the research done on

the addition of sequestering binders directly to milk for the removal of AFM1 residues is

limited (Soha et al., 2006). Thus, the application of sequestering binders directly to milk

to remediate AFM1 was studied in this chapter. This study seeks to demonstrate the

feasibility of using sequestering binders for the removal of artificially contaminated

AFM1 from milk. Additionally, we will determine if any of the sequestering binders

reduce 0.5 ng/mL AFM1 below the European Union maximum regulatory level of 0.05 ng/mL and the FDA action level (AL) of 0.5 ng/mL.

Materials and Methods

Milk preparation

Unpasteurized, raw milk, obtained from the dairy farm of Mississippi State

University’s Department of Animal and Dairy Science, was analyzed for AFM1 residue

prior to experiments being performed. Skim and raw milk were obtained from the local

grocery store. The milk, kept at 4°C for up to 5 days, was considered blank and was used

for subsequent studies. The blank milk was artificially spiked with 0.5 ng/mL AFM1

(Sigma-Aldrich).

Adsorbent materials

Several sequestering binders were examined for their proficiency to bind AFM1 directly from milk. Powdered activated carbon (PAC), Mycofix, Biofix, and MTB-100, were obtained from PhytoTechnology Laboratories (Oakland Park, KS, USA), Biomin

(Getzersdorf, Austria), Bios Agricorp (Helsinki, Finland), Alltech (Lexington, KY,

USA), and Norit (Alpharetta, GA, USA), respectively.

Sample Preparation

Four sequestering agents, PAC (0.1, 0.25, and 0.4%, w/v) and Mycofix

(bentonite), Biofix (nanoclay), and MTB-100 (an esterified glucomannan) each at concentrations of 2.0, 3.0, and 4.0% (w/v), respectively were used to determine the best adsorbent to remove spiked 0.5 ng/mL AFM1 from artificially contaminated raw, skim,

and whole milk. Blank milk, spiked with 0.5 ng/mL AFM1, was used for the controls.

Aliquots of the contaminated milk were applied to each of the 108 samples (n=3).

Treatments were homogenized with a Burrell Wrist Shaker (Figure 5.1) for 1 hour and

residual AFM1 was extracted and analyzed.

Figure 5.1 Burrell Wrist Action used for sample homogenization

A volume of 10 mL of the spiked milk (room temperature) was used for extraction. Acetonitrile (ACN), 15 mL, was added to each sample. Samples were vortexed in a Geno-Grinder (SPEX Sample Prep, Metuchen, NJ, USA) for 1 min at 1000 strokes/min. QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction salts, obtained from Agilent (Santa Clara, CA, USA) were added to each sample.

Samples were vigorously vortexed in the Geno-Grinder at 1000 strokes/min for 1 min and centrifuged for 15 min at 3200 g for separation. The organic supernatant was collected and filtered through 0.45 µm polytetrafluoroethylene (PTFE) syringe filters.

Extraction procedure is shown in Figure 5.2.

Figure 5.2 The extraction of a milk sample containing AFM1 and 0.4% powdered activated carbon (PAC) using QuEChERS extraction method.

A three-layer separation was obtained after extraction and centrifugation

Analytical instrument conditions

Samples were analyzed using the Agilent 6460 Triple Quadrupole Tandem Mass

Spectrometry (HPLC-MS/MS; Santa Clara, CA, USA). A volume of 5 μL was injected

onto a Zorbax StableBond-C18 2.1 x 50 mm, 1.8 µm column from Agilent (Santa Clara,

CA, USA) with a column temperature of 50°C. The mobile phases consisted of A: H2O

and B: MeOH both containing 5 mM ammonium acetate and 0.1% formic acid. An

isocratic gradient was 90% mobile phase A and 10% mobile phase B at a flow rate of 0.6

mL/min. The MS/MS, coupled to an electrospray ionization (ESI) operated in positive

mode parameters, was used to quantify AFM1 in samples. Data was acquired in multiple reaction monitoring (MRM) mode. The drying gas (N2) temperature was 325°C, and the desolvation gas flow rate was set to 10 L/min. The nebulizer gas pressure was set at 40 psi, and the capillary voltage was 4 kV. The sheath gas flow had an output of 12 L/min and reached a constant temperature of 400°C. The delta electron multiplier voltage

(EMV) was set to 400 V, and the dwell time lasted for 200 msec. Agilent MassHunter

Quantitative Analysis Workstation Software v. B.04.0.225.19 was used to analyze the quantitative data obtained from the samples and the calibration curve.

Linearity

Linearity was evaluated with three matrix-matched calibration curves for raw, skim and whole milk. The calibration curves were prepared by plotting the response of

the spiked sample against the expected AFM1 concentration. The calibration curves

ranged linearly from 0.039 to 2.5 ng/mL AFM1 and all curves exhibited a satisfactory linear correlation determined by the coefficient of determination, R2>0.99.

Statistical Analysis

The effect of AFM1 removal from 3 types of milk (raw, skim, and whole), 4 types

of sequestering binders (PAC, Biofix, MTB-100, and Mycofix), and 3 levels of binders

(0.1, 0.25, and 0.4% PAC and 2.0, 3.0, and 4.0% Biofix, MTB-100, and Mycofix) a total

of 36 treatments was investigated using a crossed, nested design (Appendix A). The

analysis of variance (ANOVA) was used to determine the significance of the main effects

and interactions. Least square regression was determined using general linear model

(GLM). All statistical procedures were performed using SAS software, v. 9.2 (Cary, NC,

USA). Statistical significance analyses existed when p < 0.05.

Results and Discussion

AFM1 concentrations for the controls (blank milk spiked with 0.5 ng/mL AFM1) were 0.56 ± 0.03 (112% recovery), 0.46 ± 0.1 (92% recovery), and 0.54 ± 0.07 ng/mL

(108% recovery) in raw, skim, and whole milk. All concentrations are presented as means

± standard deviation. Results from the statistical design revealed that milk type and binder type, and milk binder type nested in binder concentration significantly interacted to affect mean AFM1 concentration (Table 5.1). The “best” treatment combination

(Appendix A) shown to reduce 0.46 ng/mL AFM1 from contaminated milk was observed

at 0.25% and 0.4% PAC in skim milk, 86.7 and 90.0% reduction. A previous study

performed revealed maximum percent binding of PAC when exposed to 50 ng/mL AFM1 in skim milk for 1 h. Results indicated that the use of PAC reduced AFM1 in milk

significantly (75.2% reduction) when 0.25% PAC was applied (Figure 5.3). Reductions

of 88.8, 93.2, and 94.7% were attained when 0.5, 1.0, and 2.0% PAC were added,

respectively. 88

Table 5.1 The analysis of variance (ANOVA) used to determine the significance of the main effects and interactions

Source DF Sum of Squares Mean Square F Value Pr > F Model 35 3.3828 0.0967 30.78 <.0001 Error 72 0.2261 0.0031 Corrected Total 107 3.6088

R-Square Coeff Var Root MSE AFM1 Mean 0.9374 12.2150 0.0560 0.4587

Mean F Source DF Type III SS Pr > F Square Value MILK 2 0.1179 0.0589 18.77 <.0001 BINDER 3 2.4997 0.8332 265.37 <.0001 B_CONC(BINDER) 8 0.0649 0.0081 2.58 0.0154 MILK*BINDER 6 0.5550 0.0925 29.46 <.0001 MILK*B_CONC(BINDER) 16 0.1453 0.0091 2.89 0.0011

Figure 5.3 Maximum percent binding of PAC when exposed to 50 ng/mL AFM1 in skim milk.

In the current study, a reduction below the EU maximum regulatory level of 0.05 ng/mL and the FDA AL of 0.5 ng/mL AFM1 was only achieved when 0.4% PAC was

applied to skim milk (0.046 ± 0.01 ng/mL) suggesting that milk matrix may have an

effect on the binding the sequestering agent has on AFM1 in milk. The interference due to

the high fat and protein content (matrix) in milk is troublesome. Protein and fat contents

of milk may influence test results in various ways (e.g., fat content strongly affects

viscosity) and may interact specifically during extraction (Anfossi et al., 2011). Mean

AFM1 concentrations when varying concentrations of AC, Biofix, MTB100, and Mycofix

were applied to raw, skim, and whole milk are shown in Figure 5.4. Optimization of the

extraction method is warranted for the extraction of AFM1 from milk. Additionally, the

characterization of milk components should also be determined.

Figure 5.4 Reduction of AFM1 when sequestering binders are added to milk

For this dissertation, however, we are only interested in raw milk because if

contaminated above the FDA AL, then it would not be processed into other milk products 90

(FDA, 2000); therefore, the reduction of AFM1 in raw milk is of primary concern. A

nested design with binder type and binder concentration was carried out. In raw milk,

binder concentration nested in binder type was not significant. However, the type of

binder significantly and independently affected AFM1 reduction in raw milk (Table 5.2

and Table 5.3). Mycofix (bentonite, a clay additive) was the worst binder with a mean

AFM1 concentration of 0.59 ng/mL. PAC was the best binder with a mean AFM1 concentration of 0.25 ng/mL for the removal of AFM1 in raw milk.

Table 5.2 ANOVA to determine the significance of the main effects and nested interactions between binder type and binder concentration

Source DF Sum of Squares Mean Square F Value Pr > F Model 11 0.5868987 0.0533544 44.48 <.0001 Error 24 0.0287865 0.0011994 Corrected Total 35 0.6156852

R-Square Coeff Var Root MSE AFM1 Mean 0.953245 7.51003 0.034633 0.461156

Source DF Type III SS Mean Square F Value Pr > F BINDER 3 0.57109 0.190363 158.71 <.0001 B_CONC(BINDER) 8 0.015809 0.001976 1.65 0.1637

Table 5.3 Least squares means for the significant main effect, binder type

t Grouping Mean N BINDER A 0.59371 9 MYCO

B 0.51413 9 BIO B B 0.48211 9 MTB100

C 0.25467 9 AC

The commercial binders that consisted of clay additives and glucomannan yeast in this study had no significant effect on the reduction of AFM1 from artificially

contaminated raw milk contrary to the results obtained when these agents were applied to

naturally contaminated raw milk. Soha et al. (2006) studied the reduction of AFM1 in

naturally contaminated milk exceeding 0.05 ng/mL by the addition of HSCAS and

bentonite at 0.5 and 2%. Results indicated that bentonite reduced AFM1 levels by 90% at

both the 0.5 and 2% levels, and HSCAS obtained 47.7 and 77.8% reductions at 0.5 and

2%, respectively. This study showed that utilization of chemisorption compounds directly

in artificially contaminated milk had a high efficiency for the removal of AFM1 from

milk. Additional studies using naturally contaminated raw milk are needed in this field to

further extend our knowledge of the effects of the decontamination process of AFM1 in

milk. A limitation of this study was the discoloration of the milk when PAC was added.

Therefore, other forms of activated carbon for the removal of AFM1 from contaminated

raw milk should be explored, particularly granular activated carbon.

Conclusion

The presence of AFM1 in milk is a global concern. The possibility of PAC to

make AFM1-contaminated milk safe for human consumption is a potential avenue to

explore. The interaction between AFM1-contaminated raw milk and the tested

sequestering agents indicate the effectiveness of AFM1 removal by PAC. However, the

addition of PAC resulted in the discoloration of the milk and other forms of activated

carbon should be considered for the removal of AFM1 from milk. Furthermore, the

optimization of the extraction method is justified for the extraction of AFM1 from milk

due to matrix interference. The characterization of milk components should also be determined.

THE VALIDATION OF QuEChERS AS AN EXTRACTION METHOD FOR

AFLATOXIN M1 IN RAW MILK

Abstract

An improved, simplified, cost-effective, and time-saving extraction technique based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) for aflatoxin M1

(AFM1) determination in bovine unpasteurized, raw milk was developed at the United

States Food and Drug Administration (FDA) action level (AL) of 0.5 ng/mL. This method includes the extraction of 0.5 ng/mL AFM1 from 5 mL milk (15°C) with 10 mL

acetonitrile, centrifugation, and syringe filter (15 min/12 samples). The method was

validated according to AOAC guidelines. The matrix effect was below AOAC acceptable

criteria (<20%). The accuracy was determined by recovery (102 ± 9.2%; n=6). The

relative standard deviations were <10% in all cases. The limit of quantitation (0.0325

ng/mL) meets the AL set by the FDA (0.5 ng/mL) and European Union (0.05 ng/mL).

Specificity and linearity were also validated. This method has been successfully applied

for the analysis of AFM1 in raw milk from a local dairy farm.

Introduction

Milk is valued in the human diet for the nutrients and energy it provides to ensure proper growth and development. The increasing concern for the safety of milk is

reasonable. Aflatoxin M1 (AFM1) is the principal aflatoxin found in the milk of lactating

dairy cows that have consumed feed contaminated with aflatoxin B1 (AFB1). AFM1

presents in the milk 2-3 days after the ingestion of its parent, AFB1 (the most toxic

naturally occurring liver carcinogenic compound known), with conversion rates ranging

from 0.3 to 6.2% of AFB1 (Creppy, 2002). AFM1 has been associated with hepatotoxicity and carcinogenicity. Accordingly, AFM1 has been categorized as a Group 2B carcinogen, possible carcinogen (IARC, 2002). Due to the serious concerns AFM1 poses on the

human health, many countries have set regulatory guidelines for the management of this

toxic compound. The European Union (EU) enforces a maximum regulatory limit of 0.05

and the United States Food and Drug Administration (FDA) impose an action limit (AL)

of 0.5 ng/mL, respectively (EC, 2010; FDA, 2005). Such low regulatory levels require

highly selective extraction and sensitive analytical methods for the detection and

quantification of AFM1 in milk.

Selective extraction techniques for the determination of AFM1 in milk are

desirable. Liquid-liquid (LLE) and solid-phase extraction (SPE) are commonly utilized

for the separation of AFM1 from milk achieving acceptable recoveries ranging from 87 to

99% (Fallah, 2010a; Sorenson and Elbaek, 2005; Manetta et al., 2005; Wang et al.,

2012). Although effective, these methods are time-consuming and costly (Paya and

Anastatassiades, 2007). QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe)

has been proven to be a highly beneficial approach that vastly simplifies extraction,

cutting costs and preparation time (Paya and Anastassiades, 2007; Yogendrarajah et al.,

2013; Lehotay et al., 2010). The QuEChERS method is a relatively novel sample

preparation technique introduced by the United States Department of Agriculture

(USDA) scientists in early 2003 for the analysis of pesticide residues in foods. Although fairly new, this method has already been extended for the analyses of veterinary drug residues, antibiotics, acrylamide, and mycotoxins (including aflatoxins) in different matrices (Paya and Anastassiades, 2007; Yogendrarajah et al., 2013; Lehotay et al.,

2010). Yet, to our knowledge, this method has not been simplified, optimized, and

validated for the extraction of AFM1 from raw milk at the FDA AL of 0.5 ng/mL. The

QuEChERS procedure involves an initial extraction with acetonitrile (ACN) followed by a partitioning step with the addition of a salt mixture. Then the sample is centrifuged and analyzed by liquid or gas chromatography.

Analysis of AFM1 is challenging particularly due to its presence at very low concentrations in milk. The traditional method used for AFM1 determination is thin-layer

chromatography (TLC) possibly due to its low operating costs and ease of use. Some

authors have analyzed AFM1-contaminated milk according to TLC official method

(method 980.21) approved by AOAC International and have achieved a limit of

quantitation (LOQ) as low as 0.02 ng/mL and a limit of detection (LOD) as low as 0.01

ng/mL (AOAC 2000; Kafle et al., 2014; Fallah, 2010a). Although still accepted as an

approved reference method for the determination of aflatoxins, TLC has been largely

replaced with high performance liquid chromatography (HPLC) coupled to fluorescence

(FLD) detectors and tandem-mass spectrometry (MS/MS) for quantitative analysis of

AFM1. Several immunology-based semi-quantitative and qualitative methods such as

enzyme-linked immunosorbent assay (ELISA) are often used for screening due to

advantages such as rapidity, simplicity, and cost-effectiveness (Anfossi et al., 2008).

The HPLC-FLD method after immunoaffinity column clean-up was adopted as a standard method by ISO (International Organization for Standardization), IDF

(International Dairy Federation) and AOAC (IDF, 1995; ISO, 2007; AOAC, 2005) and

has achieved a LOD as low as 0.001 ng/mL AFM1, 50-folds lower than the AL enforced

by the EU (Manetta et al., 2005). However, the immunoaffinity column clean-up is a

multistep, expensive, and time-consuming method. The limitations of the HPLC-MS/MS

include the complexity for novices and the expense of the instrument. However, the

HPLC-MS/MS (Figure 6.1) was selected as the analytical technique for this study due to

its separation capability of the HPLC coupled to the sensitivity (improved detection

limits, ng to fg) and the specificity (high resolution of compounds) of the MS while

allowing for the avoidance of excessive clean-up of the milk sample. Many papers have

also utilized HPLC-MS/MS for the quantitative analysis of AFM1 in milk achieving

acceptable results (Sorenson, 2005; Biancardi et al., 2012). Biancardi et al. (2012) used

HPLC-MS/MS equipped with an electrospray ionization (ESI) following LLE extraction

for the analysis of AFM1 in milk. The LOQ achieved was 0.015 µg/kg.

Figure 6.1 HPLC-MS/MS used for the quantification of AFM1

The interference due to the high fat and protein content (matrix) in milk is troublesome, making extraction long and tedious, involving several multi-steps and expensive clean-up. The extraction procedure presented here details an improved, simplified, cost-effective, and time-saving analytical technique. This technique based on the QuEChERS extraction method (without an additional clean-up step) was developed for the quantitative determination of AFM1 in bovine unpasteurized, raw milk using the

HPLC-MS/MS at the FDA AL of 0.5 ng/mL. The purpose of this study is to offer an alternative for the extraction of AFM1 from milk.

Materials and Methods

Chemicals and reagents

Methanol (MeOH), ACN, and water (H2O) were HPLC grade and supplied by

Fisher Scientific (Fair Lawn, NJ, USA). Millex 0.2 µm polytetrafluoroethylene (PTFE)

syringe filters and analytical grade formic acid (≥ 99.5%) and ammonium acetate (≥

99.0%) were also obtained from Fisher Scientific. QuEChERS extraction salts (6 g

MgSO4, 1.5 g NaOAc) were acquired from Agilent (Santa Clara, CA, USA). All other chemicals and reagents used were of analytical grade.

The AFM1 standard (10 µg), purchased from Sigma-Aldrich (St. Louis, MO,

USA), was dissolved in 10 mL of 70% ACN (1000 ng/mL AFM1 stock solution) and

stored at -20°C. A working standard solution of 100 ng/mL AFM1 was prepared from the

stock solution and stored at 4°C for up to a month. The working solution was used to

prepare calibration curves in matrix, acquire the limit of detection, and used to spike

samples for recovery experiments.

Aflatoxins can cause acute toxicity including reproductive and specific target

organ toxicity; additionally, aflatoxins can cause respiratory sensitization, germ cell

mutagenicity, and carcinogenicity. Hence, all aflatoxin-contaminated glass- and plastic-

ware were handled with care and soaked in 10% aqueous sodium hypochlorite to destroy

AFM1 residue before cleaning and reuse.

Milk samples

A fresh batch of bovine unpasteurized, raw milk was obtained from Mississippi

State University’s Department of Animal and Dairy Sciences (Mississippi State, MS,

USA). The batch was analyzed for AFM1 residue before the study. The milk was

considered blank, and used to optimize and validate the analytical method.

Sample preparation

Aliquots of the blank milk were spiked with 0.25, 0.5, or 1.0 ng/mL AFM1. After

extraction optimization, a volume of 5 mL of the spiked milk was used for extraction.

Milk temperature was adjusted to 15°C and a volume of 10 mL of ACN was added to each sample. Samples were vortexed in a SPEX Sample Prep Geno-Grinder (Figure 6.2) for 1 min at 1000 strokes/min. QuEChERS extraction salts were added to each sample, vigorously vortexed in the Geno-Grinder at 1000 strokes/min for 1 min, and centrifuged for 5 min at 3200 g for separation. The organic supernatant was collected and filtered

through 0.2 µm PTFE syringe filters.

Figure 6.2 Geno-Grinder used to vigorously vortex milk samples.

Extraction optimization

A ruggedness trial for the QuEChERS extraction method of 0.5 ng/mL AFM1

from milk was investigated. From a preliminary 27-4 fractional factorial design used to

select significant main effects, factors such as time in the Geno-Grinder (30, 60, 90 sec),

strokes per min in the Geno-Grinder (500, 1000, 1500 strokes/min), and centrifugation

time (3, 5, 7 min) did not have a significant effect on the extraction method. Therefore,

60 sec at 1000 strokes/min in the Geno-Grinder with a centrifugation of 3200 g for 5 min 100

where chosen for the extraction (Table 6.1). Although not significant (p < 0.05), milk

temperature had one of the highest effects on mean AFM1 recovery as evident from the

means squares and F value. Therefore, it was included as a factor for the optimization of

the extraction method.

Table 6.1 ANOVA analysis showing significant main effect of the QuEChERS extraction method

Source DF Sum of Squares Mean Square F Value Pr > F Model 9 0.1346 0.0150 4.32 0.0074 Error 14 0.0485 0.0035

Corrected Total 23 0.1831

R-Square Coeff Var Root MSE YIELD Mean 0.73529 14.0571 0.05884 0.41858

Source DF Type III SS Mean Square F Value Pr > F REP 2 0.0032 0.0016 0.46 0.6412 VOL 1 0.0683 0.0683 19.72 0.0006 TEMP 1 0.0095 0.0095 2.74 0.1199 HOMO 1 0.0027 0.0027 0.78 0.3927 ACN 1 0.0494 0.0494 14.28 0.002 SHAKE 1 0.0013 0.0013 0.37 0.5531 STROKES 1 0.0002 0.0002 0.07 0.7948 CENTRI 1 0.0000 0.0000 0.01 0.9245

The three factors believed to affect the performance of the extraction method were

ACN volume, milk temperature, and milk volume. A volume of 10 or 20 mL ACN

(control 15 mL), milk temperature of 15 or 25°C (control 20°C), and milk volume of 5 or

15 mL (control 10 mL) were used in a 23 factorial arrangement of treatments experimental design consisting of 8 runs (Table 6.2). The experimental design was

conducted with raw blank milk spiked with 0.5 ng/mL AFM1 (n=3). A total of 9

calibration curves (0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 ng/mL) were performed for each 101

run and the control. The optimum parameters were based on the highest peak response and percent recovery and used for subsequent studies. All statistical analyses were performed with SAS software, v. 9.2 (Cary, NC, USA) (Appendix B).

Table 6.2 A 23 Factorial design of varied factor combinations using QuEChERS

milk mean peak mean AFM1 % mean ACN milk runs temp response (ng/mL) ± recovery ± (mL) (mL) (°C) (counts) standard dev standard dev 1 10 15 5 90,867 0.53 ± 0.02 105 ± 1.86 2 20 15 5 89,729 0.53 ± 0.03 105 ± 2.57 3 10 25 5 87,086 0.48 ± 0.01 96 ± 0.74 4 20 25 5 89,624 0.52 ± 0.02 104 ± 2.39 5 10 15 15 82,075 0.47 ± 0.00 94 ± 0.35 6 20 15 15 77,006 0.43 ± 0.00 86 ± 0.19 7 10 25 15 84,730 0.48 ± 0.01 96 ± 1.40 8 20 25 15 80,634 0.45 ± 0.00 89 ± 0.10 Control 15 20 10 82,038 0.46 ± 0.01 91 ± 0.62 Abbreviations: ACN (acetonitrile)

Analytical instrument conditions

Measurements were performed on an Agilent 1260 Infinity HPLC by injecting a

total volume of 5 μL on to a Zorbax Eclipse Plus-C18 2.1 x 50 mm, 1.8 µm column from

Agilent (Santa Clara, CA, USA) with a column temperature of 35°C. The mobile phases

consisted of A: H2O and B: MeOH both containing 5 mM ammonium acetate and 0.1%

formic acid. The mobile phase was pumped at a flow rate of 0.3 mL/min under gradient

elution starting from 5% B, increasing linearly to 95% B over 3 min, and decreasing back

to 5% B over 2 min. For column re-equilibration back to initial mobile phase conditions,

a 3 min delay was performed between injections. The total run time was 8 min.

