Assessment of Fumonisin B2 Contamination in Muscadine Wine and Grape Juice As a Major Risk Factor to Human Health

ASSESSMENT OF FUMONISIN B2 CONTAMINATION IN MUSCADINE WINE AND GRAPE JUICE AS A MAJOR RISK FACTOR TO HUMAN HEALTH

DEVIN C. LEWIS

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2016

To my wife, Without whom I would have never reached the unattainable and gives me the confidence to conquer anything

ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Renee Goodrich-Schneider for granting me the opportunity to pursue my doctoral degree and for all the invaluable guidance throughout this experience. I am thoroughly appreciative for the assistance and expertise shared with me by my committee members, Dr. Dennis Gray, Dr. Marty

Marshall, and Dr. Charles Sims.

I would also like to thank my lab mates, Lemâne Delva, Gayathri Balakrishnan, and Brittany Martin for all their help and suggestions. A special thank you to the Dr.

Yavuz Yagiz, Maria Ralat, and Bridget Stokes, all of whom are an indispensable bank of knowledge that I am truly grateful for. Most of all, I thank my family and close friends whose support and understanding gave me the strength to succeed through accomplishing my goals. Finally, I would like to thank coffee. Who knew that a simple fermented bean could provide such a lifeline through so many long and tireless nights.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 12

ABSTRACT ...... 15

CHAPTER

1 INTRODUCTION ...... 17

2 LITERATURE REVIEW ...... 24

Major Genera of Mycotoxigenic Fungi ...... 24 Aspergillus niger: Taxonomic Classification and Life Cycle ...... 25 Fungal Metabolism ...... 28 Primary Metabolism ...... 28 Secondary Metabolism ...... 29 Mycotoxin Classes ...... 36 Major ...... 36 Aflatoxins ...... 37 Trichothecene ...... 37 Zearalenone ...... 38 Ochratoxin A ...... 38 Ergot alkaloids ...... 39 Patulin ...... 39 Minor ...... 40 Cyclopiazonic acid ...... 40 Sterigmatocystin ...... 40 Citrinin ...... 41 Gliotoxin ...... 41 Citreoviridin ...... 41 Toxicology-Biological Effects of Mycotoxins ...... 42 Acute and Chronic Toxicity ...... 42 Cytotoxicity ...... 43 Carcinogenicity ...... 43 Antitumor Properties ...... 46 Embryogenicity / Teratogenicity ...... 49 Immunosuppressive Effects ...... 53 Mycotoxin Exposure...... 54 Preharvest and Postharvest Occurrence of Mycotoxins ...... 55

Harvest Timing Effects ...... 57 Fungicide Treatments ...... 59 Human Risk Assessment ...... 62 Hazard Characterization ...... 64 Exposure Assessment ...... 65 Risk Characterization ...... 66 Risk Management ...... 67 Fumonisins ...... 70 Structural Characteristics and Biosynthesis ...... 70 Mechanism of Toxicity ...... 73 Biodistribution and Pharmacokinetics ...... 76 Historical Relevance and Discovery ...... 80 Government Guidelines and Recommendations ...... 82 Dietary Exposure ...... 83 Measurement of Fumonisin Exposure Difficulties ...... 88 Fumonisin Effected Commodity Occurrences ...... 90 Coffee ...... 90 Dried Vine Fruit ...... 91 Maize and Corn-Based Foods ...... 92 Beer ...... 97 Wine and Grapes ...... 99 Thermal Degradation from Processing ...... 102 Muscadine Grapes ...... 104 Harvest and Post-Harvest ...... 106 Juice and Wine Considerations ...... 107 Cultivars ...... 108 Fresh market ...... 109 Wine, juice, jelly processing ...... 110 Hardiness- insect control and disease ...... 111 Breeding and Cultivation ...... 112

3 CAN ASPERGILLUS NIGER COLONIZE MUSCADINE GRAPES ...... 118

Methods and Materials...... 120 Safety ...... 120 Statistical Analysis ...... 120 Chemicals ...... 120 Fungal Hydration and Agar Production ...... 120 Grape Preparation and Extraction ...... 122 LC-MS/MS Conditions ...... 123 Result and Discussion ...... 125 Conclusion ...... 133

4 STORAGE IMPACTS ON FUMONISIN CONCENTRATIONS IN MUSCADINE GRAPE JUICE ...... 141

Methods and Materials...... 144

Safety ...... 144 Chemicals ...... 144 Juice Pasteurization and Sample Preparation ...... 144 LC-MS/MS Conditions ...... 146 Results and Discussion...... 147 Conclusion ...... 153

5 ARE COMMERCIALLY AVAILABLE MUSCADINE WINES A THREAT TO HUMANS? ...... 167

Materials and Methods...... 169 Safety ...... 169 Wine Samples ...... 169 Statistical Analysis ...... 170 Chemicals ...... 170 Sample Preparation ...... 170 Fumonisin Analysis ...... 171 Results and Discussion...... 172 Conclusion ...... 175