Analysis was performed on an Agilent 6460 Triple Quadrupole Mass

Spectrometer system coupled to an electrospray ionization (ESI) operated in positive

102

mode (Santa Clara, CA, USA). Data was acquired in multiple reaction monitoring

(MRM) mode. The desolvation or drying gas (N2) temperature was 350°C, and the desolvation gas flow rate was set to 13 L/min. The nebulizer gas pressure was set at 40 psi, and the capillary voltage was 4 kV. The sheath gas flow had an output of 12 L/min and reached a constant temperature of 400°C. The delta electron multiplier voltage

(EMV) was set to 800 V, and the dwell time lasted for 200 msec. Agilent MassHunter

Quantitative Analysis Workstation Software v. B.04.0.225.19 was used to analyze the quantitative data obtained from the samples and the calibration curve.

Method validation

The method was validated according to the single laboratory guidelines criteria of

AOAC (AOAC, 2012). Validation was based on the specificity, matrix effects, linearity,

LOD and LOQ, accuracy and precision (intra- and inter-day), and robustness of the

analytical instrument.

To confirm specificity of the method, blank milk samples (n=20) and 0.5 ng/mL

AFM1 spiked milk samples (n=3) were analyzed to check for interferences eluting at the

same retention time. Carry-over of AFM1 from the previous sample was also evaluated by analysis of a solvent blank after the highest spiked sample.

The matrix effect was evaluated by comparing triplicate peak responses of standards spiked in mobile phase A and B (50:50 v/v) solvent and standards spiked in milk matrix at three concentrations (0.05, 0.5, and 5.0 ng/mL AFM1). The differences in response between the pure solution and the post-extraction samples divided by the pure solution response and multiplied by 100 determines the degree of matrix effect occurring in the analytes in question under chromatographic conditions (Taylor, 2005). Matrix 103

effect values from 80-120% are considered suitable values indicating minor matrix effects. This range is often used as a cutoff value for use of a solvent calibration as opposed to matrix-matched standards (Kowalski et al., 2014).

Linearity was evaluated with a matrix-matched calibration curve used to reduce matrix effects. A calibration curve was prepared by plotting the response of the spiked sample against the expected AFM1 concentration of the calibration curve. The standard

solutions of the curve ranged linearly from 0.002 ng/mL to 8 ng/mL AFM1. Each

standard was replicated 3 times (Figure 6.3). The least square method was used to

estimate the linear regression. A satisfactory linear correlation was determined by the

coefficient of determination (R2). The LOD and LOQ were determined by the signal-to-

noise (S/N) ratio of more than 3 and 10, respectively using 273.1 m/z as the quantifier

product ion, and 229 m/z and 259 m/z as qualifiers.

Figure 6.3 Linearity assessed using a matrix-matched 13-point calibration curve (replicated 3 times) ranging from 0.002 to 8 ng/mL.

104

Accuracy of the method was evaluated by performing recovery experiments using blank milk spiked at 0.5 ng/mL AFM1 (n=6). Percent recovery was determined by dividing obtained concentration by the spiked concentration, and multiplied by 100.

Percent relative standard deviation (%RSD) values were used to determine the precision of the validated study under intra-day repeatability (RSDr) and inter-day reproducibility

(RSDR) conditions at spiked AFM1 concentrations of 0.25, 0.5, and 1.0 ng/mL in blank milk. Intra-day precision was performed in triplicate on the same day. Inter-day precision was performed by repeating the same procedure on three consecutive days. An acceptable

RSD was considered to be <20%.

Robustness of the HPLC-MS/MS was determined using three replicated milk samples spiked with 0.5 ng/mL AFM1. The control HPLC conditions included mobile phases A: H2O and B: MeOH both containing 5 mM ammonium acetate and 0.1% formic acid at a flow rate of 0.3 mL/min with a 35°C column temperature. Each parameter was varied from these conditions including different flow rates (0.25 and 0.35 mL/min), column temperatures (30 and 40°C), and organic mobile phase (B: ACN + 5 mM ammonium acetate and 0.1% formic acid) were utilized.

Results and Discussion

Extraction optimization

The results of the extraction optimization indicated that the volumes of ACN and milk interacted to significantly affect mean sample response; and milk volume, independently, had the biggest and most significant effect on extraction recovery (Table

6.3). Yogendrarajah et al. (2013) suggested that water within the matrix weakens the interactions between the analyte and matrix components, assisting in extraction 105

efficiency. To explore this concept, milk volume (mL), composed mostly of water, to

ACN (mL) ratios of 5:10, 5:20, 15:10, 15:20 (v/v) were examined. As shown in Table

6.2, the 5 mL volume of milk had the highest peak responses (87,086-90,867 counts) compared to the peak responses of 15 mL of milk (77,006-84,730 counts). The control samples had a mean peak response of 82,038 counts. Figure 6.4 shows the comparison of matrices by milk volume.

Table 6.3 ANOVA analysis of the 23 factorial design of varied factor combinations using QuEChERS

Sum of Source DF Mean Square F Value Pr > F Squares Model 7 0.027508 0.00393 17.02 <.0001 Error 16 0.003694 0.000231 Corrected Total 23 0.031202

R-Square Coeff Var Root MSE AFM1 Mean 0.881597 3.129392 0.015195 0.48557

Source DF Type III SS Mean Square F Value Pr > F ACN 1 0.000539 0.000539 2.33 0.146 TEMP 1 0.000269 0.000269 1.16 0.2965 ACN*TEMP 1 0.000676 0.000676 2.93 0.1063 MILK 1 0.018272 0.018272 79.14 <.0001 ACN*MILK 1 0.005194 0.005194 22.5 0.0002 TEMP*MILK 1 0.002117 0.002117 9.17 0.008 ACN*TEMP*MILK 1 0.00044 0.00044 1.9 0.1866

106

Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8

5 mL milk 5 mL milk 5 mL milk 5 mL milk 15 mL milk 15 mL milk 15 mL milk 15 mL milk 15°C milk 15°C milk 25°C milk 25°C milk 15°C milk 15°C milk 25°C milk 25°C milk 10 mL ACN 20 mL ACN 10 mL ACN 20 mL ACN 10 mL ACN 20 mL ACN 10 mL ACN 20 mL ACN

Figure 6.4 A comparison of milk matrices by milk volume using varied parameters of QuEChERS.

Runs 1-4 have 5 mL of milk and Runs 5-8 have 15 mL of milk. In order from L to R: Run 1 (15°C and 10 mL ACN), Run 2 (15°C and 20 mL ACN), Run 3 (25°C and 10 mL ACN), Run 4 (25°C and 20 mL ACN), Run 5 (15°C and 10 mL ACN), Run 6 (15°C and 20 mL ACN), Run 7 (25°C and 10 mL ACN), and Run 8 (25°C and 20 mL ACN).

Concurrently, the highest percent mean recoveries of 0.5 ng/mL AFM1 from raw

milk were obtained with the samples that had 5 mL of milk (96-105%) compared to 15

mL milk samples (86-94%). The control milk sample had a mean percent recovery of

91%. All milk samples gave acceptable percent recoveries as defined by AOAC, 2012

(70-120%). Yogendrarajah et al. (2013) investigated different water to ACN ratios,

10:15, 10:10, 10:5, 5:15, 5:10, and 5:5 mL (v/v), per 1 g of spice matrix as it relates to extraction recovery of aflatoxins and other mycotoxins. Similar to the current study,

Yogendrarajah’s data showed the highest peak responses were observed with reduced 107

water to solvent ratio. They suggested that this ratio helped to detect mycotoxins at lower

concentrations with acceptable extraction recoveries.

Temperature of the milk and milk volume also interacted to significantly affect

mean sample response. However, regardless of milk temperature (15 and 25°C), a 5 mL

volume of milk still corresponded to the highest peak response. Therefore, a 5 mL milk

sample at a temperature of 15°C was extracted with 10 mL of ACN for the optimum

parameters of the QuEChERS extraction method. After centrifugation, samples were

syringe filtered for analysis. Total extraction time per 12 samples was 15 min. This

optimized method resulted in a cost effective technique with less steps and less extraction

time compared to other studies that have analyzed AFM1 in milk (Andrade et al., 2013;

Wang et al., 2011; Wang et al., 2010). For example, Andrade et al. (2013) analyzed

mycotoxins, including AFM1, in breast milk using an optimized analytical method based

on LLE with low temperature purification. Sample preparation included sonication,

centrifugation, storage in a freezer for 12 h, syringe filter of organic phase, drying of

extract under nitrogen, and the reconstitution of sample with MeOH (Andrade et al.,

2013). Wang et al. (2011) simultaneously determined chloramphenicol and AFM1

residues in milk. Liquid milk samples (20 g) were diluted with 50 mL ACN followed by

ultrasonication for 15 min. The samples were filtered through Whatman filter paper,

filtrate was concentrated with a rotary evaporator, and 5 mL water was added to the

concentrate. The sample was then filtered through a syringe filter for analysis.

MS/MS Optimization

Data acquisition parameters for MRM were optimized on the MS for AFM1 at a

concentration of 1000 ng/mL. In positive ESI (ESI+) mode, protonated molecular ions 108

[M+H]+ were formed as the precursor ion. The use of 0.1% formic acid in the mobile phase was used to assist positive ionization of the analyte. The precursor ion of AFM1

was determined to be 329 m/z at a fragmentor voltage of 103 V. The transition ions were

273.1 m/z (quantifier), 229 m/z (qualifier), and 259 m/z (qualifier) with collision energies

of 21, 45, and 21 eV, respectively. Similarly, Biancardi et al. (2013) used HPLC-MS/MS

equipped with an ESI following LLE extraction for the analysis of AFM1 in milk.

Quantification was carried out in MRM mode using the following transitions: 329.07 >

273.00 m/z (quantifier); 329.07 > 259.00 m/z (qualifier 1), and 329.07 > 229.00 m/z

(qualifier 2). Biancardi et al. (2013) prosed a fragmentation pattern for AFM1 shown in

Figure 6.5.

Figure 6.5 Fragmentation pattern for AFM1 as proposed by Biancardi et al. (2013).

Method validation

The power of discrimination between AFM1 and matrix components was

evaluated by analyzing blank samples. Specificity was confirmed by the absence of any

chromatographic signal in the blank milk samples at the retention time of AFM1 (3.55 109

min), despite the complexity of the matrix (Figure 6.6). No carry-overs were observed at the retention time that AFM1 was eluted.

Figure 6.6 A comparison of chromatogram displaying a blank milk (top) and milk spiked with 0.5 ppb AFM1 (bottom).

This picture confirms that there are no chromatographic signals in the blank milk that interferes with the retention time of AFM1 (3.55 min).

HPLC-MS/MS coupled with ESI is a very powerful tool that allows for highly

sensitive mass detection; but, a limitation of the ESI interface is the unpredictable matrix

effect giving rise to matrix signal suppression or enhancement (Yao, et al., 2015). Matrix

effects occur as a result of competition between the co-eluting matrix components and

analyte of interest .(King et al , 2000). To assess matrix effects, the peak response from a

pure standard was compared to that of the matrix samples. At 0.05 ng/mL AFM1 concentration, the signal suppression was the highest at -12.3%. At 0.5 and 5.0 ng/mL

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AFM1 concentrations, the signal suppressions were -3.3 and -9.24%, respectively.

Although within acceptable range (< 20%), to compensate for signal suppression and

improve linearity, reliability, and accuracy, matrix-matched curves were utilized in this

study.

A satisfactory linear correlation was determined by the coefficient of

determination (R2) with a value of 0.999 for the calibration curve (Figure 6.1). This value

confirmed the best fit for the data and ensured the prediction of the AFM1 in the milk

sample was reliable. The linear regression model was y = 70675.6*x – 1887.99. The

LOD and LOQ were determined by S/N ratio method of more than 3 and 10, respectively

to determine the sensitivity of the analytical method. Wang et al. (2010) devised a sample

preparation and extraction method for AFM1 from bovine milk. Quantification was

determined using a similar system compared to the current study (Agilent 1200 series LC

coupled to an Agilent 6410 Triple Quadruple Mass Spectrometer with an ESI). However,

the isocratic mobile phase consisted of water: ACN (60:40 v/v) and 0.1% formic acid.

Matrix-matched solutions were prepared at 6 concentration levels from 0.02 to 20 ng/mL

AFM1. The LOD and LOQ were 0.006 and 0.2 ng/mL, respectively. In the current study,

the LOD value of AFM1 was 0.002 ng/mL and LOQ value was 0.03125 ng/mL (Figure

6.7). Both LOD and LOQ values meet regulatory limits set by the FDA (0.5 ng/mL

AFM1) and additionally, EU (0.05 ng/mL AFM1) (EC, 2010; FDA, 2005). The HPLC-

MS/MS method allowed analysis of AFM1 in raw milk while achieving high sensitivity at

a low concentration.

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Figure 6.7 The limit of detection (LOD) displayed (top) at a concentration of 0.002 ng/mL AFM1.

The limit of quantitation (LOQ) displayed (bottom) at a concentration of 0.0325 ng/mL AFM1.

The accuracy of the study was tested using percent mean recovery for spiked

blank milk samples at the FDA AL of 0.5 ng/mL. The mean recovery of 102 ± 9.2%

(n=6) was within the acceptable range of required performance criteria (Kowalski, 2014).

Relative standard deviations (%RSD) were calculated under intra-day repeatability

(RSDr) and inter-day reproducibility (RSDR) conditions. The RSDr values for 0.25, 0.5,

and 1.0 ppb AFM1 were 3.36, 9.02, and 1.08%, respectively. The RSDR values were

4.46, 7.07, and 2.62% for 0.25, 0.5, and 1.0 ppb AFM1, respectively (Table 6.4). The

RSDr and RSDR values were within the acceptable range of <20%.

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Table 6.4 Recovery and relative standard deviation (RSD) from spiked raw milk

within-day precision between-day precision accuracy (n=6) (n=6) (n=9) spiked AFM1 mean recovery RSDr RSDR (ng/ml) (%) (%) (%) 0.25 - 3.36 4.46 0.5 102 ± 9.2 9.02 7.07 1.0 - 1.08 2.62 Abbreviations: RSDr (relative standard deviation repeatability); RSDR (relative standard deviation reproducibility)

During the robustness of the HPLC-MS/MS, the highest mean peak response were

obtained using organic mobile phase MeOH (112,529 ± 1,108 counts) with a S/N ratio of

599 ± 51 (Table 6.5). A change in organic mobile phase to ACN resulted in a

significantly lower mean peak response of 18,721 ± 1,231 counts with a S/N ratio of 115

± 29. Using MeOH as the organic mobile phase, it was determined that 0.25 mL/min

gave the highest peak response of 138,243 ± 1,629 counts (S/N ratio 658 ± 165) of the

flow rates and 30°C gave the highest peak response of 121,578 ± 2,380 counts (S/N ratio

607 ± 207) of the column temperatures. These results indicate that a flow rate of 0.25

mL/min and a column temperature of 30°C may result in higher peak responses and

should be considered in future analyses.

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Table 6.5 HPLC optimization using peak response

mean peak response retention mean S/N (counts) (min) control 112,529 ± 1,108 599 ± 51 3.565 FR 0.25 mL/min 138,243 ± 1,629 658 ± 165 3.986 FR 0.35 mL/min 94,042 ± 2,616 573 ± 86 3.253 Column temp. 30°C 121,578 ± 2,380 607 ± 207 3.612 Column temp. 40°C 112,746 ± 1,677 579 ± 93 3.524 ACN mobile phase 18,721 ± 1,231 115 ± 29 2.903 Abbreviations: FR (flow rate); ACN (acetonitrile); S/N (signal-to-noise ratio); The control HPLC conditions included a flow rate of 0.3 mL/min and a column temperature of 35°C using an organic mobile phase of methanol + 5 mM of ammonium acetate and 0.1% formic acid. Parameters were varied from the control.

Following the validation of the study, the optimized parameters were applied to

real raw milk samples (n=10) obtained from Mississippi State University’s dairy farm.

Matrix-matched curves were developed for the raw milk matrix at a range of 0.0325 to 2

ng/mL AFM1. Quantification was carried out in MRM mode using the following

transitions: 329 > 273.1 m/z (quantifier); 329.07 > 229 m/z (qualifier) and 329 > 259 m/z

(qualifier). None of the samples were detectable for AFM1.

Conclusion

Combined with HPLC-MS/MS, a simple and reliable quantitative method based

on a QuEChERS approach for the determination of AFM1 in bovine, unpasteurized raw

milk was developed and successfully validated at the FDA AL of 0.5 ng/mL AFM1. The

QuEChERS extraction method was improved in respect to extraction time, cost, and steps involved in the technique. Additionally, this method achieved a low LOQ value that meets regulatory limits set by the FDA and EU for the quantification of AFM1 in milk.

The application to real sample milk matrix is indicative of the reliability of the method.

This simple and sensitive method provides an alternative extraction for the determination 114

of AFM1 in raw milk that can be applied for routine analyses. Further experimental

investigations should be hoped for using naturally AFM1-contaminated milk.

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THE EFFECTS OF GRANULAR ACTIVATED CARBON ON RAW MILK

CONSTITUENTS

Abstract

A potential method for the control of AFM1 in milk can be accomplished with the addition of sequestering binders added directly to milk. The effects of powdered activated carbon (PAC) proved to significantly reduce AFM1 from milk; but the addition of PAC

resulted in discoloration of the milk. Granular activated carbon (GAC) was added to the

bed of a glass column for the removal of AFM1 as contaminated unpasteurized, bovine

raw milk was pumped through. This mechanism significantly reduced discoloration of

milk compared to that of PAC. AFM1-contaminated (0.75 ng/mL) raw milk was pumped

at an optimum flow rate through the column bed with an optimum concentration of GAC.

The combination of 0.75% GAC and 0.4 mL/min were chosen as optimum conditions for

the removal of AFM1 from milk. The AFM1 level of 0.75 ng/mL was significantly

reduced to 0.012 ng/mL AFM1, a 98.4% reduction under these conditions. Additionally,

these conditions were used in the subsequent study to determine the effects of GAC on

2 milk constituents using a 2 factorial arrangement of treatments. The factors were AFM1

(0.0 and 1.0 ng/mL) and GAC (0.0 and 0.4%). The results determined that GAC had no

significant effect on major nutritive milk constituents: protein, lactose, total minerals, and

fat. There was a significant decrease in minor nutritive milk constituents: riboflavin 116

(vitamin B2) and magnesium. Additionally, there was a significant increase in percent

water from 90 to 91% in both blank milk and milk contaminated with AFM1. This

method should be considered for both the removal of AFM1 and the preservation of major

nutritive constituents. However, optimization of this method is a subject for future

research.

Introduction

Milk provides a rich source of nutrients that have a range of positive health effects

in both adults and children. A complex biological fluid, milk’s composition varies

between species depending on breed, stage of lactation, health status of the cow, and environmental factors including feed and climate (Huppertz and Kelly, 2009). The major constituent in milk is water, but milk also contains other nutrients required for growth and development; furthermore, it contains protective agents against infection and inflammation, enzymes, and growth factors for the young (Ballard and Morrow, 2014).

Milk is vital for the nourishment of humans; but, it can also be a source of contaminants.

The quality and safety of raw milk is related to the contamination of milk with

microorganisms and chemical residues such as aflatoxin M1 (AFM1). AFM1, a metabolite of aflatoxin B1, is a highly toxic, mutagenic, teratogenic and carcinogenic metabolite that has been categorized as a Group 2B possible carcinogen (IARC, 2002). The contamination of food commodities with aflatoxin continues to receive increased attention because of its potential health hazard to humans. As such, the control of AFB1

in dairy feed is a vital factor for AFM1-contamination of raw milk.

A tempting method for the reduction of aflatoxin in feed has been tried; but feed

blending exceeding regulatory action levels (AL) is unlawful and feed blending with 117

aflatoxin infected grains below regulatory AL results in animals and humans being subjected to the direct and indirect effects of low levels of aflatoxins in animal feeds and in certain human food products (Dixon et al., 2008). Dietary exposure at low aflatoxin levels can lead to cancer and aflatoxicosis (Sorenson, 1999). A practical, but not-yet-

approved-by-regulatory-officials, approach for the control of AFM1 in milk utilized the

addition of sequestering agents in contaminated feed intended for the dairy cow. When

consumed by the dairy cow, the bound aflatoxin can be excreted; and therefore, the AFB1

may not be metabolized into AFM1 (Kutz et al., 2009). Though a propitious method, studies have shown remaining AFM1 levels in milk after application of adsorbents to aflatoxin-contaminated feed (Diaz et al., 2004). Therefore, the application of a

sequestering binder, particularly granular activated carbon (GAC) directly to milk to

remediate AFM1 was studied.

Granular activated carbon is an activated carbon in a granular form widely

utilized for the removal of pollutants, contaminates, and impurities from gas, air, water,

food, beverages, and pharmaceuticals. GAC has also been widely used in the treatment of

drinking water to remove unpleasant tastes and odors (Karanfil and Kilduff, 1999). A

microporous material, GAC is produced from a number of carbonaceous materials

including wood, coal, lignin, coconut shells, and carbohydrates which are carbonized and

then activated with CO2, acids or bases, or other chemicals (Harris et al., 2008). The

resulting carbon can have a surface area of 1500 m2/g, which explains its huge adsorption

capacity. The structure of AC is comprised of carbon atoms that are ordered in parallel stacks of hexagonal layers, as well as pentagonal and heptagonal rings (Harris et al.,

2008) (Figure 7.1). These layers are extensively cross-linked with tetrahedral bonds.

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Several atoms, including oxygen, hydrogen, nitrogen, and others, can be found in the carbon matrix, in the form of single atoms and/or functional groups. Oxygen is the dominant atom found in the presence of functional groups, such as carboxyl, carbonyl, phenols, enols, lactones, and quinones, which have been postulated (Karanfil and Kilduff,

199). Surface functional groups influence adsorption properties and reactivity of GAC. A

disadvantage of GAC however, is that various researchers have shown that it can be

heavily colonized by heterotrophic microorganisms, such as bacteria.

Figure 7.1 Carbon structures containing pentagonal, hexagonal, and heptagonal rings

The prevalence of AFM1 in milk worldwide has been documented (Flores-Flores

et al., 2015; Bilandžić et al., 2010; El Khoury et al., 2011). The need for preventive

actions to reduce the risk of toxicity to humans should be considered. This study seeks to

demonstrate the feasibility of using GAC for the removal of artificially contaminated

AFM1 from bovine, unpasteurized raw milk below the maximum residue level (MRL)

imposed by the EU and the action level (AL) imposed by the FDA while assessing the

effects of the sequestering agent on milk constituents.

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Materials and Methods

Milk preparation

Unpasteurized, raw milk, obtained from the dairy farm of Mississippi State

University’s Department of Animal and Dairy Science, was analyzed for AFM1 residue prior to experiments being performed. The milk, kept at 4°C for up to 5 days, was considered blank and was used for subsequent studies.

Adsorbent materials

Granular activate carbon (GAC), obtained from Norit (Alpharetta, GA, USA), was examined for its proficiency to bind AFM1 directly from milk. The effects of GAC on milk constituents were also assessed. Before use, GAC was washed with water to remove surface impurities, followed by drying at 100°C for 24 h.

Sample Preparation

A volume of 5 mL of the spiked milk (milk temperature of 15°C) was extracted with a volume of 10 mL of acetonitrile (ACN). Samples were vortexed in a Geno-Grinder

(SPEX Sample Prep, Metuchen, NJ, USA) for 1 min at 1000 strokes/min. QuEChERS

(Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction salts, obtained from

Agilent (Santa Clara, CA, USA) were added to each sample. Samples were vigorously vortexed in the Geno-Grinder at 1000 strokes/min for 1 min and centrifuged for 5 min at

3200 g for separation. The organic supernatant was collected and filtered through 0.2 µm polytetrafluoroethylene (PTFE) syringe filters.

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Experiment 1: Optimization of granular activated carbon

The most effective concentration of GAC (%) applied to the bed of an Omnifit glass column (100 mm x 10 mm, i.d.) for the removal of 0.75 ng/mL AFM1 from milk

when pumped at an optimum flow rate (mL/min) by a Gilson peristaltic pump was

determined. Using a 52 factorial experimental design (25 treatments), two factors each at

five levels, was implemented to determine the maximum flow rate (0.2, 0.4, 0.6, 0.8, and

1.0 mL/min) in combination with percent GAC (0, 0.25, 0.5, 0.75, and 1.0%) it takes to

bind 0.75 ng/mL AFM1 from raw milk below the EU and FDA MRL (Appendix B). After

the treatments were applied, AFM1 residues were extracted and quantified using the

HPLC-MS/MS.

Calibration curve for the peristaltic pump

A calibration curve was prepared by plotting the dial setting of the peristaltic

pump against the rate of deionized water (mL/min) pumped through the peristaltic pump.

The pump dial was set at 100, 200, 400, 600, and 800. Each water sample was pumped

for 2 min (n=3), the deionized water was weighed (density 1 g/mL), and the rate was

calculated. A satisfactory linear correlation was determined by the coefficient of determination (R2).

Experiment 2: Effects of granular activated carbon on milk constituents

A 22 factorial arrangement of treatments design, 2 factors each at 2 levels (0 and

1.0 ng/mL AFM1; 0 and 0.75% GAC), was used to determine the GAC binding effect on

milk constituents. The treatment combinations were: treatment 1, milk only (control);

treatment 2, milk + 0.75% GAC; treatment 3, milk + 1.0 ng/mL AFM1; and treatment 4,

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milk + 0.75% GAC + 1.0 ng/mL AFM1) (Appendix C). Each treatment was analyzed for

AFM1, water, lactose, fat, protein, vitamins and minerals. The evaluation of water in the sample was determined using a Fisher Scientific Isotemp oven. Percent moisture was determined by the difference in weight. Milk fat was determined using the Soxhlet extraction method. Samples were weighed before and after to calculate percent fat. Total protein percent was determined using Rapid N cube (Elementar, Hanau, Germany)

according to the Dumas method (Bremner, J. M., 1996). Lactose was measured using an

Agilent 1260 Infinity evaporative light scattering detector (HPLC-ELSD). Water-soluble

vitamins (thiamine, vitamin B1; riboflavin, vitamin B2; nicotinamide, vitamin B3; calcium

pantothenate, vitamin B5; and pyridoxine, vitamin B6) in each treatment were quantified

using HPLC-MS/MS by a modified extraction method (Lu et al., 2008). Milk samples

were analyzed for minerals (sodium, magnesium, phosphorous, potassium, calcium,

manganese, iron, zinc, and selenium) with an Agilent 7900 Inductively Coupled Plasma

Mass Spectrometer (ICP-MS) by microwave sample digestion.

Statistical Analysis

Treatment differences were identified by two-way analysis of variance (ANOVA)

by the general linear models (GLM) procedure of SAS software, v. 9.2 (Cary, NC, USA).

Statistical significance analyses existed when p < 0.05.

Results and Discussion

Experiment 1: Optimization of granular activated carbon

The effectiveness of activated carbon (AC) to bind aflatoxin in feed is variable

(Galvano et al., 1998; Rao and Chopra, 2001). However, findings have shown AC to be

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more effective in aqueous solutions due to its high surface area. Di Natale et al. (2009) performed a study that examined adsorbents for AFM1 removal in ultra-high temperature

(UHT) whole milk. It was concluded that the highest removal of AFM1 (>93% removal

of 0.5 ng/mL) was obtained by 5% AC (w/v). The current study examines the effect of

GAC for the removal of AFM1 from unpasteurized, raw milk.

The peristaltic pump was calibrated before any experiments commenced. The

linear regression model was determined to be y = -3.8179 + 593.53x. The R2 was 0.9997.

The result of the dial settings at a rate of 0.2, 0.4, 0.6, and 0.8 were 115, 234, 352, and

471, respectively (Figure 7.2).

Figure 7.2 Calibration curve for peristaltic pump setting

Because the addition of PAC resulted in discoloration of the milk, GAC was chosen for the removal of AFM1 from milk. AFM1-contaminated milk (mean 0.77 ng/mL

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AFM1) was pumped at an optimum flow rate to investigate % GAC when applied to the bed of an Omnifit glass column. The schematic is shown in Figure 7.3 and the results are shown in Figure 7.4. Liquid phase adsorption processes using fixed bed operations for the removal of toxic organic compounds by carbon adsorption is a preferred method mainly

due to the ease of operation, cost, minimal attrition of adsorbent, and no carbon loss

problems (El Qada et al., 2013).

Figure 7.3 Illustration of granular activated carbon added to the bed of a glass column

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0.8

0.7

0.6

0.5 (ng/mL)

1 0.4

AFM 0.3

0.2

0.1

0.0 0.2 0.4 0.6 0.8 1 FLOW RATE (mL/min) 0% GAC 0.25% GAC 0.5% GAC 0.75% GAC 1.0% GAC

Figure 7.4 The effects of varying concentrations of granular activated carbon for the removal of a mean 0.78 ng/mL AFM1 from milk at varying flow rates.

All GAC concentrations significantly decreased AFM1 in milk below the FDA

AL (0.5 ng/mL) at all flow rates (Table 7.1; Figure 7.5). At 1.0% GAC, AFM1 was

significantly reduced to non-detectable (ND) levels in milk for all flow rates in

accordance with the MRL imposed by the EU (0.05 ng/mL) and FDA (0.5 ng/mL). For

0.75% GAC, only the 0.2 and 0.4 mL/min decreased AFM1 in milk below the EU and

FDA MRL. There was no significant difference between 1% GAC, at any flow rate, and

0.75% GAC at 0.2 (0.012 ng/mL AFM1) and 0.4 mL/min (0.010 ng/mL AFM1).

Therefore, based on percent reduction and cost, AFM1-contaminated milk was pumped at

0.4 mL/min through a column bed of 0.75% GAC for subsequent analysis. Di Natale et al. (2009) suggested that AC removed AFM1 from milk due to the high surface area,

wide micropore size, and higher affinity between AFM1 molecules and the aromatic structure of the carbon. 125

Table 7.1 One sample t-test below the FDA AL of 0.5 ng/mL

N Mean Std Dev Std Err Minimum Maximum 75 0.3017 0.2899 0.0335 0 0.8477

Mean 90% CL Mean Std Dev 90% CL Std Dev 0.3017 -Infty 0.345 0.2899 0.2558 0.3357

DF t Value Pr < t 74 -5.92 <.0001

Figure 7.5 Plot of distribution of AFM1 with 90% lower confidence

Experiment 2: Effects of granular activated carbon on milk constituents

Raw milk is composed of approximately 88.3% water, 4.52% lactose, 3.27% fat,

3.22% protein, 0.69% vitamins and minerals (USDA Nutrient Database, 2015). The

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treatment combinations (Table 7.2) for the effect of GAC on these milk components were analyzed.

Table 7.2 The effects of GAC on milk constituents when applied to blank milk and aflatoxin M1-contaminated milk.

Treatment AFM1 % % % % water % fat Combinations (ng/mL) protein lactose minerals Milk 0.0 ± 0.0 89.9 ± 0.0 2.4 ± 0.1 3.9 ± 0.2 3.9 ± 0.1 0.36 ± 0.00 Milk + 0.75% GAC 0.0 ± 0.0 90.9 ± 0.1 2.2 ± 0.2 4.0 ± 0.1 3.9 ± 0.0 0.36 ± 0.00

AFM1 Milk 1.0 ± 0.04 90.1 ± 0.0 2.2 ± 0.0 4.0 ± 0.1 4.3 ± 0.0 0.37 ± 0.01 (1.0 ng/mL)

AFM1 Milk (1.0 ng/mL) 0.1 ± 0.05 90.9 ± 0.2 2.4 ± 0.1 3.9 ± 0.1 4.1 ± 0.3 0.37 ± 0.00 + 0.75% GAC

GAC increased percent water from 90% (blank milk) to 91%. Similar results were

observed when comparing treatment 3 (90.1%) to treatment 4 (90.9%) in AFM1

contaminated milk (p=0.00). There was no significant reduction in lactose when GAC

was added to the blank milk (treatment 1, 3.9% vs. 2, 3.9%; p=0.9) or 1.0 ng/mL AFM1-

contaminated milk (treatment 3, 4.3% vs. 4, 4.1%; p=0.34). Di Natale et al. (2009)

performed a study that examined the effects of adsorbents, including four carbonaceous

materials, for AFM1 removal in milk. They also analyzed the effects of the carbon on lactose and other milk constituents. They found that there was reduction in all milk components tested, including lactose. The control milk sample had a lactose concentration of 4.39%. When 0.35% carbon was added to the milk sample, lactose concentrations ranged from 3.4 to 4.37%. When 0.5% carbon was added to the milk sample, lactose concentrations ranged from 2.94 to 4.32%. Percent GAC had no significant effect on percent fat in this study. When GAC (2.17% fat) was added to blank milk (2.35% fat), there was no significant reduction in percent fat (p=0.21). When GAC

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(2.39% fat) was added to AFM1-contaminated milk (2.21% fat), there was no significant

reduction in percent fat (p=0.07). Although AFM1 has been shown to have a high affinity

for casein, binding tightly to its hydrophobic sites (Barbiroli et al., 2007); we found that

0.75% GAC was strong enough to release AFM1 from these sites while significantly

reducing AFM1 levels from 1.0 ng/mL to 0.1 ng/mL. A future study involving naturally

AFM1-contaminated milk is needed to verify this observation. When GAC (3.97%

protein) was added to blank milk (3.92%), there was no significant reduction in percent

protein (p=0.64). When GAC (3.94% protein) was added to AFM1-contaminated milk

(3.99% protein), there was no significant reduction in percent protein (p=0.59).

Milk provides essential nutrients including vitamins and minerals. Results indicated that water-soluble vitamins, thiamine (vitamin B1), nicotinamide (vitamin B3), and pyridoxine (vitamin B6) did not appear in raw milk at detectable levels. There was no significant difference when GAC (2.84 µg/mL vitamin B5) was added to blank milk (2.80

µg/mL vitamin B5) with respect to calcium pantothenate (vitamin B5). There was also no

significant difference when GAC (2.64 µg/mL vitamin B5) was added to AFM1- contaminated milk (2.64 µg/mL vitamin B5). Both p-values were > 0.05. However,

0.75% GAC was shown to significantly reduce riboflavin (vitamin B2) from 1.64 to 1.36

µg/mL in blank milk (p=0.02) and from 1.85 to 1.36 µg/mL in AFM1-contaminated milk

(p=0.00). No significant differences (p>0.05) were shown between blank milk (0.36% mineral) and milk with GAC added (0.36% mineral) and between milk with AFM1

(0.37% mineral) and milk with AFM1 + GAC (0.37% mineral). However, magnesium was significantly reduced with added GAC when comparing treatment 1 with treatment 2

(p=0.036) and treatment 3 with treatment 4 (p=0.037). There was no significant reduction

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in sodium, phosphorous, potassium, calcium, manganese, iron, zinc, and selenium

(p>0.05).

Conclusion

GAC may prevent the discarding of AFM1-contaminated milk by reducing AFM1 contamination in milk below FDA AL and EU MRL while still providing major nutritional values for human consumption. This method should be optimized to assure both removal and preservation of all milk constituents to allow the decontaminated milk to be used as a food product or as raw material for other dairy products. Further experiments using naturally contaminated milk should be performed in the future for application of this treatment industrially.

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GENERAL DISCUSSION AND SUMMARY

Of all the mycotoxins, aflatoxins, produced by two species in the genus of

Aspergillus, are of the greatest concern due to their toxicity and carcinogenicity.

Aflatoxins, and the fungi responsible for them, occur worldwide in many agricultural

commodities such as corn, cotton, peanuts, tree nuts, and certain animal products,

including milk and milk products. Moisture content, temperature, insect damage and

length of storage are the most critical environmental factors influencing fungal growth

and aflatoxin production.

Aflatoxin B1 (AFB1), the most potent mycotoxin, is also the most toxic naturally

occurring liver carcinogen known (McKean et al., 2006). AFB1, metabolized in the liver,

produces a number of metabolites including hydroxylated derivative, aflatoxin M1

(AFM1). AFM1, a possible human carcinogen, although less carcinogenic, appears to be

as toxic as its parent compound. Yet, little attention has been given to the remediation of

AFM1 contamination from milk. Instead, attention has been focused on the carryover of

AFB1 from feed to AFM1 in milk when dietary sequestering agents were added to feed.

However, a significant amount of AFM1 has been shown to remain in the milk.

Provisions to limit AFM1 in milk are necessary and introducing sequestering binders

directly to milk is a promising avenue for the removal of AFM1 residues.

130

The hypothesis for this research stated that if a sequestering agent is added to milk, then the agent will bind strongly enough to AFM1 to prevent the toxicity of raw

milk. The main objectives were to: (1) first examine the proficiency of adsorbents

(Biofix, Mycofix, MTB-100, and powdered activated carbon) for the removal of AFM1 from raw milk; (2) develop and validate a simple and reliable extraction method for

AFM1 in bovine, unpasteurized milk using QuEChERS as an extraction method; and (3)

determine the effectiveness of the “best” adsorbent (which happens to be granular

activated carbon) on milk fat, protein, carbohydrate, moisture, vitamins, and minerals.

The removal of aflatoxin M1 (AFM1) bovine milk by adsorption using

sequestering binders, powdered activated carbon (PAC) and several industrial sorbents

(Mycofix, Biofix, and MTB-100 binders) for the removal of 0.5 ng/mL AFM1 from

artificially contaminated milk was investigated. The industrial sorbents had no significant

effect on the reduction of AFM1 from raw milk; but, the PAC resulted in the highest

reduction removing 57.2% of AFM1 from raw milk at a 0.4% concentration. The

effectiveness of PAC to remove AFM1 from raw milk was determined. However, the

addition of PAC resulted in the discoloration of the milk. This problem was remediated

with objective 3 with the addition of granular activated carbon. There was also a need for

extraction optimization of this method. An improved, simplified, cost-effective, and

timesaving extraction technique based on QuEChERS for AFM1 determination in bovine

unpasteurized, raw milk was developed at the FDA action level (AL) of 0.5 ng/mL. The

optimized extraction parameters were 5 mL of AFM1-contaminated milk (15°C) extracted

with 10 mL acetonitrile, centrifuged at 3200 g, and syringe filtered with PTFE for a total

extraction time of 15 min per 12 samples. The method was validated according to AOAC

131

guidelines. The matrix effect was below acceptable criteria (<20%). The accuracy was determined by recovery (102 ± 9.2%; n=6). The relative standard deviations were <10% in all cases. The limit of quantitation (0.0325 ng/mL) meets the AL set by the FDA (0.5 ng/mL) and European Union (0.05 ng/mL). Specificity and linearity were also validated.

This method has been successfully applied for the analysis of AFM1 in raw milk from a

local dairy farm. The third objective examined the effects of granular activated carbon

(GAC) on milk constituents in blank milk and AFM1-contaminated milk (1.0 ng/mL).

Before this objective was carried out, optimization of the concentration of GAC and the flow rate in which the contaminated milk was pumped was achieved. The combination of

0.75% GAC and 0.4 mL/min were chosen as optimum conditions for the removal of

AFM1 from milk. The results also obtained from the third objective determined that GAC

had no significant effect on major nutritive milk constituents: protein, lactose, total

minerals, and fat. There was a significant decrease in minor nutritive milk constituents:

riboflavin (vitamin B2) and magnesium. Additionally, there was a significant increase in percent water from 90 to 91% in both blank milk and milk contaminated with AFM1. It

was concluded that this method should be considered for both the removal of AFM1 and

the preservation of major nutritive milk constituents.

In summary, the findings of these investigations have answered many questions,

but as also succeeded in creating new queries such as: can GAC be applied industrially,

will this method have a significant effect globally, and what will be the effects of GAC

when applied to naturally contaminated milk? The results of this project demonstrate the

significance of removal of AFM1 from raw milk. Hopefully, this dissertation has offered

132

an alternative avenue for the removal of AFM1 from milk and given the reader an appreciation of the problem AFM1 imposes on the dairy industry.

133

REFERENCES

Abbas, H. K., Williams, W. P., Windham, G. L., Pringle, III, H. C., Xie, W., and Shier, W. T., 2002. Aflatoxin and fumonisin contamination of commercial corn (Zea mays) hybrids in Mississippi. Journal of Agricultural and Food Chemistry 50: 5246-5254.

Abbas, H. K., Zablotowicz, R. M., Horn, B. W., Phillips, N. A., Johnson, B. J., Jin, X., and Abel, C. A., 2011. Comparison of major biocontrol strains of non- aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize. Food Additives and Contaminants 28: 198-208.

Abdullah, A. H., Kassim, A., Zainal, Z., Hussien, M. Z., Kuang, D., Ahmad, F., and Wooi, O. S., 2001. Preparation and characterization of activated carbon from gelam wood bark (Melaleuca cajuputi). Malaysian Journal of Analytical Sciences 7: 65-68.

Abdulrazzaq, Y. M., Osman, N., Yousif, Z. M., and Trad, O., 2004. Morbidity in neonates of mothers who have ingested aflatoxins. Annals of Tropical Paediatrics 24: 145-151.

Akbas, M. Y., and Ozdemir, M., 2006. Effect of different ozone treatments on aflatoxin degradation and physicochemical properties of pistachios. Journal of the Science of Food and Agriculture 86: 2099-2104.

Aksoy, A., Yavuz, O., Guvenc, D., Das, Y. K., Terzi, G., and Celik, S., 2010. Determination of aflatoxin levels in raw milk, cheese and dehulled hazelnut samples consumed in Samsun province, Turkey. Kafkas Universitesi Veteriner Fakultesi Dergisi 16(Suppl. A): 513-516.

Allameh, A., Safamehr, A., Mirhadi, S. A., Shivazad, M., Razzaghi-Abyaneh, M., and Afshar-Naderi, A., 2005. Evaluation of biochemical and production parameters of broiler chicks fed ammonia treated aflatoxin contaminated maize grains. Animal Feed Science and Technology 122: 289-301.

Ali, M., El Zubeir, I., and Fadel Elseed, A., 2014. Aflatoxin M1 in raw and imported powdered milk sold in Khartoum State, Sudan. Food Additives & Contaminants: Part B 7: 208-212.

134

Alonso, V. A., Monge, M. P., Larriestra, A., Dalcero, A. M., Cavaglieri, L. R., and Chiacchiera, S. M., 2010. Naturally occurring aflatoxin M1 in raw bulk milk from farm cooling tanks in Argentina. Food Additives & Contaminants 27: 373-379.

Andrade, P. D., de Silva, J. L. G., and Caldas, E. D., 2013. Simultaneous analysis of aflatoxins B1, B2, G1, G2, M1 and ochratoxin A in breast milk by high- performance liquid chromatography/fluorescence after liquid-liquid extraction with low temperature purification (LLE–LTP). Journal of Chromatography A 1304: 61-68.

Amr Amer, A. and Ekbal Ibrahim, M. A., 2010. Determination of aflatoxin M1 in raw milk and traditional. Journal of Toxicology and Environmental Health Sciences 2: 50-53.

Andrade, P. D., da Silva, J. L. G., and Caldas, E. D., 2013. Simultaneous analysis of aflatoxins B1, B2, G1, G2, M1 and ochratoxin A in breast milk by high- performance liquid chromatography/fluorescence after liquid-liquid extraction with low temperature purification (LLE–LTP). Journal of Chromatography A 1304: 61-68.

Akkaya, L., Birdane Y., Oguz, H., and Çemek, M., 2006. Occurrence of aflatoxin M1 in yogurt samples from Afyonkarahisar, Turkey. Bulletin of the Veterinary Institute in Pulawy 50: 517-519.

Anfossi, L., D’Arco, G., Calderara, M., Baggiani, C., Giovannoli, C., and Giraudi, G., 2011. Development of a quantitative lateral flow immunoassay for the detection of aflatoxins in maize. Food Additives and Contaminants 28: 226-234.

Anfossi, L., Calderara, M., Baggiani, C., Giovannoli, C., Arletti, E., and Giraudi, G., 2008. Development and application of solvent-free extraction for the detection of aflatoxin M1 in dairy products by enzyme immunoassay. Journal of Agricultural and Food Chemistry 56: 1852-1857.

Anfossi, L., Baggiani, C., Giovannoli, C., Biagioli, F., D’Arco, G., and Giraudi, G., 2013. Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk. Analytica Chimica Acta 772: 75-80.

AOAC official method 2000.08, 2005. Aflatoxin M1 in liquid milk, immunoaffinity column by liquid chromatography. In: Official Methods of Analysis of AOAC International, 18th edition, AOAC International. Gaithersburg, Maryland 20877- 2417, USA, 45-47.

AOAC official method 980.21, 2000. Aflatoxin M1 in milk and cheese, thin-layer chromatographic method. In: Official Methods of Analysis of AOAC International. 17th edition, volume II, AOAC International. Gaithersburg, Maryland 20877- 2417, USA, 37-38.

135

AOAC, 2012. AOAC guidelines for single laboratory validation of chemical methods for dietary supplements and botanicals. http://www.aoac.org/, Accessed May 2015.

Arast, Y., Mohammadian, M., and Behnamipour, S., 2012. Occurrence of aflatoxin M1 in two dairy products by ELISA in central part of Iran. Life Science Journal 9: 1831- 1833.

Arizona Geographical Alliance. http://www.geoalliance.asu.edu/azga, Accessed August 2015.

Asao, T., Büchi, G., Abdel-Kader, M. M., Chang, S. B., Wick, E. L., and Wogan, G. N., 1965. The structures of aflatoxins B1 and G1. Journal of the American Chemical Society 87: 882-886 1965.

Ashraf, M. W., 2012. Determination of aflatoxin levels in some dairy food products and dry nuts consumed in Saudi Arabia. Food and Public Health 2: 39-42.

Atalia, M. M., Hassanein, N. M., El-Beih, A. A., and Youssef, Y. A., 2004. Effect of Fluorescent and UV Light on Mycotoxm Production under Different Relative Humidities in Wheat Grains. Acta Pharmaceuíica Turcica 46: 205-222.

Atasever, M. A., Atasever, M., Özturan, K., and Urçar, S., 2010a. Determination of aflatoxin M1 in butter samples consumed in Erzurum, Turkey. Journal of the Faculty of Veterinary Medicine S159-S162.

Atasever, M., Atasever, M., Özturan, K., and Urcar, S., 2010b. Determination of aflatoxin M1 in UHT milk in Erzurum-Turkey. Journal of the Faculty of Veterinary Medicine 16: S119-S122.

Atehnkeng, J., Ojiambo, P. S., Ikotun, T., Sikora, R. A., Cotty, P. J., and Bandyopadhyay, R., 2008. Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize. Food Additives and Contaminants 25: 1264-1271.

Atehnkeng, J., Ojiambo, P. S., Cotty, P. J., and Bandyopadhyay, R., 2014. Field efficacy of a mixture of atoxigenic Aspergillus flavus Link: Fr vegetative compatibility groups in preventing aflatoxin contamination in maize (Zea mays L.). Biological Control 72: 62-70.

Bahamón, A. L., 2014. Evaluation of the prevalence of aflatoxin M1 (AFM1) in breast milk and its relation to dietary aflatoxin source Case study: Nabón, Ecuador. Universidad del Tolima.

Bailey, C. A., Latimer, G. W., Barr, A. C., Wigle, W. L., Haq, A. U., Balthrop, J. E., and Kubena, L. F., 2006. Efficacy of montmorillonite clay (NovaSil PLUS) for protecting full-term broilers from aflatoxicosis. The Journal of Applied Poultry Research 15: 198-206. 136

Ballard, O., and Morrow, A. L., 2014. Human milk composition: Nutrients and bioactive factors. Pediatric Clinics of North America 61: 49-74.

Banu, N., and Muthumary, J., 2010. Taxol as chemical detoxificant of aflatoxin produced by Aspergillus flavus isolated from sunflower seed. Health 2: 789.

Barbiroli, A., Bonomi, F., Benedetti, S., Mannino, S., Monti, L., Cattaneo, T., and Lametti, S., 2007. Binding of aflatoxin M1 to different protein fractions in ovine and caprine milk. Journal of Dairy Science 90: 532-540.

Barjesteh, M. H., Gholampour Azizi, I., and Noshfar, E., 2010. Occurrence of aflatoxin M1 in pasteurized and local yogurt in Mazandaran Province (Northern Iran) using ELISA. Global Veterinaria 4: 459-462.

Bascarán, V., de Rojas, A. H., Choucino, P., and Delgado, T., 2007. Analysis of ochratoxin A in milk after direct immunoaffinity column clean-up by high- performance liquid chromatography with fluorescence detection. Journal of Chromatography A 1167: 95-101.

Belhassen, H., Jiménez-Díaz, I., Arrebola, J. P., Ghali, R., Ghorbel, H., Olea, N., and Hedili, A., 2015. Zearalenone and its metabolites in urine and breast cancer risk: A case-control study in Tunisia. Chemosphere 128: 1-6.

Bennet, J. W., 2010. An overview of the genus Aspergillus. In: Machida, M., Gomi, K., eds. Aspergillus: Molecular Biology and Genomics. Norfolk, UK: Caster Academic Press, p. 1-18.

Bennett, J. W., and Klich, M., 2003. Mycotoxins. Clinical Microbiology Review 16: 497- 516.

Bennett, J.W., Lee, L.S., and Vinnett, C.H., 1971. The correlation of aflatoxin and norsolorinic acid production. Journal of American Oil Chemistry Society.48: 368- 370.

Bennett, R. A., Essigman, J. M., and Wogan, G. N., 1981. Excretion of an aflatoxin guanine adduct in the urine of aflatoxin B1-treated rats. Cancer Research 41: 650- 654.

Berthiller, F., Burdaspal, P., Crews, C., Iha, M., Krsha, R., Lattanzio, V., MacDonald, S., Malone, R. J., Maragos, C., Solfrizzo, M., Stroka, J., and Whitaker, T. B., 2014. Developments in mycotoxin analysis: an update for 2012-2013. World Mycotoxin Journal 7: 3-33.

Berthiller, F., Brera, C., Crews, C., Iha, M., Krsha, R., Lattanzio, V., MacDonald, S., Malone, R. J., Maragos, C., Solfrizzo, M., Stroka, J., and Whitaker, T. B., 2015. Developments in mycotoxin analysis: an update for 2013-2014. World Mycotoxin Journal 8: 5-35. 137

Bhatnagar, D., Cleveland, T. E., and Payne, G. A., 2000. Aspergillus flavus. In: Encyclopedia of Food Microbiology. Edited by: Robinson, R. K., Batt, C. A., and Patel, P. D., London: Academic Press. 72-79.

Biancardi, A., Piro, R., Dall’Asta, C., and Galaverna, G., 2013. A simple and reliable liquid chromatography-tandem mass spectrometry method for the determination of aflatoxin M1 in milk. Food Additives & Contaminants: Part A 30: 381-388.

Bilandžić, N., Varenina, I., and Solomun, B., 2010. Aflatoxin M1 in raw milk in Croatia. Food Control 21: 1279-1281.

Blount, W. P., 1961. Turkey “X” disease. Journal of British Turkey Federation 9: 55-58.

Boudra, H., Barnouin, J., Dragacci, S., and Morgavi, D. P., 2007. Aflatoxin M1 and ochratoxin A in raw bulk milk from French dairy herds. Journal of Dairy Science 90: 3197-3201.

Boutrif, E. 1998. Prevention of aflatoxin in pistachios. Food, Nutrition and Agriculture (FAO).

Bovo, F., Corassin, C. H., Rosim, R. E., and de Oliveira, C. A., 2013. Efficiency of lactic acid bacteria strains for decontamination of aflatoxin M1 in phosphate buffer saline solution and in skimmed milk. Food and Bioprocess Technology 6: 2230- 2234.

Bremner, J. M., 1996. Nitrogen-total. Methods of soil analysis. Part, 3. Chemical Methods, SSSA, Inc. Madison, WI, USA. 1085-1121.

Burgos-Hernandez, A., Price, R. L., Jorgensen-Kornman, K., López-García, R., Njapau, H., Park, D. L., 2002. Decontamination of AFB1-contaminated corn by ammonium persulphate during fermentation. Journal of the Science of Food and Agriculture 82: 546-552.

Busman, M., Bobell, J., and Maragos, C., 2015. Determination of the aflatoxin M1 from milk by direct analysis in real time-mass spectrometry (DART-MS). Food Control 47: 592-598.

Caloni, F., Cortinovis, C., Pizzo, F., and De Angelis, I., 2012. Transport of aflatoxin M1 in human intestinal Caco-2 cells. Frontiers in Pharmacology 3: 1-8.

Caloni, F., Stammati, A., Friggè, G., and De Angelis, I., 2006. Aflatoxin M1 absorption and cytotoxicity on human intestinal in vitro model. Toxicon 47: 409-415.

Cano-Sancho, G., Marin, S., Ramos, A. J., Peris-Vicente, J., and Sanchis, V., 2010. Occurrence of aflatoxin M1 and exposure assessment in Catalonia (Spain). Revista Iberoamericana de Micología 27: 130-135.

138

CAST, 2003. Mycotoxins: Risks in plant, animal, and human systems. Report 139. Ames, IA: Council for Agricultural Science and Technology.

Cavaliere, C., Foglia, P., Guarino, C., and Marzioni, F., 2006. Aflatoxin M1 determination in cheese by liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 1135: 135-141.

Centers for Disease Control, 2004. Outbreak of aflatoxins poisoning-eastern and central provinces, Kenya. Morbidity and Mortality Weekly Report. 53: 790-793.

Chaytor, A., See, M., Hansen, J., De Souza, A., Middleton, T., and Kim, S., 2011. Effects of chronic exposure of diets with reduced concentrations of aflatoxin and deoxynivalenol on growth and immune status of pigs. Journal of Animal Science 89: 124-135.

Chen, C. Y., Li, W. J., and Peng, K. Y., 2005. Determination of aflatoxin M1 in milk and milk powder using high-flow solid-phase extraction and liquid chromatography- tandem mass spectrometry. Journal of Agricultural and Food Chemistry 53: 8474- 8480.

Chen, R., Ma, F., Li, P. W., Zhang, W., Ding, X. X., Zhang, Q. I., Li, M., Wang, Y., and Xu, B. C., 2014. Effect of ozone on aflatoxins detoxification and nutritional quality of peanuts. Food Chemistry 146: 284-288.

Cleveland, T.E., and Bhatnagar, D., 1991. Molecular regulation of aflatoxin biosynthesis. In: Mycotoxins, Cancer and Health. Edited by: Bray, G. A., Ryan, D. H., Baton Rouge, LA, LSU Press 270-287.

Cole, R. J., Sanders, T. J., Dorner, J. W., and Blankenship, P. D., 1989. Environmental conditions required to induce pre-harvest concentration in groundnut. Summary of six years research. In: Proceedings of international workshop on aflatoxin concentration in groundnut. International Crops Research Institute for the Semi- Arid Tropics, Palancheru India. 6-9: 279-287.

Cotty, P. J., and Bhatnagar, D., 1994. Variability among atoxigenic Aspergillus flavus strains in ability to prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes. Applied and Environmental Microbiology 60: 2248-2251.

Creppy, E. E., 2002. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology Letters 127: 19–28.

Dai, J., and Mumper, R. J., 2010. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 15: 7313-7352.

139

Diao, E., Hou, H., Chen, B., Shan, C., and Dong, H., 2013. Ozonolysis efficiency and safety evaluation of aflatoxin B1 in peanuts. Food and Chemical Toxicology 55: 519-525.

Diaz, D. E., Hagler Jr., W. M., Blackwelder, J. T., Eve, J. A., Hopkins, B. A., Anderson, K. L., Jones, F. T., and Whitlow, L. W., 2004. Aflatoxin binders II: Reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed. Mycopathologia 157: 233-241.

Diaz, G. J., and Sánchez, M. P., 2015. Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia. Food Additives & Contaminants: Part A 32: 1192-1198.

Dixon, J. B., Kannewischer, I., Arvide, M. T., and Velazquez, A. B., 2008. Aflatoxin sequestration in animal feeds by quality-labeled smectite clays: An introductory plan. Applied Clay Science 40: 201-208.

De Mello, F. R., and Scussel, V. M., 2007. Characteristics of in-shell Brazil nuts and their relationship to aflatoxin contamination: criteria for sorting. Journal of Agricultural and Food Chemistry 55: 9305-9310.

De Mello, F. R., and Scussel, V. M., 2009. Development of physical and optical methods for in-shell Brazil nuts sorting and aflatoxin reduction. Journal of Agricultural Science 1: 3.

Di Natale, F., Gallo, M., and Nigro, R., 2009. Adsorbents selection for aflatoxins removal in bovine milks. Journal of Food Engineering, 95, 186-191.

Dohnal, V., Wu, Q., and Kuča, K., 2014. Metabolism of aflatoxins: key enzymes and interindividual as well as interspecies differences. Archives of Toxicology 88: 1635-1644.

Doko, B., and Park, D. L., 2008. Mycotoxin reduction and decontamination, in Mycotoxin Contamination and Control, ed. by Njapau H, et al. Bloomington: AuthorHouse, pp. 231-244.

Dorner, J. W., 2009. Biological Control of aflatoxin contamination in corn using a nontoxigenic strain of A. flavus. Journal of Food Protection 4: 696-914.

Dorner, J. W., Cole, R. J., Connick, W. J., Daigle, D. J., McGuire, M. R., and Shasha, B. S., 2003. Evaluation of biological control formulations to reduce aflatoxin contamination in peanuts. Biological Control 26: 318-324.

Dorner, J. W., Cole, R.J., Sanders, T.H., and Blankenship, P. D., 1989. Inter-relationship of kernel water activity, soil temperature, maturity, and phytoalexin production in pre-harvest aflatoxin concentration of drought-stressed peanuts. Mycopathologia 105: 117-128. 140

Dorner, J. W., Cole, R. J., and Wicklow, D. T., 1999. Aflatoxin reduction in corn through field application of competitive fungi. Journal of Food Protection 62: 567-697.

Dorner, J. W., and Horn, B. W., 2007. Separate and combined applications of nontoxigenic Aspergillus flavus and A. parasiticus for biocontrol of aflatoxin in peanuts. Mycopathologia 163: 215-223.

Duarte, S. C., Almeida, A. M., Teixeira, A. S., Falcao, A. C., Pena, A., and Lino, C. M., 2013. Aflatoxin M1 in marketed milk in Portugal: Assessment of human and animal exposure. Food Control 30: 411-417.

Duraković, L., Tudić, A., Delaš, F., and Huić-Babić, K., 2012. Schiff base: A high affinity chemical agent to decrease the concentration of aflatoxin M1 in raw milk contaminated artificially. Mljekarstvo 62: 24-34.

Edrington, T. S., Sarr, A. B., Kubena, L. F., Harvey, R. B., and Phillips, T. D., 1996. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M1 in turkey poults. Lack of effect by activated charcoal on aflatoxicosis. Toxicology Letters 89: 115-122.

Egner, P. A., Groopman, J. D., Wang, J. S., Kensler, T. W., and Friesen, M. D., 2006. Quantification of aflatoxin B1-N7-guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chemical Research in Toxicology 46: 3924-3931.

El Khoury, A., Atoui, A., and Yaghi, J., 2011. Analysis of aflatoxin M1 in milk and yogurt and AFM1 reduction by lactic acid bacteria used in Lebanese industry. Food Control 22: 1695-1699.

El Marnissi, B., Belkhou, R., Morgavi, D. P., Bennani, L., and Boudra, H., 2012. Occurrence of aflatoxin M1 in raw milk collected from traditional dairies in Morocco. Food and Chemical Toxicology 50: 2819-2821.

El Qada, E. N., Abdelghany, E., Magdy, Y. H., 2013. Utilization of activated carbon for the removal of basic dyes in fixed-bed microcolumn. International Journal of Energy and Environment 4: 815-824.

El-Tras, W. F., El-Kady, N. N., and Tayel, A. A., 2011. Infants’ exposure to aflatoxin M1 as a novel foodborne zoonosis. Food and Chemical Toxicology 49: 2816-2819.

Elsanhoty, R. M., Salam, S. A., Ramadan, M. F., and Badr, F. H., 2014. Detoxification of aflatoxin M1 in yoghurt using probiotics and lactic acid bacteria. Food Control 43: 129-134.

Elzupir, A. O., and Elhussein, A. M., 2010. Determination of aflatoxin M1 in dairy cattle milk in Khartoum State, Sudan. Food Control 21: 945-946.

141

European Commission, 2010. Commission Regulation (EU) No 165/2010 of 26 February 2010 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins. Official Journal of the European Union 50:8-12.

Fallah, A. A., 2010a. Assessment of aflatoxin M1 contamination in pasteurized and UHT milk marketed in central part of Iran. Food and Chemical Toxicology 48: 988– 991.

Fallah, A. A., 2010b. Aflatoxin M1 contamination in dairy products marketed in Iran during winter and summer. Food Control 21: 1478-1481.

Fakhoury, A. M., and Woloshuk, C. P., 1999. Amy1, the α-amylase gene of Aspergillus flavus: involvement in aflatoxin biosynthesis in maize kernels. Phytopathology 89:908–914.

Fandohan, P., Zoumenou, D., Hounhouigan, D. J., Marasas, W. F. O., Wingfield, M. J., and Hell, K., 2005. Fate of aflatoxins and fumonisins during the processing of maize into food products in Benin. International Journal of Food Microbiology 98: 249-259.

FAO, 1997. Worldwide regulations for mycotoxins in 1995: A Compendium. FAO Food and Nutrition Paper 64.

FAO, Food and Agriculture Organization, 2003. Worldwide regulations for mycotoxins in food and feed in 2003: Mycotoxin regulations in 2003 and current developments. Available at: http://www.fao.org/3/a-y5499e/y5499e07.htm. Accessed 28 September 2014.

FDA, 2000. Guidance for industry: action levels for poisonous or deleterious substances in human food and animal feed. Food and Drug Administration. Retrieved August 2015 from http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInf ormation/ChemicalContaminantsMetalsNaturalToxinsPesticides/ucm077969.htm

FDA, 2004. Office of Regulatory Affairs (ORA) Compliance Policy Guides (CPG) 7126.33 Sec. 683.100—Action levels for aflatoxin in animal feeds, Issued Nov 21, 1979, Reissued Oct 1, 1980, Revised Aug 15, 1982, May 18, 1989, and Aug 28.

FDA, 2005. Compliance Policy Guide, CPG Sec. 527.400 Whole milk, low-fat milk, skim milk: Aflatoxin M1. U.S. Food and Drug Administration. Retrieved February 25, 2015 from http://www.fda.gov/ICECI/ComplianceManuals/CompliancePolicyGuidanceMan ual/ucm074482.htm.

142

Filbert, M. E., and Brown, D. L., 2012. Aflatoxin contamination in Haitian and Kenyan peanut butter and two solutions for reducing such contamination. Journal of Hunger and Environmental Nutrition 7: 321-332.

Fink-Gremmels, J., 2008. The role of mycotoxins in the health and performance of dairy cows. The Veterinary Journal 176: 84-92.

Flores-Flores, M. E., Lizarraga, E., de Cerain, A. L., and González-Peñas, E., 2015. Presence of mycotoxins in animal milk: A review. Food Control 53: 163-176.

Fuchs, B., Süß, R., Teuber, K., Eibisch, M., and Schiller, J., 2011. Lipid analysis by thin- layer chromatography: A review of the current state. Journal of Chromatography A 1218: 2754-2774.

Gajecki, M., 2001. Zearalenone-undesirable substances in feed. Polish Journal of Veterinary sciences 5: 117-122.

Gallo, A., Masoero, F., Bertuzzi, T., Piva, G., and Pietri, A., 2010. Effect of the inclusion of adsorbents on aflatoxin B1 quantification in animal feedstuffs. Food Additives and Contaminants 27: 54-63.

Galvano, F., Pietri, A., Bertuzzi, T., Piva, A., Chies, L., and Galvano, M., 1998. Activated carbons: in vitro affinity for ochratoxin A and deoxynivalenol and relation of adsorption ability to physicochemical parameters. Journal of Food Protection 61: 469-475.

Galvano, F., Piva, A., Ritieni, A., and Galvano. G., 2001. Dietary strategies to counteract the effects of mycotoxins: A review. Journal of Food Protection J Food Prot. 64:120-131.

García-Cela, E., Ramos, A. J., Sanchis, V., and Marin, S., 2012. Emerging risk management metrics in food safety: FSO, PO. How do they apply to the mycotoxin hazard? Food Control 25: 797-808.

Garmakhany, A., Zighamian, H., Sarhangpour, R., Rasti, M., and Aghajani, N., 2011. Occurrence of aflatoxin Ml in raw and pasteurized milk in Esfahan province of Iran. Minerva Biotecnologica 23: 53-57.

Ghanem, I., Orfi, M., and Shamma, M., 2008. Effect of gamma radiation on the inactivation of aflatoxin B1 in food and feed crops. Brazilian Journal of Microbiology 39: 787-791.

Gomaa, M. N. E., Ayesh, A. M., Galil, M. A., and Naguib, K., 1997. Effect of high pressure ammoniation procedure on the detoxification of aflatoxins. Mycotoxin Research 13: 23-34.

143

González Pereyra, M. L., Carvalho, E. C. Q., Tissera, J. L., Keller, K. M., Magnoli, C. E., Rosa, C. A. R., Dalcero, A. M., and Cavaglieri, L. R., 2008. An outbreak of acute aflatoxicosis on a chinchilla (Chinchilla lanigera) farm in Argentina. Journal of Veterinary Diagnostic Investigation 20: 853-856.

Guan, D., Li, P., Zhang, Q., Zhang, W., Zhang, D., and Jiang, J., 2011. An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products. Food Chemistry 125: 1359-1364.

Guo, Y., Yuan, Y., and Yue, T., 2013. Aflatoxin M1 in milk products in China and dietary risk assessment. Journal of Food Protection 76: 849-853.

Gürbay, A., Engin, A. B., Çağlayan, A., and Şahin, G., 2006. Aflatoxin M1 levels in commonly consumed cheese and yogurt samples in Ankara, Turkey. Ecology of Food and Nutrition 45: 449-459.

Gutiérrez, R., Rosell, P., Vega, S., Pérez, J., Ramírez, A., and Coronado, M., 2013. Self and foreign substances in organic and conventional milk produced in the eastern region of Mexico. Food and Nutrition Sciences 4: 586-593.

Hadavi, E., 2005. Several physical properties of aflatoxin-contaminated pistachio nuts: Application of BGY fluorescence for separation of aflatoxin-contaminated nuts. Food additives and contaminants 22: 1144-1153.

Han, R. W., Zheng, N., Wang, J. Q., Zhen, Y. P., Xu, X. M., and Li, S.L., 2013. Survey of aflatoxin in dairy cow feed and raw milk in China. Food Control 34:35-39.

Harris, P. J., Liu, Z., and Suenaga, K., 2008. Imaging the atomic structure of activated carbon. Journal of Physics: Condensed Matter 20: 1-5.

Hassan, H. F., and Kassaify, Z., 2014. The risks associated with aflatoxins M1 occurrence in Lebanese dairy products. Food Control 37: 68-72.

Herzallah, S. M., 2009. Determination of aflatoxins in eggs, milk, meat, and meat products using HPLC fluorescent and UV detectors. Food Chemistry 114: 1141- 1146.

Herzallah, S., Alshawabkeh, K., and Fataftah, A. A., 2008. Aflatoxin decontamination of artificially contaminated feeds by sunlight, γ-Radiation, and microwave heating. The Journal of Applied Poultry Research 17: 515-521.

Hruska, Z., Rajasekaran, K., Yao, H., Kincaid, R., Darlington, D., Brown, R. L., Bhatnagar, D., and Cleveland, T. E., 2014. Co-inoculation of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus to study fungal invasion, colonization, and competition in maize kernels. Frontiers in Microbiology 5.

144

Hsu, I. C., Metcalf, A., Sun, T., Welsh, J. A., Wang, N. J., and Harris, C. C., 1991. Mutational hot spot in the p53 gene in human hepatocellular carcinomas. Nature 350: 427-428.

Huang, L. C., Zheng, N., Zheng, B. Q., Wen, F., Cheng, J. B., Han, R. W., Xu, X. M., Li, S. L., and Wang, J. Q., 2014. Simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and α-zearalenol in milk by UHPLC-MS/MS. Food Chemistry 146: 242-249.

Hussain, I., 2011. Aflatoxin measurement and analysis. In: Torres-Pacheco, I., (ed.) Aflatoxins: Detection, measurement, and control. InTech Open Access Publisher 130-146.

Huppertz, T., and Kelly, A. L., 2009. Properties and constituents of cow’s milk. In: Tamime, A. Y., (ed.), Milk Processing and Quality Management. Blackwell Publishing: Malden, MA pp. 23-47.

Iha, M. H., Barbosa, C. B., and Favaro, R. M. D., 2011. Chromatographic method for the determination of aflatoxin M1 in cheese, yogurt, and dairy beverages. Food Chemical Contaminants 94: 1513-1518.

Iha, M. H., Barbosa, C. B., Okada, I. A., and Trucksess, M. W., 2013. Aflatoxin M1 in milk and distribution and stability of aflatoxin M1 during production and storage of yoghurt and cheese. Food Control 29: 1-6.

Inan, F., Pala, M., and Doymaz, I., 2007. Use of ozone in detoxification of aflatoxin B1 in red pepper. Journal of Stored Products Research 43: 425-429.

International Agency for Research on Cancer (IARC), 1993. Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monographs of Carcinogenic Risks to Humans 56: 397-444.

International Agency for Research on Cancer (IARC), 2002. Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. IARC Monographs Evaluation of Carcinogenic Risks to Humans. 82: 1-556.

International Dairy Federation, IDF 171:1995. Milk and milk powder. Determination of aflatoxin M1 content. Clean-up by immunoaffinity chromatography and determination by HPLC. International IDF Standard No. 171: 1995. Brussels: IDF.

International Organization for Standardization, EN ISO 14501:2007. Milk and milk powder. Determination of aflatoxin M1 content. Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography, ISO/DIS 14501, IDF 171. Geneva: ISO.

145

Iqbal, S. Z., and Asi, M. R., 2013. Assessment of aflatoxin M1 in milk and milk products from Punjab, Pakistan. Food Control 30: 235-239.

Isman, B., and Biyik, H., 2009. The aflatoxin contamination of fig fruits in Aydin City (Turkey. Journal of Food Safety 29: 318-330.

Jacobson, W. C., Harmeyer, W. C., and Wiseman, H. G., 1971. Determination of aflatoxins B1 and M1 in milk. Journal of Dairy Science 54: 21-24.

Jafarian-Dehkordi, A., and Pourradi, N., 2013 Aflatoxin M1 contamination human breast milk in Isfahan, Iran. Advanced Biomedical Research 2: 86.

Jager, A. V., Tedesco, M. P., Souto, P. C. M. C., and Oliveira, C. A. F., 2013. Assessment of aflatoxin intake in in São Paulo Brazil. Food Control 33: 87-92.

Jaime-Garcia, R., and Cotty, P.J., 2003. Aflatoxin contamination in commercial cottonseed in South Texas. Phytopathology 93, 1190–1200.

Jalili, M., Jinap, S., and Noranizan, M. A., 2012. Aflatoxins and ochratoxin a reduction in black and white pepper by gamma radiation. Radiation Physics and Chemistry 81: 1786-1788.

Jalili, M., Jinap, S., and Noranizan, A., 2010. Effect of gamma radiation on reduction of mycotoxins in black pepper. Food Control 21: 1388-1393.

Jard, G., Liboz, T., Mathieu, F., Guyonvarc’h, A., and Lebrihi, A., 2011. Review of mycotoxin reduction in food and feed: from prevention in the field to detoxification by adsorption or transformation. Food Additives and Contaminants: Part A 28: 1590-1609.

Jasutiene, I., Kulikauskiene, M., and Garmiene, G., 2007. Stability of aflatoxin M1 during production of fermented dairy products. Veterinary Medicine and Zootechnics 37: 20-23.

Jaynes, W. F., and Zartman, R. E., 2011. Aflatoxin toxicity reduction in feed by enhanced binding to surface-modified clay additives. Toxins 3: 551-565.

Johnston, C. I., Singleterry, R., Reid, C., Sparks, D., Brown, A., Baldwin, B., Ward, S. H., and Williams, W. P., 2012. The fate of aflatoxin in corn fermentation. Natural Resources 3: 126-136.

Kabak, B., and Dobson, A. D. W., 2009. Biological strategies to counteract the effects of mycotoxins. Journal of Food Protection 9: 1812-2016.

Kafle, P., Sedai, D., Rai, K. P., and Pokharel, B. B., 2014. Study on the level of aflatoxin M1 contamination in raw and processed milk marketed in Kathmandu Valley. Journal of Food Science and Technology Nepal 7: 52-56. 146

Kaleibar, M. T. and Helan J. A., 2013. A field outbreak of aflatoxicosis with high fatality rate I feedlot calves in Iran. Comparative Clinical Pathology 22: 1155-1163.

Kamkar, A. 2005. A study on the occurrence of aflatoxin M1 in raw milk produced in Sarab city of Iran. Food Control 16: 593-599.

Kara, R., and Ince, S., 2014. Aflatoxin M1 in buffalo and cow milk in Afyonkarahisar, Turkey. Food Additives & Contaminants: Part B 7: 7-10.

Karanfil, T., and Kilduff, J. E., 1999. Role of granular activated carbon surface chemistry on the adsorption of organic compounds. 1. Priority pollutants. Environmental Science & Technology 33: 3217-3224.

Karaseva, N. M., Amelin, V. G., and Tret’yakov, A. V., 2014. QuEChERS coupled to dispersive liquid-liquid microextraction for the determination of aflatoxins B1 and M1 in dairy foods by HPLC. Journal of Analytical Chemistry 69: 461-466.

Kav, K., Col, R., and Tekinsen, K. K., 2011. Detection of aflatoxin M1 levels by ELISA in white-brined Urfa cheese consumed in Turkey. Food Control 22: 1883-1886.

Kensler, T. W., Egner, P. A., Davidson, N. E., Davidson, N. E., Roebuck, B. D., Pikul, A., and Groopman, J.D., 1986. Modulation of aflatoxin metabolism, aflatoxin-N7- guanineformation, and hepatic tumorigenesis in rats fed ethoxyquin: Role of induction of glutathione s-transferases. Cancer Research 46: 3924-3931.

Kensler, T. W., Roebuck, B. D., Wogan, G. N., and Groopman, J. D., 2011. Aflatoxin: A 50-year odyssey of mechanistic and translational toxicology. Toxicological Sciences 120: S28-S48.

Khoshnevis, S. H., Azizi, I. G., Shateri, S., and Mousavizadeh, M., 2012. Determination of the aflatoxin M1 in ice cream in Babol City (Northern Iran). Global Veterinaria 8: 205-208.

Khosravi, A. R., Shokri, H., Eshghi, S., and Darvishi, S., 2013. Global occurrence of aflatoxin M1 in milk with particular reference to Iran. Food Security 5: 533-539.

Klich, M. A., 2002. Biogeography of Aspergillus species in soil and litter. Mycologia 94: 21-27.

Klich, M. A., Tiffany, L. H., and Knaphus, G., 1992. Ecology of the Aspergilli of soils and litter. In: Bennet J. W., Klich, M. A., eds. Aspergillus: biology and industrial applications. Boston, MA: Nutterworth Heineman p. 329-354.

King, J. M., Prudente, Jr., A. D., 2005. Chemical detoxification of aflatoxins in food and feeds, in Aflatoxin and Food Safety, ed. Abbas HK. Boca Raton: CRC Press, pp. 543-550.

147

King, R., Bonfiglio, R., Fernandez-Metzler, C., Miller-Stein, C., Olah, T., 2000. Mechanistic investigation of ionization suppression in electrospray ionization. Journal of the American Society for Mass Spectrometry 11: 942-950.

Kocasari, F. S., Tasci, F., and Mor, F., 2012. Survey of aflatoxin M1 in milk and dairy products consumed in Burdur, Turkey. International Journal of Dairy Technology 65: 365-371.

Kos, J., Lević, J., Đuragić, O., Kokić, B., and Miladinović, I., 2014. Occurrence and estimation of aflatoxin M1 exposure in milk in Serbia. Food Control 38: 41-46.

Kowalski, J., Lupo, S., Cochran, J., 2014. Mitigating matrix effects: Examination of dilution, QuEChERS, and calibration strategies for LC-MS/MS analysis of pesticide residues in diverse food types. http://www.restek.com, Accessed August 2015.

Kozakiewicz, Z., and Smith, D., 1994. Physiology of Aspergillus. In: Aspergillus. Edited by: Smith, J. K., New York: Plenum Press. 23-38.

Krishnamachari, K. A. V. R., Nagarajan, V., Bhat, R. V., and Tilak, T. B. G., 1975. Hepatitis due to aflatoxicosis: An outbreak in Western India. Lancet 305: 1061- 1063.

Kutz, R. E., Sampson, J. D., and Pompeu, L. B., 2009. Efficacy of Solis, NovasiPlus, and MTB-100 to reduce aflatoxin M1 levels in milk of early to mid-lactation dairy cows fed aflatoxin B1. Journal of Dairy Science 92: 3959-3963.

Latha, R., Manonmani, H. K., and Rati, E. R., 2008. Multiplex PCR assay for the detection of aflatoxigenic and non-aflatoxigenic Aspergilli. Research Journal of Microbiology 3: 136-142.

Le Blanc, J. C., Tard, A., Volatier, J. L., and Verger, P., 2005. Estimated dietary exposure to principal food mycotoxins from The First French Total Diet Study. Food Additives & Contaminants 22: 652-672.

Lee, B. N., and Adams, T. H., 1994. Overexpression of flbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and premature initiation of development. Molecular Microbiology 14: 323-334.

Lee, D., and Lee, K., 2015. Analysis of aflatoxin M1 and M2 in commercial dairy products using high-performance liquid chromatography with a fluorescence detector. Food Control 50: 467-471.

Lehotay, S. J., Son, K.A., Kwon, H., Koesukwiwat, U., Fu, W., Mastovska, K., Hoh, E., Leepipatpiboon, N. Comparison of QuEChERS sample preparation methods for the analysis of pesticide residues in fruits and vegetables. Journal of Chromatography A. 2010, 1217, 2548-2560. 148

Lewis, L., Onsongo, M., Njapau, H., Schurz-Rogers, H., Luber, G., Kieszak, S., Nyamongo, J., Backer, L., Dahiye, A. M., Misore, A., DeCock, K., and Rubin, C., 2005. Aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in eastern and central Kenya. Environmental Health Perspectives 113:1763-1767.

Li, J. J., Suo, D. C., and Su, X. O., 2010. Binding capacity for aflatoxin b1 by different adsorbents. Agricultural Sciences in China 9: 449-456.

Lillard, D. A., and Lantin, R. S., 1970. Some chemical characteristics and biological effects of photomodified aflatoxins. Journal of the Association of Official Chemists 53: 1060-1063.

Liu, Y., and Wu, F., 2010. Global burden of aflatoxin-induced hepatocellular carcinoma: A risk assessment. Environmental Health Perspectives 118: 818-824.

Liu, D., Huang, Y., Wang, S., Liu, K., Chen, M., Xiong, Y., Yang, W., and Lai, W. 2015. A modified lateral flow immunoassay for the detection of trace aflatoxin M1 based on immunomagnetic nanobeads with different antibody concentrations. Food Control 51: 218-224.

Liu, H., Liu, D., Yuan, P., Tan, D., Cai, J., He, H., Zhu, J., and Song, Z., 2013. Studies on the solid acidity of heated and cation-exchanged montmorillonite using n- butylamine titration in non-aqueous system and diffuse reflectance Fourier transform infrared (DRIFT) spectroscopy. Physics and Chemistry of Minerals 40: 479-489.

Liu, R., Jin, Q., Tao, G., Shan, L., Liu, Y., and Wang, X., 2010. LC–MS and UPLC– quadrupole time-of-flight MS for identification of photodegradation products of aflatoxin B1. Chromatographia 71: 107-112.

Lu, B., Ren, Y., Huang, B., Liao, W., Cai, Z., and Tie, X., 2008. Simultaneous determination of four water-soluble vitamins in fortified infant foods by ultra- performance liquid chromatography coupled with triple quadrupole mass spectrometry. Journal of Chromatography Science 46: 225-232.

Lunadei, L., Ruiz-Garcia, L., Bodria, L., and Guidetti, R., 2013. Image-based screening for the identification of bright greenish yellow fluorescence on pistachio nuts and cashews. Food and Bioprocess Technology 6: 1261-1268.

Magan, N., and Aldred, D., 2007. Post-harvest control strategies: Minimizing mycotoxins in the food chain. International Journal of Food Microbiology 119: 131-139.

Magbanua, Z. V., De Moraes, C. M., Brooks, T. D., Williams, W. P., and Luthe, D. S., 2007. Is catalase activity one of the factors associated with maize resistance to Aspergillus flavus? Molecular Plant-Microbe Interactions Journal 20: 697-706.

149

Magnoli, A. P., Monge, M. P., Miazzo, R. D., Cavaglieri, L. R., Magnoli, C. E., Merkis, C. I., ... and Chiacchiera, S. M., 2011. Effect of low levels of aflatoxin B1 on performance, biochemical parameters, and aflatoxin B1 in broiler liver tissues in the presence of monensin and sodium bentonite. Poultry Science 90: 48-58.

Magoha, H., Kimanya, M., De Meulenaer, B., Roberfroid, D., Lachat, C., and Kolsteren, P., 2014. Association between aflatoxin M1 exposure through breast milk and growth impairment in infants from Northern Tanzania. World Mycotoxin Journal 7: 277-284.

Manafi, M., and Khosravinia, H., 2012. Effects of aflatoxin on the performance of broiler breeders and its alleviation through herbal mycotoxin binder. Journal of Agricultural Science and Technology 15: 55-63.

Manetta, A. C., 2011. Aflatoxins: Their measure and analysis. In: Torres-Pacheco, I., ed., Aflatoxins: Detection, measurement, and control. ISBN 978-953-307-711-6, InTech Open Access Publisher, 93-108.

Manetta, A. C., Di Giuseppe, L., Giammarco, M., Fusaro, I., Simonella, A., Gramenzi, A., and Formigoni, A., 2005. High-performance liquid chromatography with post- column derivatisation and fluorescence detection for sensitive determination of aflatoxin M1 in milk and cheese. Journal of Chromatography A 1083: 219-222.

Manetta, A.C., Giammarco, M., Di Giuseppe, L., Fusaro, I., Gramenzi, A., Formigoni, A., Vignola, G., and Lambertini, L., 2009. Distribution of aflatoxin M1 during Grana Padano cheese production from naturally contaminated milk. Food Chemistry 113: 595-599.

Marin, S., Ramos, A. J., Cano-Sancho, G., and Sanchis, V., 2013. Mycotoxins: Occurrence, toxicology, and exposure assessment. Food and Chemical Toxicology 60: 218-237.

Martins, H. M., Magalhaes, S. A., Almeida, I., Marques, M., Guerra, M. M., and Bernardo, F., 2007. Aflatoxin M1 determination in cheese by immunoaffinity column clean-up coupled to high-performance liquid chromatography. Ciência Veterinarias 102: 321-325.

Masoero, F., Moschini, M., Gallo, A., and Diaz, D., 2010. In vivo release of aflatoxin B1 bound to different sequestering agents in dairy cows. Italian Journal of Animal Science 6: 315-317.

Mateo, R., Medina, Á., Mateo, E. M., Mateo, F., and Jiménez, M., 2007. An overview ochratoxin A in beer and wine. International Journal of Food Microbiology 119: 79-83.

150

McKean, C., Tang, L., Tang, M., Billam, M., Wang, Z., Theodorakis, C. W., Kendall, R. J., and Wang, J. S., 2006. Comparative acute and combinative toxicity of aflatoxin B1 and fumonisin B1 in animals and human cells. Food and Chemical Toxicology 44: 868–876.

McKenzie, K. S., Sarr, A. B., Mayura, K., Bailey, R. H., Miller, D. R., Rogers, T. D., Nored, W. P., Voss, K. A., Plattner, R. D., Kubena, L. F. and Phillips, T. D., 1997. Oxidative degradation and detoxification of mycotoxins using a novel source of ozone. Food and Chemical Toxicology 35: 807-820.

Mclean, M., and Dutton, M. F., 1995. Cellular interactions and metabolism of aflatoxin: an update. Pharmacology & Therapeutic 65: 163-192.

McMillian, W. W., 1987. Relation of insects to aflatoxin contamination in maize grown in the southeastern USA. In: Zuber, M. S., Lillehoj, E. B., Renfro, B. L., eds., Aflatoxin in maize: A proceeding of the workshop. Mexico, Mexico: The International Maize and Wheat Improvement Centre p. 194-200.

McMillian, W.W., Wilson, D. M., and Widstrom, N. W., 1985. Aflatoxin contamination of preharvest corn in Georgia: A six-year study of insect damage and visible Aspergillus flavus. Journal of Environmental Quality 14: 200-202.

Meunier, B., deVisser, S. P., and Shaik, S., 2004. Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes. Chemical Reviews 104: 3947-3980.

Milani JM, 2013. Ecological conditions affecting mycotoxin production in cereals: a review. Veterinarni Medicina 58: 405-411.

Mishra, H. N., and Das, C., 2003. A review of biological control and metabolism of aflatoxin. Critical Reviews in Food Science and Nutrition 43: 245-264.

Mitchell, N. J., Xue, K. S., Lin, S., Marroquin‐Cardona, A., Brown, K. A., Elmore, S. E., Tang, L., Romoser, A., Gelderblom, W. C. A., Wang, J., and Phillips, T. D., 2014. Calcium montmorillonite clay reduces AFB1 and FB1 biomarkers in rats exposed to single and co‐exposures of aflatoxin and fumonisin. Journal of Applied Toxicology 34: 795-804.

Mobeen, A. K., 2011. Aflatoxins B1 and B2 contamination of peanut and peanut products and subsequent microwave detoxification. Journal of Pharmacy and Nutrition Sciences 1: 1-3.

Monaci, L., Vatinno, R., and De Benedetto. G., 2007. Fast detection of cyclopiazonic acid in cheese using Fourier transform mid-infrared ATR spectroscopy. European Food Research and Technology 225: 585-588.

151

Montaseri, H., Arjmandtalab, S., Dehghanzadeh, G., Karami, S., Razmjoo, M. M., Sayadi, M., and Oryan, A., 2014. Effect of production and storage of probiotic yogurt on aflatoxin M1 residue. Journal of Food Quality and Hazards Control 1: 7- 14.

Nachtmann, C., Gallina, S., Rastelli, M., Ferro, G. L., and Decastelli, L., 2007. Regional monitoring plan regarding the presence of aflatoxin M1 in pasteurized and UHT milk in Italy. Food Control 18: 623-629.

Narasaiah, K. V., Sashidhar, R. B., and Subramanyam, C., 2006. Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates. Mycopathologia 162:179-189.

Navas, S. A., Sabino, B., and Rodriguez-Amaya, D. B., 2005. Aflatoxin M1 and ochratoxin A in a human milk bank in the city of Sao Paulo, Brazil. Food Additives & Contaminants 22: 457-462.

Neal, G. E., Eaton, D. L., Judah, D. J., and Verma, A., 1998. Metabolism and toxicity of aflatoxins M1 and B1 in human-derived in vitro systems. Toxicology and Applied Pharmacology 151: 152-158.

Nebert, D. W., and Vasiliou, V., 2004. Analysis of the glutathione S-transferase (GST) gene family. Human Genomics 1: 460-464.

Nemati, M., Mehran, M. A., Hamed, P. K., and Masoud, A., 2010. A survey on the occurrence of aflatoxin M1 in milk samples in Ardabil, Iran. Food Control 21: 1022-1024.

Nesci, A., Rodriguez, M., and Etcheverry, M., 2003. Control of Aspergillus growth and aflatoxin production using antioxidants at different conditions of water activity and pH. Journal of Applied Microbiology 95: 279-287.

Newman, S. J., Smith, J. R., Stenske, K. A., Newman, L. B., Dunlap, J. R., Imerman, P. M., and Kirk, C. A., 2007. Aflatoxicosis in nine dogs after exposure to contaminated commercial dog food. Journal of Veterinary Diagnostic Investigation 19:168-175.

Nuryono, N., Agus, A., Wedhastri, S., Maryudhani, Y. M. S., Pranowo, D., Yunianto, Y., and Razzazi-Fazeli, E., 2012. Adsorption of aflatoxin B1 in corn on natural zeolite and bentonite. Indonesian Journal of Chemistry 12: 279-286.

Nyamete, F. A., 2013. Potential of lactic acid fermentation in reducing aflatoxin B1 and fumonisin B1 in Tanzanian maize-based complementary gruel (Master’s Thesis). Retrieved from ProQuest Dissertations and Theses. (UMI Number: 1547710).

Oluwafemi, F., Odebiyi, T., and Kolapo, A., 2012. Occupational aflatoxin exposure among feed mill workers in Nigeria. World Mycotoxin Journal 5: 385-289. 152

Omar, S. S., 2012. Incidence of aflatoxin M1 in human and animal milk in Jordan. Journal of Toxicology and Environmental Health, Part A 75: 1404-1409.

Ortatali, M., Oğuz, H., Hatipoğlu, F., and Karaman, M., 2005. Evaluation of pathological changes in broilers during chronic aflatoxin (50 and 100 ppb) and clinoptilolite exposure. Research in Veterinary Science 78: 61-68.

Öztürk, B., Celik, F., Celik, Y., Kabaran, S., and Ziver, T., 2014. To determine the occurrence of aflatoxin M1 (AFM1) in samples of Cyprus traditional cheese (halloumi): A cross-sectional study. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 20: 773-778.

Pasha, T. N., Farooq, M. U., Khattak, F. M., Jabbar, M. A., and Khan, A. D., 2007. Effectiveness of sodium bentonite and two commercial products as aflatoxin absorbents in diets for broiler chickens. Animal Feed Science and Technology 132: 103-110.

Paterson, R. R. M., and Lima, N., 2011. Further mycotoxin effects from climate change. Food Research International 44: 2555-2566.

Patterson, D. S. P., Glancy, E. M., and Roberts, B. A., 1980. The ‘carry over’ of aflatoxin M1 into the milk of cows fed rations containing a low concentration of aflatoxin B1. Food and Cosmetics Toxicology 18: 35-37.

Paya, P., and Anastassiades, M., 2007. Analysis of pesticide residues using the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) pesticide multiresidue method in combination with gas and liquid chromatography and tandem mass spectrometric detection. Analytical and Bioanalytical Chemistry 389: 1697-1714.

Pearson, T. C., Wicklow, D. T., and Brabec, D. L., 2010. Technical Note: Characteristics and Sorting of White Food Corn Contaminated with Mycotoxins. Applied Engineering in Agriculture 26: 109-113.

Pearson, T. C., Wicklow, D. T., and Pasikatan, M. C., 2004. Reduction of aflatoxin and fumonisin contamination in yellow corn by high-speed dual-wavelength sorting. Cereal Chemistry 81: 490-498.

Pérez‐Flores, G. C., Moreno‐Martínez, E., and Méndez‐Albores, A., 2011. Effect of microwave heating during alkaline‐cooking of aflatoxin contaminated maize. Journal of Food Science 76: T48-T52.

Pestka, J. J., and Smolinski, A. T., 2005. Deoxynivalenol: toxicology and potential effects on humans. Journal of Toxicology and Environmental Health, Part B 8: 39-69.

153

Pfeiffer, E., Fleck, S. C., and Metzler, M., 2014. Catechol formation: A novel pathway in the metabolism of sterigmatocystin and 11-methoxysterigmatocystin. Chemical Research in Toxicology 27: 2093-2099.

Pfohl‐Leszkowicz, A., and Manderville, R. A., 2007. Ochratoxin A: An overview on toxicity and carcinogenicity in animals and humans. Molecular nutrition & food research 51: 61-99.

Phillips, T. D., 1999. Dietary clay in the chemoprevention of aflatoxin-induced disease. Toxicological Sciences 52(suppl 1): 118-126.

Phillips, T. D., Afriyie-Gyawu, E., Williams, J., Huebner, H., Ankrah, N., Ofori-Adjei, D., Jolly, P., Johnson, N., Taylor, J., Marroquin-Cardona, A., Xu, L., Tang, L., and Wang J., 2008. Reducing human exposure to aflatoxin through the use of clay: A review. Food Additives and Contaminants: Part A 25: 134-135.

Picinin, L. C. A., Cerqueira, M. M. O. P., Vargas, E. A., Lana, Â. M. Q., Toaldo, I. M., and Bordignon-Luiz, M. T., 2013. Influence of climate conditions on aflatoxin M1 contamination in raw milk from Minas Gerais State, Brazil. Food Control 31: 419-424.

Pimpukdee, K. L. F. K., Kubena, L. F., Bailey, C. A., Huebner, H. J., Afriyie-Gyawu, E., and Phillips, T. D., 2004. Aflatoxin-induced toxicity and depletion of hepatic vitamin A in young broiler chicks: Protection of chicks in the presence of low levels of NovaSil PLUS in the diet. Poultry Science 83: 737-744.

Pitt, J. I., Taniwaki, M. H., and Cole, M. B., 2013. Mycotoxin production in major crops as influenced by growing, harvesting, storage and processing, with emphasis on the achievement of Food Safety Objectives. Food Control 32: 205-215.

Pittet, A., 2005. Modern methods and trends in mycotoxin analysis. Mitteilungen aus Lebensmitteluntersuchung und Hygiene 96: 424-444.

Prandini, A., Tansini, G., Sigolo, S., Fillippi, L., Laporta, M., and Piva, G., 2009. On the occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology 47: 984-991.

Prawat Tanboon-ek, 1991. Control of aflatoxin in maize. In: Semple, R. L., Firo, A. S., Hicks, P. A., and Lozare, J. V., (eds.). Mycotoxin prevention and control in food- grains. UNDP/FAO.

Piva, G., Galvano, F., and Pietri A, 1995. Detoxification method of aflatoxins: A review. Nutrition research 15: 767-76

Proctor, A. D., Ahmedna, M., Kumar, J. V., and Goktepe, I., 2004. Degradation of aflatoxins in peanut kernels/flour by gaseous ozonation and mild heat treatment. Food Additives and Contaminants 21: 786-793. 154

Rafiei, H., Dehghan, P., Pakshir, K., Pour, M. C., an Akbari, M., 2014. The concentration of aflatoxin M1 in the mothers’ milk in Khorrambid City, Fars, Iran. Advanced Biomedical Research 3: 152.

Rao, S. N., and Chopra, R. C., 2007. Effect of supplementing sodium bentonite or activated charcoal on mineral balances in growing male goats receiving diets with or without added aflatoxin B1. Indian Journal of Animal Sciences 77: 617-620.

Rao, S. N., and Chopra, R. C., 2001. Influence of sodium bentonite and activated charcoal on aflatoxin M 1 excretion in milk of goats. Small Ruminant Research, 41(3), 203-213.

Reddy, K. R. N., Reddy, C. S., and Muralidharan, K., 2009. Potential of botanicals and biocontrol agents on growth and aflatoxin production by Aspergillus flavus infecting rice grains. Food Control 20: 173-178.

Reverberi, M., Ricelli, A., Zjalic, S., Fabbri, A. A., and Fanelli, C., 2010. Natural functions of mycotoxins and control of their biosynthesis in fungi. Applied Microbiology and Biotechnology 87: 899-911.

Reverberi, M., Zjalic, S., Ricelli, A., Punelli, F., Camera, E., Fabbri, C., Picardo, M., Fanelli, C., and Fabbri, A. A., 2008 Modulation of antioxidant defense in Aspergillus parasiticus is involved in aflatoxin biosynthesis: a role for the ApyapA gene. Eukaryotic Cell 7:988-1000.

Reverberi, M., Zjalic, S., Ricelli, A., Fabbri, A. A., and Fanelli, C., 2006. Oxidant/antioxidant balance in Aspergillus parasiticus affects aflatoxin biosynthesis. Mycotoxin Research, 22: 39-47.

Reybroeck, W., Ooghe, S., Saul, S. J., and Salter, R. S., 2014. Validation of a lateral flow test (MRLAFMQ) for the detection of aflatoxin M1 at 50 ng/L in raw commingled milk. Food Additives & Contaminants: Part A 31: 2080-2089.

Richard, J. L., 2007. Some major mycotoxins and their mycotoxicoses: An overview. Journal of Food Microbiology 119: 3-10.

Robens, J., and Cardwell, K., 2003. The costs of mycotoxin management to the USA: Management of aflatoxins in the United States. Journal of Toxicology 22:139- 152.

Ruangwises, N., and Ruangwises, S., 2010. Aflatoxin M1 contamination in raw milk within the central region of Thailand. Bulletin of Environmental Contamination and Toxicology 85: 195-198.

Ruangwises. N., Saipan, P., Ruangwises, S., 2011. Estimated daily intake of aflatoxin M1 in Thailand. In: Guevara-González R. G., (ed.). Aflatoxins: Biochemistry and molecular biology. , InTech Open Access Publisher, 439-446. 155

Ruangwises, S., and Ruangwises, N., 2009. Occurrence of aflatoxin M1 in pasteurized milk of the school milk project in Thailand. Journal of Food Protection 72: 1761- 1763.

Rubert, J., León, N., Sáez, C., Martins, C. P., Godula, M., Yusà, V., Mañes, J., Soriano, J. M., and Soler, C., 2014. Evaluation of mycotoxins and their metabolites in human breast milk using liquid chromatography coupled to high resolution mass spectrometry. Analytica Chimica Acta 820: 39-46.

Rubio, R., Licon, C. C., Berruga, M. I., Molina, M. P., and Molina, A., 2011. Short communication: Occurrence of aflatoxin M1 in the Manchego cheese supply chain. Journal of Dairy Science 94: 2775-2778.

Sabater-Vilar, M., Malekinejad, H., Selman, M. H. J., Van der Doelen, M. A. M., and Fink-Gremmels, J., 2007. In vitro assessment of adsorbents aiming to prevent deoxynivalenol and zearalenone mycotoxicoses. Mycopathologia 163: 81-90.

Sani, A., Khezri, M., and Moradnia, H., 2012. Determination of aflatoxin in milk by ELISA technique in Mashad (Northeast of Iran). ISRN Toxicology 2012: Article ID 121926.

Santini, A., Raiola, A., Ferrantelli, V., Giangrosso, G., Macaluso, A., Bognanno, M., Galvano, F, and Ritieni, A., 2013. Aflatoxin M1 in raw, UHT milk and dairy products in Sicily (Italy). Food Additives & Contaminants: Part B 6: 181-186.

Santos, A., Bando, E., and Machinski Jr., M., 2014. Occurrence of aflatoxin M1 in bovine milk commercialized in the Parana State, Brazil. Semina: Ciências Agrárias (Londrina) 35: 371-374.

Sargeant, K., Sheridan, A., O’Kelly, J., and Carnaghan, R. B. A., 1961. Toxicity associated with certain samples of groundnuts. Nature 192: 1096-1097.

Sarr, A.B., Clement, B.A., and Phillips, T.D., 1990. Effects of molecular structure on the chemisorption of aflatoxin B1 and related compounds by hydrated sodium calcium aluminosilicate. Toxicologist 10: 163.

Scarpari, M., Punelli, M., Scala, V., Zaccaria, M., Nobili, C., Ludovici, M., Camera, E., Fabbri, A. A., Reverberi, M., and Fanelli, C., 2014. Lipids in Aspergillus flavus- maize interaction. Frontiers in Microbiology 5.

Schaafsma, A. W., & Hooker, D. C. (2007). Climatic models to predict occurrence of Fusarium toxins in wheat and maize. International Journal of Food Microbiology 119: 116-125.

Schatzki, T. F., and Ong, M. S., 2000. Distributions in almonds with heavy insect damage. Journal of Agricultural and Food Chemistry 48: 489-492.

156

Scheidegger, K. A., and Payne, G. A., 2003. Unlocking the secrets behind secondary metabolism: A review of Aspergillus flavus from pathogenicity to functional genomics. Journal of Toxicology 22: 423-459.

Scott, B., and Eaton, C. J., 2008. Role of reactive oxygen species in fungal cellular differentiations. Current Opinion in Microbiology 11:488-493.

Sefidgar, S. A. A., Mirzae, M., Assmar, M., and Naddaf, S. R., 2011. Aflatoxin M1 in pasteurized milk in Babol city, Mazandaran Province, Iran. Iranian Journal of Public Health, 40: 115-118.

Segal, B. H., 2009. Aspergillosis. New England Journal of Medicine 360: 1870-1884.

Shan, X., and Williams, W. P., 2014. Toward elucidation of genetic and functional genetic mechanisms in corn host resistance to Aspergillus flavus infection and aflatoxin contamination. Frontiers in Microbiology 5.

Shebl, M. A., Naglaa, A. H., and Motawe, H. F. A., 2010. Genotoxic studies of yeast cell wall and hydrated sodium calcium aluminosilicate (HSCAS) on the DNA damage chromosomal aberrations induced by aflatoxin in broiler. Journal of American Science 6: 961-967.

Shephard, G. S., 2008. Impact of mycotoxins on human health in developing countries. Food Additives & Contaminants 25: 146-151.

Shephard, G.S., 2009. Aflatoxin analysis at the beginning of the twenty-first century. Analytical and Bioanalytical Chemistry 395: 1215-1224.

Shephard, G. S., Berthiller, F., Dorner, J., Krska, R., Lombaert, G. A., Malone, B., Maragos, C., Sabino, M., Solfrizzo, M., Trucksess, M. W., Van Egmond, H.P., and Whitaker, T. B., 2010. Developments in mycotoxin analysis: an update for 2008-2009. World Mycotoxin Journal, 3: 3-23.

Shephard, G.S., Berthiller, F., Burdaspal, P.A., Crews, C., Jonker, M.A., Krska, R., MacDonald, S., Malone, R.J., Maragos, C., Sabino, M., Solfrizzo, M., Van Egmond, H.P., and Whitaker, T.B., 2011. Developments in mycotoxin analysis: an update for 2009-2010. World Mycotoxin Journal 4: 3-28.

Shephard, G.S., Berthiller, F., Burdaspal, P.A., Crews, C., Jonker, M.A., Krska, R., MacDonald, S., Malone, R.J., Maragos, C., Sabino, M., Solfrizzo, M., Van Egmond, H.P., and Whitaker, T.B., 2012. Developments in mycotoxin analysis: an update for 2010-2011. World Mycotoxin Journal 5: 3-30.

Shephard, G.S., Berthiller, F., Burdaspal, P.A., Crews, C., Jonker, M.A., Krska, R., Lattanzio, V.M.T., MacDonald, S., Malone, R.J., Maragos, C., Sabino, M., Solfrizzo, M., Van Egmond, H.P. and Whitaker, T.B., 2013. Developments in mycotoxin analysis: an update for 2011-2012. World Mycotoxin Journal 6: 3-30. 157

Shundo, L., and Sabino, M., 2006. Aflatoxin M1 in milk by immunoaffinity column cleanup with TLC/HPLC determination. Brazilian Journal of Microbiology 37: 164-167.

Shundo, L., Navas, S. A., Lamardo, L. C. A., Ruvieri, V., and Sabino, M., 2009. Estimate of aflatoxin M1 exposure in milk and occurrence in Brazil. Food Control 20: 655- 657.

Siddappa, V., Nanjegowda, D. K., and Viswanath, P., 2012. Occurrence of aflatoxin M1 in some samples of UHT, raw & pasteurized milk from Indian states of Karnataka and Tamilnadu. Food and Chemical Toxicology 50: 4158-4162.

Silva, M. V., Janeiro, V., Bando, E., and Machinski, M., 2015. Occurrence and estimative of aflatoxin M1 intake in UHT cow milk in Paraná State, Brazil. Food Control 53: 222-225.

Simas, M. M., Albuquerque, R., Oliveira, C. A., Rottinghaus, G. E., and Correa, B., 2010. Influence of gamma radiation on productivity parameters of chicken fed mycotoxin-contaminated corn. Applied Radiation and Isotopes 68: 1903-1908.

Sinha, K., 2009.Detoxification of mycotoxins and food safety in Mycotoxins in Agriculture and Food Safety ed. by Sinha KK and Bhatnagar D, CRC Press pp. 381-399.

Smela, M. E., Currier, S. S., Bailey, E. A., and Essigmann, J. M., 2001. The chemistry and biology of aflatoxin B1: from mutational spectrometry to carcinogenesis. Carcinogenesis 22: 535-545.

Smith, G. S., 1992. Toxification and detoxification of plant compounds by ruminants: an overview. Journal of Range Management 45: 25-30.

Sobrova, P., Adam, V., Vasatkova, A., Beklova, M., Zeman, L., and Kizek, R., 2010. Deoxynivalenol and its toxicity. Interdisciplinary Toxicology 3: 94-99.

Soha, S., Mazloumi, M. T., and Borji, M., 2006. Reduction of aflatoxin M1 residue in milk utilizing chemisorption compounds and its effect on quality of milk. Journal of Arab Neonatal Forum 3: 53-58.

Sørensen, L. K., and Elbaek, T. H., 2005. Determination of mycotoxins in bovine milk by liquid chromatography tandem mass spectrometry. Journal of Chromatography B 820: 183-196.

Sorenson, W. G., 1999. Fungal spores: hazardous to health? Environmental Health Perspectives 107: 469-472.

158

Stockmann-Juvala, H., & Savolainen, K., 2008. A review of the toxic effects and mechanisms of action of fumonisin B1. Human & experimental toxicology 27: 799-809.

Stroud, J., 2006. The effect of feed additives on aflatoxin in milk of dairy cows fed aflatoxin-contaminated diets. MS Thesis. North Carolina State Univ., Raleigh.

Suriyasathaporn, W., and Nakprasert, W., 2012. Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand. Food Additives & Contaminants: Part B 5: 145-149.

Suzuki, T., Munakata, Y., Morita, K., Shinoda, T., and Ueda, H., 2007. Sensitive detection of estrogenic mycotoxin zearalenone by open sandwich immunoassay. Analytical sciences 23: 65-70.

Sweeney, M. J., White, S., and Dobson, A. D. W., 2000. Mycotoxin in Agriculture and Food Safety. Irish Journal of Agricultural and Food Research 39:235-244.

Tavakoli, H. R., Riazipour, M., Kamkar, A., Shaldehi, H. R., and Nejad, A., 2012. Occurrence of aflatoxin M1 in white cheese samples from Tehran, Iran. Food Control 23: 293-295.

Taylor, P. J., 2005. Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography electrospray tandem mass spectrometry. Clinical Biochemistry 38: 328-334.

Temamogullari, F., and Kanici, A., 2014. Aflatoxin M1 in dairy products sold in Sanliurfa, Turkey. Journal of Dairy Science 97: 162-165.

Titato, G., and Lanças, F., 2005. Comparison between different extraction (LLE and SPE) and determination (HPLC and capillary-LC) techniques in the analysis of selected PAHs in water samples. Journal of Liquid Chromatography & Related Technologies 28:3045-3056.

Tripathi, S., and Mishra, H. N., 2009. Studies on the efficacy of physical, chemical and biological aflatoxin B1 detoxification approaches in red chilli powder. International Journal of Food Safety, Nutrition and Public Health, 2(1), 69-77.

Trombete, F. M., Castro, I. M. D., Teixeira, A. D. S., Saldanha, T., and Fraga, M. E., 2014. Aflatoxin M1 contamination in grated parmesan cheese marketed in Rio de Janeiro-Brazil. Brazilian Archives of Biology and Technology 57: 269-273.

Tsakiris, I. N., Tzatzarakis, M. N., Alegakis, A. K., Vlachou, M. I., Renieri, E. A., and Tsatsakis, A. M., 2013. Risk assessment scenarios of children’s exposure to aflatoxin M1 residues in different milk types from the Greek market. Food and Chemical Toxicology 56: 261-265.

159

Turner, N. W., Subrahmanyam, S. and Piletsky, S. A., 2009. Analytical methods for determination of mycotoxins: a review. Analytica Chimica Acta 632: 168-180.

Turner, P. C., Collinson, A. C., Cheung, Y. B., Gong, Y., Hall, A. J., Prentice, A. M., and Wild, C. P., 2007. Aflatoxin exposure in utero causes growth faltering in Gambian infants. International Journal of Epidemiology, 36:1119-1125.

USDA, 2015. Basic Report 01211, milk, whole, 3.25% milkfat, without added vitamin A and vitamin D. USDA National Nutrient Database for Standard Reference. Report date, August 10, 2015.

Vagef, R., and Mahmoudi, R., 2013. Occurrence of aflatoxin M1 in raw and pasteurized milk produced in west region of Iran (during summer and winter). International Food Research Journal 20: 1421-1425.

Van Egmond, H. P., and Jonker, M. A., 2000. Case study: International inquiry into mycotoxin regulation in 2002/2003. In: Magan, N., and Olsen, M., (eds.) Mycotoxin in Food. Woodhead Publishing, Cambridge, England, 55-62.

Vardon, P. J., 2003. Potential economic costs of mycotoxins in the United States. In: Richard, J., and Payne, G. A., (eds.) Mycotoxins: Risks in Plant, Animal and Human Systems, Council for Agricultural Science and Technology (CAST), Ames, Iowa, 36-142.

Vdovenko, M.M., Lu, C.C., Yu, F.Y. and Sakharov, I.Y., 2014. Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin M1 in milk. Food Chemistry 158: 310-314.

Veldman, A., Meijs, J.A.C., Borggreve, J., and Heeres-van der Tol, J.J., 1992. Carry-over of aflatoxin from cows’ food to milk. Animal Production 55: 163-168.

Villers, P., 2014. Aflatoxins and safe storage. Frontiers in Microbiology 5: 158.

Wang, H., Zhou, X. J., Liu, Y. Q., Yang, H. M., and Guo, Q. L., 2010. Determination of aflatoxin M1 in milk by triple quadrupole liquid chromatography-tandem mass spectrometry. Food Additives & Contaminants 9: 1261-1265.

Wang, H., Zhou, X. J., Liu, Y. Q., Yang, H. M., and Guo, Q. L., 2011. Simultaneous determination of chloramphenicol and aflatoxin M1 residues in milk by triple quadrupole liquid chromatography-tandem mass spectrometry. Journal of Agricultural and Food Chemistry 59: 3532-3538.

Wang, Y., Liu, X., Xiao, C., Wang, Z., Wang, J., Xiao, H., Cui, L., Xiang, Q., and Yue, T. 2012. HPLC determination of aflatoxin M1 in liquid milk and milk powder using solid phase extraction on OASIS HLB. Food Control 28:131-134.

160

Wannop, C. C., 1961. The histopathology of Turkey “X” disease in Great Britain. Avian Diseases 5: 371-381.

Warburton, M. L., and Williams, W. P., 2014. Aflatoxin resistance in maize: What have we learned lately? Advance in Botany http://dx.doi.org/10.1155/2014/352831.

Whitlow, L. W., 2006. Evaluation of mycotoxin binders. In Proceedings of the 4th Mid- Atlantic Nutrition Conference pp. 132-143.

Wicklow, D. T., 1988. Epidemiology of Aspergillus flavus in corn. In: Shotwell, O. L., ed. Aflatoxin in Corn: New Perspectives. Ames, Iowa: North Central Regional Research Publication p. 315-323.

Wild, C. P., and Gong, Y. Y., 2010. Mycotoxins and human disease: a largely ignored global health issue. Carcinogenesis 31:71-82.

Wild, C. P., and Montesano R., 2009. A model of interaction: aflatoxins and hepatitis viruses in liver cancer aetiology and prevention. Cancer Letters 286: 22-28.

Williams, J. H., Phillips, T. D., Jolly, P. E., Stiles, J. K., Jolly, C. M., and Aggarwal, D., 2004. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. The American Journal of Clinical Nutrition. 80: 1106-1122.

Williams, S. B., Baributsa, D., and Woloshuk, C., 2014. Assessing Purdue Improved Crop Storage (PICS) bags to mitigate fungal growth and aflatoxin contamination. Journal of Stored Products Research 59: 190-196.

Williams, W. P., Buckley, P. M., and Windham, P. M., 2002a. Southwestern corn borer (Lepidoptera: crambidae) damage and aflatoxin accumulation in maize. Journal of Economic Entomology 95:1049–1053

Williams, W. P., Davis, F. M., Windham, G. L., and Buckley, P. M., 2002. Southwestern corn borer damage and aflatoxin accumulation in a diallel cross of maize. Journal of Genetics and Breeding 56: 165-170.

Windham, G. L., Williams, W. P., and Davis, F. M., 1999. Effects of the southwestern corn borer on Aspergillus flavus kernel infection and aflatoxin accumulation in maize hybrids. Plant Disease 83: 535-540.

Woloshuk, C. P., Cavaletto, J. R., and Cleveland, T. E., 1997. Inducers of aflatoxin biosynthesis from colonized maize kernels are generated by an amylase activity from Aspergillus flavus. Phytopathology 87: 164-169.

Woloshuk, C. P., and Shim, W. B., 2012. Aflatoxins, fumonisins and trichothecenes: a convergence of knowledge. Microbiology Reviews. 37: 94-109.

161

Wood, G. E., 1992. Mycotoxins in foods and feeds in the United States. Journal of Animal Science 70: 3941-3949.

World Health Organization, 2002. Evaluation of certain mycotoxins in food: Fifty-sixth report of the Joint FAO/WHO Expert Committee of Food Additives. WHO Technical Report Series 906: 1-62.

Wu, F., 2004. Mycotoxin risk assessment for the purpose of setting international regulatory standards. Environmental Science Technology 38: 4049-4055.

Wu, F., 2012. Aflatoxin regulations in a network of global maize trade. PloS ONE: e45151.

Wu, F., Liu, Y., and Bhatnagar D, 2008. Cost-effectiveness of aflatoxin control methods: economic incentives. Toxin Reviews 27: 203-225.

Xiong, J. L., Wang, Y. M, Ma M. R., and Liu, J. X., 2013. Seasonal variation of aflatoxin M1 in raw milk from the Yangtze River Delta region of China. Food Control 34:703-706.

Yao, H., Hruska, Z., and Di Manvungu, J., 2015. Developments in detection and determination of aflatoxins. World Mycotoxin Journal 8: 181-191.

Yao, H., Hruska, Z., Kincaid, R., Brown, R., Cleveland, T., and Bhatnagar, D., 2010. Correlation and classification of single kernel fluorescence hyperspectral data with aflatoxin concentration in corn kernels inoculated with Aspergillus flavus spores. Food Additives and Contaminants 27: 701-709.

Yener, N., Bicer, C., Önal, M., and Sarıkaya, Y., 2012. Simultaneous determination of cation exchange capacity and surface area of acid activated bentonite powders by methylene blue sorption. Applied Surface Science 258: 2534-2539.

Yiannikouris, A., Jouany, J. P., 2002. Mycotoxins in feeds and their fate in animals: A review. Animal Research 51:81-99.

Yogendrarajah, P., Van Poucke, C., De Meulanaer, B., and De Saeger, S, 2013. Development and validation of a QuEChERS based liquid chromatography tandem mass spectrometry method for the determination of multiple mycotoxins in spices. Journal of Chromatography A 1297: 1-11.

Yu, J., 2012. Current understanding on aflatoxin biosynthesis and future perspective in reducing aflatoxin contamination. Toxins 4: 1024-1057.

Yu, J., Chang, P. K., Ehrlich, K. C., Cary, J. W., Bhatnagar, D., Cleveland, T. E., Payne, G. A., Linz, J. E., Woloshuk, C. P., and Bennet, J. W., 2006. Clustered pathway genes in aflatoxin biosynthesis. Applied and Environmental Microbiology 70: 1253-1262. 162

Yu, J., Mohawed, S. M., and Bhatnagar, D., Cleveland, T. E., 2003. Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus. Journal of Applied Microbiology 95: 1334-1342.

Yu, J., Bhatnagar, D., and Ehrlich, K. C., 2002. Aflatoxin biosynthesis. Revista Iberoamericana de Micología 19: 191-200.

Yu, J. H., Butchko, R. A. E., Fernandes. M., Keller, N. P., Leonard, T. J., and Adams, T. H., 1996. Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus. Current Genetics 29:549–555.

Yunus, A. W., Ghareeb, K., Abd-El-Fattah, A. A. M., Twaruzek, M., and Böhm, J., 2011. Gross intestinal adaptations in relation to broiler performance during chronic aflatoxin exposure. Poultry Science 90: 1683-1689.

Zain, 2011. Impact of mycotoxins on humans and animals. Journal of Saudi Chemical Society 15: 129-144.

Zhang, D. H., Li, P. W., Zhang, Q., Yang, Y., Zhang, W., Guan, D., and Ding, X. X., 2012. Extract-free immunochromatographic assay for on-site tests of aflatoxin M1 in milk. Analytical Methods 4: 3307-3313.

Zheng, N., Wang, J. Q., Zhen, Y. P., Han, R. W., and Xu, X. M., 2013. Occurrence of aflatoxin M1 in UHT milk and pasteurized milk in China market. Food Control 29: 198-201.

Zmeili, O. S., and Soubani, A. O., 2007. Pulmonary aspergillosis: a clinical update. Quarterly International Journal of Medicine 100: 317-334.

163

RAW DATA AND STATISTICAL ANALYSES FOR CHAPTER V

164

SAS Statistical Codes for crossed, nested design

INFILE 'E:\Statistics\STATISTICS\Orginal Binder Data1.txt'; INPUT MILK $ REP BINDER $ B_CONC AFM1; RUN; PROC PRINT; RUN; PROC MEANS MEAN STD VAR MAXDEC=3 FW=10; VAR AFM1; CLASS MILK BINDER B_CONC; WAYS 1 2 3; RUN; PROC GLM; CLASS MILK BINDER B_CONC; MODEL AFM1 = MILK BINDER B_CONC(BINDER) MILK*BINDER MILK*B_CONC(BINDER)/SS3; LSMEANS MILK*B_CONC(BINDER)/STDERR PDIFF LINES; LSMEANS MILK*BINDER/STDERR PDIFF LINES; RUN; QUIT;

165

Table A.1 Raw data of the crossed, nested statistical design

Obs MILK REP BINDER B_CONC AFM1

1 R 1 AC 0.1 0.2936 2 R 2 AC 0.1 0.2784 3 R 3 AC 0.1 0.1526 4 R 1 AC 0.25 0.2976 5 R 2 AC 0.25 0.29 6 R 3 AC 0.25 0.2635 7 R 1 AC 0.4 0.2681 8 R 2 AC 0.4 0.2154 9 R 3 AC 0.4 0.2328 10 S 1 AC 0.1 0.1946 11 S 2 AC 0.1 0.128 12 S 3 AC 0.1 0.1781 13 S 1 AC 0.25 0.0622 14 S 2 AC 0.25 0.0771 15 S 3 AC 0.25 0.0444 16 S 1 AC 0.4 0.04 17 S 2 AC 0.4 0.0588 18 S 3 AC 0.4 0.0394 19 W 1 AC 0.1 0.3006 20 W 2 AC 0.1 0.3889 21 W 3 AC 0.1 0.3185 22 W 1 AC 0.25 0.2378 23 W 2 AC 0.25 0.1883 24 W 3 AC 0.25 0.232 25 W 1 AC 0.4 0.1333 26 W 2 AC 0.4 0.2673 27 W 3 AC 0.4 0.1312 28 R 1 MTB100 2 0.5389 29 R 2 MTB100 2 0.5113 30 R 3 MTB100 2 0.5212 31 R 1 MTB100 3 0.483 32 R 2 MTB100 3 0.4976 33 R 3 MTB100 3 0.3887 34 R 1 MTB100 4 0.4847 35 R 2 MTB100 4 0.491

166

Table A.1 (Continued)

36 R 3 MTB100 4 0.4226 37 S 1 MTB100 2 0.6182 38 S 2 MTB100 2 0.7318 39 S 3 MTB100 2 0.7127 40 S 1 MTB100 3 0.6759 41 S 2 MTB100 3 0.765 42 S 3 MTB100 3 0.662 43 S 1 MTB100 4 0.7502 44 S 2 MTB100 4 0.7612 45 S 3 MTB100 4 0.6063 46 W 1 MTB100 2 0.5 47 W 2 MTB100 2 0.4864 48 W 3 MTB100 2 0.5052 49 W 1 MTB100 3 0.6283 50 W 2 MTB100 3 0.6885 51 W 3 MTB100 3 0.4864 52 W 1 MTB100 4 0.5228 53 W 2 MTB100 4 0.5307 54 W 3 MTB100 4 0.4465 55 R 1 MYCO 2 0.5441 56 R 2 MYCO 2 0.6185 57 R 3 MYCO 2 0.5969 58 R 1 MYCO 3 0.5917 59 R 2 MYCO 3 0.5902 60 R 3 MYCO 3 0.587 61 R 1 MYCO 4 0.6148 62 R 2 MYCO 4 0.5998 63 R 3 MYCO 4 0.6004 64 S 1 MYCO 2 0.4761 65 S 2 MYCO 2 0.5096 66 S 3 MYCO 2 0.4399 67 S 1 MYCO 3 0.6104 68 S 2 MYCO 3 0.6222 69 S 3 MYCO 3 0.5795 70 S 1 MYCO 4 0.5679 71 S 2 MYCO 4 0.71 72 S 3 MYCO 4 0.5353 73 W 1 MYCO 2 0.4944

167

Table A.1 (Continued)

74 W 2 MYCO 2 0.5654 75 W 3 MYCO 2 0.4275 76 W 1 MYCO 3 0.3972 77 W 2 MYCO 3 0.4866 78 W 3 MYCO 3 0.3444 79 W 1 MYCO 4 0.4304 80 W 2 MYCO 4 0.198 81 W 3 MYCO 4 0.4465 82 R 1 BIO 2 0.498 83 R 2 BIO 2 0.487 84 R 3 BIO 2 0.5067 85 R 1 BIO 3 0.4938 86 R 2 BIO 3 0.4872 87 R 3 BIO 3 0.5297 88 R 1 BIO 4 0.5358 89 R 2 BIO 4 0.5371 90 R 3 BIO 4 0.5519 91 S 1 BIO 2 0.6182 92 S 2 BIO 2 0.485 93 S 3 BIO 2 0.7225 94 S 1 BIO 3 0.6274 95 S 2 BIO 3 0.5905 96 S 3 BIO 3 0.5169 97 S 1 BIO 4 0.7156 98 S 2 BIO 4 0.7222 99 S 3 BIO 4 0.7709 100 W 1 BIO 2 0.5088 101 W 2 BIO 2 0.4771 102 W 3 BIO 2 0.4847 103 W 1 BIO 3 0.5106 104 W 2 BIO 3 0.4197 105 W 3 BIO 3 0.4621 106 W 1 BIO 4 0.4525 107 W 2 BIO 4 0.4356 108 W 3 BIO 4 0.4824

168

Table A.2 Summary statistics of the crossed, nested statistical design

Analysis Variable : AFM1

B_CONC N Obs Mean Std Dev Variance

0.1 9 0.248 0.088 0.008 0.25 9 0.188 0.101 0.01 0.4 9 0.154 0.095 0.009 2 27 0.54 0.082 0.007 3 27 0.545 0.101 0.01 4 27 0.553 0.13 0.017

Analysis Variable : AFM1

BINDER N Obs Mean Std Dev Variance

AC 27 0.197 0.099 0.01 BIO 27 0.542 0.095 0.009 MTB100 27 0.571 0.112 0.012 MYCO 27 0.525 0.107 0.011

Analysis Variable : AFM1

BINDER B_CONC N Obs Mean Std Dev Variance

AC 0.1 9 0.248 0.088 0.008 0.25 9 0.188 0.101 0.01 0.4 9 0.154 0.095 0.009 BIO 2 9 0.532 0.083 0.007 3 9 0.515 0.063 0.004 4 9 0.578 0.126 0.016 MTB100 2 9 0.57 0.095 0.009 3 9 0.586 0.125 0.016 4 9 0.557 0.124 0.015 MYCO 2 9 0.519 0.067 0.004 3 9 0.534 0.101 0.01 4 9 0.523 0.149 0.022

Analysis Variable : AFM1 MILK N Obs Mean Std Dev Variance R 36 0.461 0.133 0.018 S 36 0.498 0.254 0.065 W 36 0.417 0.133 0.018

169

Table A.2 (Continued)

Analysis Variable : AFM1

MILK B_CONC N Obs Mean Std Dev Variance

R 0.1 3 0.242 0.077 0.006 0.25 3 0.284 0.018 0 0.4 3 0.239 0.027 0.001 2 9 0.536 0.045 0.002 3 9 0.517 0.067 0.004 4 9 0.538 0.063 0.004 S 0.1 3 0.167 0.035 0.001 0.25 3 0.061 0.016 0 0.4 3 0.046 0.011 0 2 9 0.59 0.116 0.013 3 9 0.628 0.07 0.005 4 9 0.682 0.088 0.008 W 0.1 3 0.336 0.047 0.002 0.25 3 0.219 0.027 0.001 0.4 3 0.177 0.078 0.006 2 9 0.494 0.036 0.001 3 9 0.492 0.109 0.012 4 9 0.438 0.097 0.009

Analysis Variable : AFM1

MILK BINDER N Obs Mean Std Dev Variance

R AC 9 0.255 0.047 0.002 BIO 9 0.514 0.025 0.001 MTB100 9 0.482 0.048 0.002 MYCO 9 0.594 0.021 0 S AC 9 0.091 0.06 0.004 BIO 9 0.641 0.099 0.01 MTB100 9 0.698 0.06 0.004 MYCO 9 0.561 0.082 0.007 W AC 9 0.244 0.086 0.007 BIO 9 0.47 0.031 0.001 MTB100 9 0.533 0.077 0.006 MYCO 9 0.421 0.105 0.011

170

Table A.2 (Continued)

Analysis Variable : AFM1

MILK BINDER B_CONC N Obs Mean Std Dev Variance

R AC 0.1 3 0.242 0.077 0.006 0.25 3 0.284 0.018 0 0.4 3 0.239 0.027 0.001 BIO 2 3 0.497 0.01 0 3 3 0.504 0.023 0.001 4 3 0.542 0.009 0 MTB100 2 3 0.524 0.014 0 3 3 0.456 0.059 0.003 4 3 0.466 0.038 0.001 MYCO 2 3 0.587 0.038 0.001 3 3 0.59 0.002 0 4 3 0.605 0.008 0 S AC 0.1 3 0.167 0.035 0.001 0.25 3 0.061 0.016 0 0.4 3 0.046 0.011 0 BIO 2 3 0.609 0.119 0.014 3 3 0.578 0.056 0.003 4 3 0.736 0.03 0.001 MTB100 2 3 0.688 0.061 0.004 3 3 0.701 0.056 0.003 4 3 0.706 0.086 0.007 MYCO 2 3 0.475 0.035 0.001 3 3 0.604 0.022 0 4 3 0.604 0.093 0.009 W AC 0.1 3 0.336 0.047 0.002 0.25 3 0.219 0.027 0.001 0.4 3 0.177 0.078 0.006 BIO 2 3 0.49 0.017 0 3 3 0.464 0.045 0.002 4 3 0.457 0.024 0.001 MTB100 2 3 0.497 0.01 0 3 3 0.601 0.104 0.011 4 3 0.5 0.047 0.002 MYCO 2 3 0.496 0.069 0.005 3 3 0.409 0.072 0.005 4 3 0.358 0.139 0.019 171

Table A.3 ANOVA analysis of the crossed, nested statistical design

Source DF Sum of Squares Mean Square F Value Pr > F Model 35 3.3828 0.0967 30.78 <.0001 Error 72 0.2261 0.0031 Corrected Total 107 3.6088

R-Square Coeff Var Root MSE AFM1 Mean 0.9374 12.2150 0.0560 0.4587

Source DF Type III SS Mean Square F Value Pr > F MILK 2 0.1179 0.0589 18.77 <.0001 BINDER 3 2.4997 0.8332 265.37 <.0001 B_CONC(BINDER) 8 0.0649 0.0081 2.58 0.0154 MILK*BINDER 6 0.5550 0.0925 29.46 <.0001 MILK*B_CONC(BINDER) 16 0.1453 0.0091 2.89 0.0011

172

Table A.4 Least squares means of milk*binder concentration nested in binder type

AFM1 Standard LSMEAN MILK B_CONC BINDER Pr > |t| LSMEAN Error Number R 0.1 AC 0.2415 0.0324 <.0001 1 R 0.25 AC 0.2837 0.0324 <.0001 2 R 0.4 AC 0.2388 0.0324 <.0001 3 S 0.1 AC 0.1669 0.0324 <.0001 4 S 0.25 AC 0.0612 0.0324 0.0624 5 S 0.4 AC 0.0461 0.0324 0.1588 6 W 0.1 AC 0.3360 0.0324 <.0001 7 W 0.25 AC 0.2194 0.0324 <.0001 8 W 0.4 AC 0.1773 0.0324 <.0001 9 R 2 BIO 0.4972 0.0324 <.0001 10 R 3 BIO 0.5036 0.0324 <.0001 11 R 4 BIO 0.5416 0.0324 <.0001 12 S 2 BIO 0.6086 0.0324 <.0001 13 S 3 BIO 0.5783 0.0324 <.0001 14 S 4 BIO 0.7362 0.0324 <.0001 15 W 2 BIO 0.4902 0.0324 <.0001 16 W 3 BIO 0.4641 0.0324 <.0001 17 W 4 BIO 0.4568 0.0324 <.0001 18 R 2 MTB100 0.5238 0.0324 <.0001 19 R 3 MTB100 0.4564 0.0324 <.0001 20 R 4 MTB100 0.4661 0.0324 <.0001 21 S 2 MTB100 0.6876 0.0324 <.0001 22 S 3 MTB100 0.7010 0.0324 <.0001 23 S 4 MTB100 0.7059 0.0324 <.0001 24 W 2 MTB100 0.4972 0.0324 <.0001 25 W 3 MTB100 0.6011 0.0324 <.0001 26 W 4 MTB100 0.5000 0.0324 <.0001 27 R 2 MYCO 0.5865 0.0324 <.0001 28 R 3 MYCO 0.5896 0.0324 <.0001 29 R 4 MYCO 0.6050 0.0324 <.0001 30 S 2 MYCO 0.4752 0.0324 <.0001 31 S 3 MYCO 0.6040 0.0324 <.0001 32 S 4 MYCO 0.6044 0.0324 <.0001 33 W 2 MYCO 0.4958 0.0324 <.0001 34 W 3 MYCO 0.4094 0.0324 <.0001 35 W 4 MYCO 0.3583 0.0324 <.0001 36

173

Table A.4 (Continued)

T Comparison Lines for Least Squares Means of MILK*B_CONC(BINDER) LS-means with the same letter are not significantly different.

AFM1 LSMEAN MILK B_CONC BINDER LSMEAN Number A 0.736233 S 4 BIO 15 A A 0.7059 S 4 MTB100 24 A A 0.700967 S 3 MTB100 23 A B A 0.687567 S 2 MTB100 22 B B C 0.608567 S 2 BIO 13 B C B C 0.605 R 4 MYCO 30 B C B C 0.6044 S 4 MYCO 33 B C B C 0.604033 S 3 MYCO 32 B C B C 0.601067 W 3 MTB100 26 C D C 0.589633 R 3 MYCO 29 D C D C E 0.5865 R 2 MYCO 28 D C E D F C E 0.578267 S 3 BIO 14

174

Table A.4 (Continued)

D F C E G D F C E 0.5416 R 4 BIO 12 G D F C E G D F C E 0.5238 R 2 MTB100 19 G D F E G D F E 0.503567 R 3 BIO 11 G D F E G D F H E 0.5 W 4 MTB100 27 G F H E G F H E 0.497233 R 2 BIO 10 G F H E G F H E 0.4972 W 2 MTB100 25 G F H E G F H E 0.495767 W 2 MYCO 34 G F H G F H 0.4902 W 2 BIO 16 G H G H 0.4752 S 2 MYCO 31 G H G H 0.4661 R 4 MTB100 21 G H G H 0.464133 W 3 BIO 17 G H G H 0.456833 W 4 BIO 18 G H G H 0.456433 R 3 MTB100 20 H I H 0.4094 W 3 MYCO 35 I I J 0.3583 W 4 MYCO 36 I J I J 0.336 W 0.1 AC 7 J K J 0.2837 R 0.25 AC 2 K K L 0.241533 R 0.1 AC 1 K L K L 0.238767 R 0.4 AC 3 K L K L 0.219367 W 0.25 AC 8 L L 0.177267 W 0.4 AC 9 L L 0.1669 S 0.1 AC 4

M 0.061233 S 0.25 AC 5 M M 0.046067 S 0.4 AC 6

175

Table A.5 Least squares means of milk*binder type

AFM1 Standard LSMEAN MILK BINDER Pr > |t| LSMEAN Error Number R AC 0.254667 0.018678 <.0001 1 R BIO 0.514133 0.018678 <.0001 2 R MTB100 0.482111 0.018678 <.0001 3 R MYCO 0.593711 0.018678 <.0001 4 S AC 0.0914 0.018678 <.0001 5 S BIO 0.641022 0.018678 <.0001 6 S MTB100 0.698144 0.018678 <.0001 7 S MYCO 0.561211 0.018678 <.0001 8 W AC 0.244211 0.018678 <.0001 9 W BIO 0.470389 0.018678 <.0001 10 W MTB100 0.532756 0.018678 <.0001 11 W MYCO 0.421156 0.018678 <.0001 12

Least Squares Means for effect MILK*BINDER Pr > |t| for H0: LSMean(i)=LSMean(j) Dependent Variable: AFM1 i/j 1 2 3 4 5 6 7 8 9 10 11 12 1 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.6934 <.0001 <.0001 <.0001 2 <.0001 0.2294 0.0036 <.0001 <.0001 <.0001 0.0789 <.0001 0.1021 0.4831 0.0008 3 <.0001 0.2294 <.0001 <.0001 <.0001 <.0001 0.0038 <.0001 0.6585 0.0592 0.0239 4 <.0001 0.0036 <.0001 <.0001 0.0775 0.0002 0.2226 <.0001 <.0001 0.0239 <.0001 5 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 6 <.0001 <.0001 <.0001 0.0775 <.0001 0.0339 0.0035 <.0001 <.0001 0.0001 <.0001 7 <.0001 <.0001 <.0001 0.0002 <.0001 0.0339 <.0001 <.0001 <.0001 <.0001 <.0001 8 <.0001 0.0789 0.0038 0.2226 <.0001 0.0035 <.0001 <.0001 0.001 0.285 <.0001 9 0.6934 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 10 <.0001 0.1021 0.6585 <.0001 <.0001 <.0001 <.0001 0.001 <.0001 0.0209 0.0664 11 <.0001 0.4831 0.0592 0.0239 <.0001 0.0001 <.0001 0.285 <.0001 0.0209 <.0001 12 <.0001 0.0008 0.0239 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.0664 <.0001

176

Table A.5 (Continued)

T Comparison Lines for Least Squares Means of MILK*BINDER LS-means with the same letter are not significantly different. AFM1 LSMEAN MILK BINDER LSMEAN Number A 0.6981444 S MTB100 7

B 0.6410222 S BIO 6 B C B 0.5937111 R MYCO 4 C C D 0.5612111 S MYCO 8 D E D 0.5327556 W MTB100 11 E D E D F 0.5141333 R BIO 2 E F E F 0.4821111 R MTB100 3 F G F 0.4703889 W BIO 10 G G 0.4211556 W MYCO 12

H 0.2546667 R AC 1 H H 0.2442111 W AC 9

I 0.0914 S AC 5

177

SAS Statistical Codes for nested design

OPTIONS PS=55 LS=85 NODATE; DATA BINDER; INFILE 'C:\USERS\erika\DROPBOX\MSU\DISSERTATION\STATISTICS\BINDER DATA RAW.TXT'; INPUT REP BINDER $ B_CONC AFM1; RUN; PROC PRINT; RUN; PROC MEANS MEAN STD VAR MAXDEC=3 FW=10; VAR AFM1; CLASS BINDER B_CONC; WAYS 1 2; RUN; PROC GLM; CLASS BINDER B_CONC; MODEL AFM1 = BINDER B_CONC(BINDER)/SS3; MEANS BINDER/LSD LINES; RUN; QUIT;

178

Table A.6 Raw data for nested design

Obs REP BINDER B_CONC AFM1

1 1 AC 0.1 0.2936 2 2 AC 0.1 0.2784 3 3 AC 0.1 0.1526 4 1 AC 0.25 0.2976 5 2 AC 0.25 0.29 6 3 AC 0.25 0.2635 7 1 AC 0.4 0.2681 8 2 AC 0.4 0.2154 9 3 AC 0.4 0.2328 10 1 MTB100 2 0.5389 11 2 MTB100 2 0.5113 12 3 MTB100 2 0.5212 13 1 MTB100 3 0.483 14 2 MTB100 3 0.4976 15 3 MTB100 3 0.3887 16 1 MTB100 4 0.4847 17 2 MTB100 4 0.491 18 3 MTB100 4 0.4226 19 1 MYCO 2 0.5441 20 2 MYCO 2 0.6185 21 3 MYCO 2 0.5969 22 1 MYCO 3 0.5917 23 2 MYCO 3 0.5902 24 3 MYCO 3 0.587 25 1 MYCO 4 0.6148 26 2 MYCO 4 0.5998 27 3 MYCO 4 0.6004 28 1 BIO 2 0.498 29 2 BIO 2 0.487 30 3 BIO 2 0.5067 31 1 BIO 3 0.4938 32 2 BIO 3 0.4872 33 3 BIO 3 0.5297 34 1 BIO 4 0.5358 35 2 BIO 4 0.5371 36 3 BIO 4 0.5519

179

Table A.7 Summary statistics of the nested statistical design

Analysis Variable : AFM1

B_CONC N Obs Mean Std Dev Variance

0.1 3 0.242 0.077 0.006 0.25 3 0.284 0.018 0 0.4 3 0.239 0.027 0.001 2 9 0.536 0.045 0.002 3 9 0.517 0.067 0.004 4 9 0.538 0.063 0.004

Analysis Variable : AFM1

BINDER N Obs Mean Std Dev Variance

AC 9 0.255 0.047 0.002 BIO 9 0.514 0.025 0.001 MTB100 9 0.482 0.048 0.002 MYCO 9 0.594 0.021 0

Analysis Variable : AFM1

BINDER B_CONC N Obs Mean Std Dev Variance

AC 0.1 3 0.242 0.077 0.006 0.25 3 0.284 0.018 0 0.4 3 0.239 0.027 0.001 BIO 2 3 0.497 0.01 0 3 3 0.504 0.023 0.001 4 3 0.542 0.009 0 MTB100 2 3 0.524 0.014 0 3 3 0.456 0.059 0.003 4 3 0.466 0.038 0.001 MYCO 2 3 0.587 0.038 0.001 3 3 0.59 0.002 0 4 3 0.605 0.008 0

180

Table A.8 ANOVA analysis of the nested statistical design

Source DF Sum of Squares Mean Square F Value Pr > F Model 11 0.586899 0.053354 44.48 <.0001 Error 24 0.028787 0.001199 Corrected Total 35 0.615685

R-Square Coeff Var Root MSE AFM1 Mean 0.953245 7.51003 0.034633 0.461156

Source DF Type III SS Mean Square F Value Pr > F BINDER 3 0.57109 0.190363 158.71 <.0001 B_CONC(BINDER) 8 0.015809 0.001976 1.65 0.1637

Table A.9 Least squares means of the nested statistical design

Alpha 0.05 Error Degrees of Freedom 24 Error Mean Square 0.001199 Critical Value of t 2.0639 Least Significant Difference 0.0337

Means with the same letter are not significantly different.

t Grouping Mean N BINDER

A 0.59371 9 MYCO

B 0.51413 9 BIO B B 0.48211 9 MTB100

C 0.25467 9 AC

181

RAW DATA AND STATISTICAL ANALYSES FOR CHAPTER VI

182

SAS Statistical Codes for the 27-4 fractional factorial design for QuEChERS main effects

OPTIONS PS=55 LS=85 NODATE; DATA ROBUSTNESS; INFILE 'c:\users\erika\dropbox\msu\dissertation\validation paper\old results\ROBUSTNESS2015.txt'; INPUT RUN REP VOL TEMP HOMO ACN SHAKE STROKES CENTRI AFM1; IF VOL = 5 THEN VOL = -1; IF VOL = 15 THEN VOL = 1; IF TEMP = 15 THEN TEMP = -1; IF TEMP = 25 THEN TEMP = 1; IF HOMO = 5 THEN HOMO = -1; IF HOMO = 25 THEN HOMO = 1; IF ACN = 10 THEN ACN = -1; IF ACN = 20 THEN ACN = 1; IF SHAKE = 30 THEN SHAKE = -1; IF SHAKE = 90 THEN SHAKE = 1; IF STROKES = 500 THEN STROKES = -1; IF STROKES = 1500 THEN STROKES = 1; IF CENTRI = 3 THEN CENTRI = -1; IF CENTRI = 7 THEN CENTRI = 1; RUN; PROC PRINT; RUN; PROC MEANS sum MEAN STD VAR MAXDEC=4; VAR AFM1; CLASS RUN REP VOL TEMP HOMO ACN SHAKE STROKES CENTRI; TYPES RUN REP VOL TEMP HOMO ACN SHAKE STROKES CENTRI; RUN; PROC GLM; CLASS REP VOL TEMP HOMO ACN SHAKE STROKES CENTRI; MODEL AFM1 = REP VOL TEMP HOMO ACN SHAKE STROKES CENTRI/SS3; RUN;

183

Table B.1 Raw data of the 27-4 fractional factorial design

Obs RUN REP VOL TEMP HOMO ACN SHAKE STROKES CENTRI AFM1 1 1 1 -1 -1 -1 1 1 1 -1 0.3417 2 1 2 -1 -1 -1 1 1 1 -1 0.2932 3 1 3 -1 -1 -1 1 1 1 -1 0.2612 4 2 1 1 -1 -1 -1 -1 1 1 0.5417 5 2 2 1 -1 -1 -1 -1 1 1 0.4257 6 2 3 1 -1 -1 -1 -1 1 1 0.4842 7 3 1 -1 1 -1 -1 1 -1 1 0.4965 8 3 2 -1 1 -1 -1 1 -1 1 0.3313 9 3 3 -1 1 -1 -1 1 -1 1 0.4478 10 4 1 1 1 -1 1 -1 -1 -1 0.4134 11 4 2 1 1 -1 1 -1 -1 -1 0.4107 12 4 3 1 1 -1 1 -1 -1 -1 0.4485 13 5 1 -1 -1 1 1 -1 -1 1 0.2599 14 5 2 -1 -1 1 1 -1 -1 1 0.3982 15 5 3 -1 -1 1 1 -1 -1 1 0.2456 16 6 1 1 -1 1 -1 1 -1 -1 0.5541 17 6 2 1 -1 1 -1 1 -1 -1 0.5248 18 6 3 1 -1 1 -1 1 -1 -1 0.454 19 7 1 -1 1 1 -1 -1 1 -1 0.4397 20 7 2 -1 1 1 -1 -1 1 -1 0.4611 21 7 3 -1 1 1 -1 -1 1 -1 0.4067 22 8 1 1 1 1 1 1 1 1 0.4315 23 8 2 1 1 1 1 1 1 1 0.4469 24 8 3 1 1 1 1 1 1 1 0.5276

184

Table B.2 Summary statistics of the 27-4 fractional factorial design

Analysis Variable : AFM1 CENTRI N Obs Sum Mean Std Dev Variance -1 12 5.0091 0.4174 0.0856 0.0073 1 12 5.0369 0.4197 0.0965 0.0093

Analysis Variable : AFM1 STROKES N Obs Sum Mean Std Dev Variance -1 12 4.9848 0.4154 0.0962 0.0093 1 12 5.0612 0.4218 0.0858 0.0074

Analysis Variable : AFM1 SHAKE N Obs Sum Mean Std Dev Variance -1 12 4.9354 0.4113 0.0841 0.0071 1 12 5.1106 0.4259 0.0973 0.0095

Analysis Variable : AFM1 ACN N Obs Sum Mean Std Dev Variance -1 12 5.5676 0.464 0.0623 0.0039 1 12 4.4784 0.3732 0.091 0.0083

Analysis Variable : AFM1 HOMO N Obs Sum Mean Std Dev Variance -1 12 4.8959 0.408 0.0853 0.0073 1 12 5.1501 0.4292 0.0955 0.0091

Analysis Variable : AFM1 TEMP N Obs Sum Mean Std Dev Variance -1 12 4.7843 0.3987 0.1158 0.0134 1 12 5.2617 0.4385 0.0486 0.0024

Analysis Variable : AFM1 VOL N Obs Sum Mean Std Dev Variance -1 12 4.3829 0.3652 0.0878 0.0077 1 12 5.6631 0.4719 0.0523 0.0027

185

Table B.2 (Continued)

Analysis Variable : AFM1 REP N Obs Sum Mean Std Dev Variance 1 8 3.4785 0.4348 0.0994 0.0099 2 8 3.2919 0.4115 0.0731 0.0053 3 8 3.2756 0.4095 0.1024 0.0105

Analysis Variable : AFM1 RUN N Obs Sum Mean Std Dev Variance 1 3 0.8961 0.2987 0.0405 0.0016 2 3 1.4516 0.4839 0.058 0.0034 3 3 1.2756 0.4252 0.0849 0.0072 4 3 1.2726 0.4242 0.0211 0.0004 5 3 0.9037 0.3012 0.0843 0.0071 6 3 1.5329 0.511 0.0515 0.0026 7 3 1.3075 0.4358 0.0274 0.0008 8 3 1.406 0.4687 0.0516 0.0027

186

Table B.3 ANOVA analysis of the 27-4 fractional factorial design

Sum of Mean Source DF F Value Pr > F Squares Square Model 9 0.1346 0.0150 4.32 0.0074 Error 14 0.0485 0.0035

Corrected 23 0.1831 Total

R-Square Coeff Var Root MSE AFM1 Mean

0.7353 14.0571 0.0588 0.4186

Type III Mean Source DF F Value Pr > F SS Square REP 2 0.0032 0.0016 0.46 0.6412 VOL 1 0.0683 0.0683 19.72 0.0006 TEMP 1 0.0095 0.0095 2.74 0.1199 HOMO 1 0.0027 0.0027 0.78 0.3927 ACN 1 0.0494 0.0494 14.28 0.002 SHAKE 1 0.0013 0.0013 0.37 0.5531 STROKES 1 0.0002 0.0002 0.07 0.7948 CENTRI 1 0.0000 0.0000 0.01 0.9245

187

SAS Statistical Codes for the 23 factorial design for peak response using QuEChERS OPTIONS PS=55 LS=85 NODATE; DATA OPTIMIZE3; INFILE 'C:\USERS\ERIKA\DROPBOX\MSU\EXPERIMENTS\VALIDATION EXPERIMENTS\QuEChERS_Y_07192015.TXT'; INPUT RUN REP ACN TEMP MILK Y; RUN; PROC PRINT; RUN; PROC MEANS MEAN STD VAR MAXDEC=4; VAR Y; CLASS ACN TEMP MILK; Ways 2 3; RUN; PROC GLM; CLASS ACN TEMP MILK; MODEL Y = ACN |TEMP |MILK /SS3; LSMEANS ACN*MILK /STDERR PDIFF LINES; LSMEANS MILK*TEMP/STDERR PDIFF LINES; RUN; PROC MEANS SUM MEAN NOPRINT; VAR Y; CLASS ACN MILK; WAYS 2; OUTPUT OUT=MILK2 MEAN=Y_MEAN; RUN; SYMBOL1 INTERPOL=JOIN; PROC GPLOT DATA=MILK2; PLOT Y_MEAN*ACN=MILK; RUN; QUIT;

OPTIONS PS=55 LS=85 NODATE; DATA OPTIMIZE3; INFILE 'C:\USERS\ERIKA\DROPBOX\MSU\EXPERIMENTS\VALIDATION EXPERIMENTS\QuEChERS_Y_07192015.TXT'; INPUT RUN REP ACN TEMP MILK Y; RUN; /*PROC PRINT; RUN; PROC MEANS MEAN STD VAR MAXDEC=4; VAR Y; CLASS ACN TEMP MILK; Ways 2 3; RUN; PROC GLM; CLASS ACN TEMP MILK; MODEL Y = ACN |TEMP |MILK /SS3; LSMEANS ACN*MILK /STDERR PDIFF LINES; LSMEANS MILK*TEMP/STDERR PDIFF LINES; 188

RUN;*/ PROC MEANS SUM MEAN NOPRINT; VAR Y; CLASS TEMP MILK; WAYS 2; OUTPUT OUT=MILK3 MEAN=Y_MEAN; RUN; SYMBOL1 INTERPOL=JOIN; PROC GPLOT DATA=MILK3; PLOT Y_MEAN*TEMP=MILK; RUN; QUIT;

Table B.4 Raw data of the 23 factorial design

Obs RUN REP ACN TEMP MILK Y 1 1 1 10 15 5 94266 2 1 2 10 15 5 88542 3 1 3 10 15 5 89794 4 2 1 20 15 5 88136 5 2 2 20 15 5 86592 6 2 3 20 15 5 94460 7 3 1 10 25 5 85644 8 3 2 10 25 5 84062 9 3 3 10 25 5 91552 10 4 1 20 25 5 91316 11 4 2 20 25 5 91204 12 4 3 20 25 5 86352 13 5 1 10 15 15 83165.68 14 5 2 10 15 15 81408.53 15 5 3 10 15 15 81650.5 16 6 1 20 15 15 77023.07 17 6 2 20 15 15 77148.74 18 6 3 20 15 15 76847.41 19 7 1 10 25 15 86166.05 20 7 2 10 25 15 83175.68 21 7 3 10 25 15 84848.85 22 8 1 20 25 15 80487.32 23 8 2 20 25 15 82256.61 24 8 3 20 25 15 79159.35

189

Table B.5 Summary statistics of the 23 factorial design

Analysis Variable : Y TEMP MILK N Obs Mean Std Dev Variance 15 5 6 90298.33 3310.997 10962700 15 6 79540.66 2842.333 8078854 25 5 6 88355 3373.322 11379302 15 6 82682.31 2626.184 6896841

Analysis Variable : Y ACN MILK N Obs Mean Std Dev Variance 10 5 6 88976.67 3761.036 14145395 15 6 83402.55 1837.503 3376418 20 5 6 89676.67 3188.814 10168534 15 6 78820.42 2218.945 4923716

Analysis Variable : Y ACN TEMP N Obs Mean Std Dev Variance 10 15 6 86471.12 5213.144 27176872 25 6 85908.1 2966.011 8797222 20 15 6 83367.87 7451.388 55523178 25 6 85129.21 5331.282 28422572

Analysis Variable : Y ACN TEMP MILK N Obs Mean Std Dev Variance 10 15 5 3 90867.33 3009.166 9055077 15 3 82074.9 952.3564 906982.7 25 5 3 87086 3947.727 15584548 15 3 84730.19 1498.712 2246138 20 15 5 3 89729.33 4168.979 17380389 15 3 77006.41 151.3545 22908.19 25 5 3 89624 2834.188 8032624 15 3 80634.43 1553.861 2414485

190

Table B.6 ANOVA analysis of the 23 factorial design

Source DF Sum of Squares Mean Square F Value Pr > F

Model 7 521177303.70 74453901 10.7 <.0001 Error 16 111286304.80 6955394 Corrected Total 23 632463608.50

R-Square Coeff Var Root MSE Y Mean

0.824043 3.094739 2637.308 85219.07

Mean F Source DF Type III SS Pr > F Square Value ACN 1 22606419.4 22606419 3.25 0.0903 TEMP 1 2153962.2 2153962 0.31 0.5856 ACN*TEMP 1 8104009 8104009 1.17 0.2964 MILK 1 404935505.4 4.05E+08 58.22 <.0001 ACN*MILK 1 41851372.4 41851372 6.02 0.026 TEMP*MILK 1 38785659.5 38785660 5.58 0.0312 ACN*TEMP*MILK 1 2740375.8 2740376 0.39 0.5391

191

Table B.7 Least squares means of ACN volume*milk volume

Standard LSMEAN ACN MILK Y LSMEAN Pr > |t| Error Number

10 5 88976.6667 1076.677 <.0001 1 10 15 83402.5483 1076.677 <.0001 2 20 5 89676.6667 1076.677 <.0001 3 20 15 78820.4167 1076.677 <.0001 4

Least Squares Means for effect ACN*MILK Pr > |t| for H0: LSMean(i)=LSMean(j) Dependent Variable: Y i/j 1 2 3 4 1 0.0021 0.6519 <.0001 2 0.0021 0.0008 0.0083 3 0.6519 0.0008 <.0001 4 <.0001 0.0083 <.0001

T Comparison Lines for Least Squares Means of ACN*MILK LS-means with the same letter are not significantly different. LSMEAN Y LSMEAN ACN MILK Number A 89677 20 5 3 A A 88977 10 5 1

B 83403 10 15 2

C 78820 20 15 4

192

Table B.8 Least squares means of milk temperature*milk volume

Standard LSMEAN TEMP MILK Y LSMEAN Pr > |t| Error Number

15 5 90298.3333 1076.677 <.0001 1 15 15 79540.655 1076.677 <.0001 2 25 5 88355 1076.677 <.0001 3 25 15 82682.31 1076.677 <.0001 4

Least Squares Means for effect TEMP*MILK Pr > |t| for H0: LSMean(i)=LSMean(j) Dependent Variable: Y i/j 1 2 3 4 1 <.0001 0.2201 0.0001 2 <.0001 <.0001 0.0557 3 0.2201 <.0001 0.0018 4 0.0001 0.0557 0.0018

T Comparison Lines for Least Squares Means of TEMP*MILK LS-means with the same letter are not significantly different. LSMEAN Y LSMEAN TEMP MILK Number A 90298 15 5 1 A A 88355 25 5 3

B 82682 25 15 4 B B 79541 15 15 2

193

Figure B.1 Plot of ACN volume*milk volume

Figure B.2 Plot of milk temperature*milk volume

194

3 SAS Statistical Codes for the 2 factorial design for AFM1 (ng/mL) concentration using QuEChERS OPTIONS PS=55 LS=85 NODATE; DATA OPTIMIZE3; INFILE 'C:\USERS\ERIKA\DROPBOX\MSU\EXPERIMENTS\VALIDATION EXPERIMENTS\QuEChERS_AFM1_07192015.TXT'; INPUT RUN REP ACN TEMP MILK AFM1; RUN; PROC PRINT; RUN; PROC MEANS MEAN STD MAXDEC=4; VAR AFM1; CLASS ACN TEMP MILK; WAYS 2 3; RUN; PROC GLM; CLASS ACN TEMP MILK; MODEL AFM1 = ACN |TEMP |MILK /SS3; LSMEANS ACN*MILK /STDERR PDIFF LINES; LSMEANS MILK*TEMP/STDERR PDIFF LINES; RUN; PROC MEANS SUM MEAN NOPRINT; VAR AFM1; CLASS ACN MILK; WAYS 2; OUTPUT OUT=MILK2 MEAN=Y_MEAN; RUN;

195

Table B.9 Raw data of the 23 factorial design

Obs RUN REP ACN TEMP MILK AFM1 1 1 1 10 15 5 0.54597 2 1 2 10 15 5 0.52088 3 1 3 10 15 5 0.50971 4 2 1 20 15 5 0.52645 5 2 2 20 15 5 0.50047 6 2 3 20 15 5 0.55193 7 3 1 10 25 5 0.47963 8 3 2 10 25 5 0.47412 9 3 3 10 25 5 0.48884 10 4 1 20 25 5 0.49983 11 4 2 20 25 5 0.5464 12 4 3 20 25 5 0.51372 13 5 1 10 15 15 0.47548 14 5 2 10 15 15 0.47308 15 5 3 10 15 15 0.46868 16 6 1 20 15 15 0.43027 17 6 2 20 15 15 0.43366 18 6 3 20 15 15 0.43042 19 7 1 10 25 15 0.49567 20 7 2 10 25 15 0.46772 21 7 3 10 25 15 0.48393 22 8 1 20 25 15 0.44623 23 8 2 20 25 15 0.4445 24 8 3 20 25 15 0.44607

196

Table B.10 Summary statistics of the 23 factorial design

Analysis Variable : AFM1 TEMP MILK N Obs Mean Std Dev 15 5 6 0.5259 0.0201 15 6 0.4519 0.0226 25 5 6 0.5004 0.0266 15 6 0.464 0.0221

Analysis Variable : AFM1 ACN MILK N Obs Mean Std Dev 10 5 6 0.5032 0.0275 15 6 0.4774 0.0107 20 5 6 0.5231 0.0225 15 6 0.4385 0.0079

Analysis Variable : AFM1 ACN TEMP N Obs Mean Std Dev 10 15 6 0.499 0.0314 25 6 0.4817 0.0101 20 15 6 0.4789 0.0544 25 6 0.4828 0.0435

Analysis Variable : AFM1 ACN TEMP MILK N Obs Mean Std Dev 10 15 5 3 0.5255 0.0186 15 3 0.4724 0.0035 25 5 3 0.4809 0.0074 15 3 0.4824 0.014 20 15 5 3 0.5263 0.0257 15 3 0.4315 0.0019 25 5 3 0.52 0.0239 15 3 0.4456 0.001

197

Table B.11 ANOVA analysis of the 23 factorial design

Sum of Source DF Mean Square F Value Pr > F Squares Model 7 0.027508 0.00393 17.02 <.0001 Error 16 0.003694 0.000231 Corrected 23 0.031202 Total

R- Coeff Root AFM1 Mean Square Var MSE 0.881597 3.129392 0.015195 0.48557

Type III Mean F Source DF Pr > F SS Square Value ACN 1 0.000539 0.000539 2.33 0.146 TEMP 1 0.000269 0.000269 1.16 0.2965 ACN*TEMP 1 0.000676 0.000676 2.93 0.1063 MILK 1 0.018272 0.018272 79.14 <.0001 ACN*MILK 1 0.005194 0.005194 22.5 0.0002 TEMP*MILK 1 0.002117 0.002117 9.17 0.008 ACN*TEMP*MILK 1 0.00044 0.00044 1.9 0.1866

198

Table B.12 Least squares means of ACN volume*milk volume

AFM1 Standard LSMEAN ACN MILK Pr > |t| LSMEAN Error Number 10 5 0.50319 0.006203 <.0001 1 10 15 0.477428 0.006203 <.0001 2 20 5 0.523135 0.006203 <.0001 3 20 15 0.438527 0.006203 <.0001 4

Least Squares Means for effect ACN*MILK Pr > |t| for H0: LSMean(i)=LSMean(j) Dependent Variable: AFM1 i/j 1 2 3 4 1 0.0097 0.0371 <.0001 2 0.0097 <.0001 0.0004 3 0.0371 <.0001 <.0001 4 <.0001 0.0004 <.0001

T Comparison Lines for Least Squares Means of ACN*MILK LS-means with the same letter are not significantly different. AFM1 LSMEAN ACN MILK LSMEAN Number A 0.523135 20 5 3

B 0.50319 10 5 1

C 0.477428 10 15 2

D 0.438527 20 15 4

199

Table B.13 Least squares means of milk temperature*milk volume

AFM1 Standard LSMEAN TEMP MILK Pr > |t| LSMEAN Error Number

15 5 0.525902 0.006203 <.0001 1 15 15 0.451933 0.006203 <.0001 2 25 5 0.500423 0.006203 <.0001 3 25 15 0.464021 0.006203 <.0001 4

Least Squares Means for effect TEMP*MILK Pr > |t| for H0: LSMean(i)=LSMean(j) Dependent Variable: AFM1 i/j 1 2 3 4 1 <.0001 0.0104 <.0001 2 <.0001 <.0001 0.1872 3 0.0104 <.0001 0.0008 4 <.0001 0.1872 0.0008

T Comparison Lines for Least Squares Means of TEMP*MILK LS-means with the same letter are not significantly different. AFM1 LSMEAN TEMP MILK LSMEAN Number A 0.525902 15 5 1

B 0.500423 25 5 3

C 0.464021 25 15 4 C C 0.451933 15 15 2

200

RAW DATA AND STATISTICAL ANALYSES FOR CHAPTER VII

201

SAS codes for contact time study

OPTIONS PS=55 LS=85 NODATE; DATA Contact Time; INFILE 'c:\users\erika\dropbox\msu\experiments\optimum contact time\contact time raw data.txt'; INPUT Rate AC AFM1_CONC; RUN; PROC PRINT; RUN; PROC MEANS SUM MEAN STD VAR MAXDEC=4; VAR AFM1_CONC; CLASS Rate AC; TYPES Rate AC Rate*AC; run; PROC GLM; CLASS Rate AC; MODEL AFM1_CONC = Rate|AC/SS3; LSMEANS Rate*AC/STDERR TDiff PDIFF Lines; run; Quit;

202

Table C.1 Raw data for 52 factorial design for contact study

Obs Rate AC AFM1_CONC 1 0.2 0 0.8148 2 0.2 0 0.8477 3 0.2 0 0.8288 4 0.4 0 0.8108 5 0.4 0 0.8220 6 0.4 0 0.7782 7 0.6 0 0.7631 8 0.6 0 0.7982 9 0.6 0 0.7767 10 0.8 0 0.7398 11 0.8 0 0.7714 12 0.8 0 0.7393 13 1 0 0.7196 14 1 0 0.7369 15 1 0 0.7467 16 0.2 0.25 0.5566 17 0.2 0.25 0.3208 18 0.2 0.25 0.3454 19 0.4 0.25 0.4951 20 0.4 0.25 0.4116 21 0.4 0.25 0.4257 22 0.6 0.25 0.5303 23 0.6 0.25 0.5512 24 0.6 0.25 0.3238 25 0.8 0.25 0.2804 26 0.8 0.25 0.4931 27 0.8 0.25 0.5168 28 1 0.25 0.4955 29 1 0.25 0.4647 30 1 0.25 0.4596 31 0.2 0.5 0.1428 32 0.2 0.5 0.1331 33 0.2 0.5 0.0977 34 0.4 0.5 0.1691 35 0.4 0.5 0.1641 36 0.4 0.5 0.1394 37 0.6 0.5 0.2110 38 0.6 0.5 0.2295 39 0.6 0.5 0.0799 40 0.8 0.5 0.2225 41 0.8 0.5 0.2355 42 0.8 0.5 0.2145 43 1 0.5 0.1503 44 1 0.5 0.2325 45 1 0.5 0.2141 46 0.2 0.75 0.0000 47 0.2 0.75 0.0238 48 0.2 0.75 0.0107

203

Table C.1 (Continued)

49 0.4 0.75 0.0253 50 0.4 0.75 0.0000 51 0.4 0.75 0.0049 52 0.6 0.75 0.0717 53 0.6 0.75 0.0680 54 0.6 0.75 0.0792 55 0.8 0.75 0.2342 56 0.8 0.75 0.1489 57 0.8 0.75 0.1750 58 1 0.75 0.3818 59 1 0.75 0.1753 60 1 0.75 0.1595 61 0.2 1 0.0000 62 0.2 1 0.0000 63 0.2 1 0.0000 64 0.4 1 0.0000 65 0.4 1 0.0000 66 0.4 1 0.0000 67 0.6 1 0.0000 68 0.6 1 0.0000 69 0.6 1 0.0038 70 0.8 1 0.0000 71 0.8 1 0.0000 72 0.8 1 0.0000 73 1 1 0.0047 74 1 1 0.0349 75 1 1 0.0241

204

Table C.2 Summary statistics for 52 factorial design for contact study

The MEANS Procedure Analysis Variable : AFM1_CONC AC N Obs Sum Mean Std Dev Variance 0 15 11.6939 0.7796 0.0392 0.0015 0.25 15 6.6704 0.4447 0.0897 0.0081 0.5 15 2.6359 0.1757 0.051 0.0026 0.75 15 1.5583 0.1039 0.1086 0.0118 1 15 0.0675 0.0045 0.0105 0.0001

Analysis Variable : AFM1_CONC Rate N Obs Sum Mean Std Dev Variance 0.2 15 4.1221 0.2748 0.3292 0.1083 0.4 15 4.2462 0.2831 0.3171 0.1006 0.6 15 4.4863 0.2991 0.3036 0.0922 0.8 15 4.7714 0.3181 0.2697 0.0728 1 15 5 0.3333 0.2606 0.0679

Analysis Variable : AFM1_CONC Rate AC N Obs Sum Mean Std Dev Variance 0.2 0 3 2.4912 0.8304 0.0165 0.0003 0.25 3 1.2228 0.4076 0.1296 0.0168

0.5 3 0.3736 0.1245 0.0237 0.0006

0.75 3 0.0345 0.0115 0.0119 0.0001

1 3 0 0 0 0

0.4 0 3 2.411 0.8037 0.0227 0.0005 0.25 3 1.3324 0.4441 0.0447 0.002

0.5 3 0.4727 0.1576 0.0159 0.0003

0.75 3 0.0302 0.0101 0.0134 0.0002

1 3 0 0 0 0

0.6 0 3 2.338 0.7793 0.0177 0.0003 0.25 3 1.4053 0.4684 0.1257 0.0158

0.5 3 0.5203 0.1734 0.0816 0.0067

0.75 3 0.2189 0.073 0.0057 0

1 3 0.0038 0.0013 0.0022 0

0.8 0 3 2.2505 0.7502 0.0184 0.0003 0.25 3 1.2903 0.4301 0.1302 0.0169

0.5 3 0.6725 0.2242 0.0106 0.0001

0.75 3 0.5581 0.186 0.0437 0.0019

1 3 0 0 0 0

1 0 3 2.2032 0.7344 0.0137 0.0002 0.25 3 1.4197 0.4732 0.0194 0.0004

0.5 3 0.5969 0.199 0.0431 0.0019

0.75 3 0.7166 0.2389 0.124 0.0154

1 3 0.0636 0.0212 0.0153 0.0002

205

Table C.3 ANOVA for 52 factorial design for contact study

Source DF Sum of Squares Mean Square F Value Pr > F Model 24 6.0577669 0.252407 78.02 <.0001 Error 50 0.1617478 0.003235

Corrected Total 74 6.2195147

Coeff Root R-Square AFM1_CONC Mean Var MSE 0.973994 18.85336 0.056877 0.301679

Source DF Type III SS Mean Square F Value Pr > F Rate 4 0.0351933 0.0087983 2.72 0.0398 AC 4 5.8823259 1.4705815 454.59 <.0001 Rate*AC 16 0.1402477 0.0087655 2.71 0.0037

Table C.4 Least squares for 52 factorial design for contact study

AFM1_CONC Standard LSMEAN Rate AC Pr > |t| LSMEAN Error Number 0.2 0 0.8303936 0.0328378 <.0001 1 0.2 0.25 0.4075873 0.0328378 <.0001 2 0.2 0.5 0.1245256 0.0328378 0.0004 3 0.2 0.75 0.011511 0.0328378 0.7274 4 0.2 1 0 0.0328378 1 5 0.4 0 0.8036568 0.0328378 <.0001 6 0.4 0.25 0.4441188 0.0328378 <.0001 7 0.4 0.5 0.1575588 0.0328378 <.0001 8 0.4 0.75 0.0100751 0.0328378 0.7603 9 0.4 1 0 0.0328378 1 10 0.6 0 0.7793184 0.0328378 <.0001 11 0.6 0.25 0.4684354 0.0328378 <.0001 12 0.6 0.5 0.1734274 0.0328378 <.0001 13 0.6 0.75 0.0729699 0.0328378 0.0308 14 0.6 1 0.0012786 0.0328378 0.9691 15 0.8 0 0.7501774 0.0328378 <.0001 16 0.8 0.25 0.4300978 0.0328378 <.0001 17 0.8 0.5 0.2241656 0.0328378 <.0001 18 0.8 0.75 0.1860179 0.0328378 <.0001 19 0.8 1 0 0.0328378 1 20 1 0 0.7344101 0.0328378 <.0001 21 1 0.25 0.4732234 0.0328378 <.0001 22 1 0.5 0.1989605 0.0328378 <.0001 23 1 0.75 0.2388573 0.0328378 <.0001 24 1 1 0.0212134 0.0328378 0.5212 25

206

Table C.4 (Continued)

207

Table C.4 (Continued)

T Comparison Lines for Least Squares Means of Rate*AC LS-means with the same letter are not significantly different. AFM1_CONC LSMEAN Rate AC LSMEAN Number A 0.8303936 0.2 0 1 A B A 0.8036568 0.4 0 6 B A B A 0.7793184 0.6 0 11 B A B A 0.7501773 0.8 0 16 B B 0.7344101 1 0 21

C 0.4732234 1 0.25 22 C C 0.4684354 0.6 0.25 12 C C 0.4441188 0.4 0.25 7 C C 0.4300978 0.8 0.25 17 C C 0.4075873 0.2 0.25 2

D 0.2388573 1 0.75 24 D D 0.2241656 0.8 0.5 18 D E D 0.1989605 1 0.5 23 E D E D 0.1860179 0.8 0.75 19 E D E D 0.1734274 0.6 0.5 13 E D E D F 0.1575588 0.4 0.5 8 E F E F 0.1245256 0.2 0.5 3 F G F 0.0729699 0.6 0.75 14 G G 0.0212134 1 1 25 G G 0.011511 0.2 0.75 4 G G 0.0100751 0.4 0.75 9 G G 0.0012786 0.6 1 15 G G 0 0.2 1 5 G G 0 0.4 1 10 G G 0 0.8 1 20

208

SAS codes

DATA Contact time; INFILE 'c:\users\erika\dropbox\msu\experiments\optimum contact time\contact time raw data.txt'; INPUT Rate AC AFM1_CONC; RUN; ODS GRAPHICS ON; PROC TTEST H0=0.5 PLOTS (SHOWH0) SIDES=L ALPHA=0.1; VAR AFM1_CONC; RUN; ODS GRAPHICS OFF; QUIT;

Table C.5 One sample t-test below the FDA AL of 0.5 ng/mL

N Mean Std Dev Std Err Minimum Maximum 75 0.3017 0.2899 0.0335 0 0.8477

Mean 90% CL Mean Std Dev 90% CL Std Dev 0.3017 -Infty 0.345 0.2899 0.2558 0.3357

DF t Value Pr < t 74 -5.92 <.0001

209

Figure C.1 Plot of distribution of AFM1 with 90% lower confidence

210