6 DOES VINIFICATION EFFECT MYCOTOXIN CONCENTRATIONS IN MUSCADINE WINES? ...... 181

Materials and Methods...... 184 Safety ...... 184 Statistical Analysis ...... 184 Sample Preparation ...... 185 Wine lees extraction ...... 186 Yeast sediment extraction ...... 186 Titratable acidity, pH, and °Brix ...... 186 Alcohol content ...... 187 Noble wine production ...... 187 Carlos wine production ...... 189 Fumonisin Analysis ...... 190 Results and Discussion...... 191 Conclusion ...... 196

7 OVERALL CONCLUSIONS AND FUTURE WORK ...... 207

LIST OF REFERENCES ...... 212

BIOGRAPHICAL SKETCH ...... 240

LIST OF TABLES

Table page

2-1 Summary of vegetative characteristics of select muscadine ...... 117

3-1 Fumonisin B2 production by Aspergillus niger on Carlos and Noble muscadine grape cultivars after artificial inoculation with pre and post inoculation masses ...... 139

4-1 Mean fumonisin B2 content of pasteurized Carlos grape juice over six month period storage in three different storage environments after 350 ppb artificial inoculation ...... 156

4-2 Mean fumonisin B2 content of pasteurized Carlos grape juice over six month period in three different storage environments after 530 ppb artificial inoculation ...... 156

4-3 Mean fumonisin B2 content of non-pasteurized Carlos grape juice over six month period in three different storage environments aftert 350 ppb artificial inoculation ...... 157

4-4 Mean fumonisin B2 content of non-pasteurized Carlos grape juice over six month period in three different storage environemnts after 530 ppb artificial inoculation ...... 157

5-1 Mean fumonisin B2 concentrations in contaminated wine by grape cultivar (n=68) ...... 177

5-2 Mean fumonisin B2 concentrations in contaminated wine by sampled state ... 179

6-1 Noble and Carlos muscadine wine physical attributes before and after production ...... 198

6-2 Mean fumonisin B2 concentrations in wine and wine fractions during Carlos wine production after artificial inoculation with 200 and 1500ppb FB2 ...... 199

6-3 One-way analysis of variance applied to fumonisin B2 content (ppb) of samples collected in different Carlos winemaking steps ...... 199

6-4 Fumonisin B2 concentrations in wine and wine fractions during Noble wine production after artificial inoculation with 200 and 1500 ppb FB2 ...... 202

6-5 One-way analysis of variance applied to fumonisin B2 content (ppb) of samples collected in different Noble winemaking steps ...... 202

LIST OF FIGURES

Figure page

2-1 Fully formed Aspergillus niger with conidia (spores) for dissemination ...... 115

2-2 Fumonisin B2 structure ...... 115

2-3 Molecular structure of “fumonisin-like” sphingolipids ...... 116

3-1 Aspergillus niger on CYAS-20 ...... 134

3-2 Mean fumonisin B2 concnetrations extracted from CYAS-20 agar plug samples after A. niger cultivation ...... 134

3-3 Control Carlos Muscadine after incubation at 25-30°C for 7 days ...... 135

3-4 Surface Inoculated Carlos Muscadine after incubation at 25-30°C for 7 days .. 135

3-5 Wounded Carlos Muscadine after incubation at 25-30°C for 7 days ...... 136

3-6 Control Noble Muscadine after incubation at 25-30°C for 7 days ...... 136

3-7 Surface Inoculated Noble Muscadine after incubation at 25-30°C for 7 days ... 137

3-8 Wounded Noble Muscadine after incubation at 25-30°C for 7 days ...... 137

3-9 Healthy Noble grape on vine ...... 138

3-10 Healthy Carlos grape on vine ...... 138

3-11 Plate 4 chromatogram ...... 140

3-12 Plate 4 total ion chromatogram, FB2 daughter ions 336.23 and 512.28 ...... 140

4-1 Pasteurization apparatus 1 for Carlos grape juice processing ...... 155

4-2 Pasteurization apparatus 2 for Carlos grape juice processing ...... 155

4-3 Mean fumonisin B2 retention percentages in pasteurized Carlos grape juice over six months after storage in three different temperaturesfollowing 350 ppb artificial inoculation ...... 158

4-4 Mean fumonisin B2 retention percentages in pasteurized Carlos grape juice over six months after storage in three different temperatures following 530 ppb artificial inoculation ...... 158

4-5 Mean fumonisin B2 retention percentages in non-pasteurized Carlos grape juice over six months after storage in three different temperatures following 350 ppb spike level artificial inoculation ...... 159

4-6 Mean fumonisin B2 retention percentages in non-pasteurized Carlos grape juice over six months after storage in three different temperatures following 530 ppb artificial inoculation ...... 159

4-7 Non-pasteurized (20°C) 530 ppb month 6 chromatogram ...... 160

4-8 Non-pasteurized (20°C) 530 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28 ...... 160

4-9 Non-pasteurized (-20°C) 530 ppb month 6 chromatogram ...... 161

4-10 Non-pasteurized (-20°C) 530 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28 ...... 161

4-11 Non-pasteurized (3°C) 530 ppb month 6 chromatogram ...... 162

4-12 Non pasteurized (3°C) 530 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28 ...... 162

4-13 Pasteurized (-20°C) 350 ppb month 6 chromatogram ...... 163

4-14 Pasteurized (-20°C) 350 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28 ...... 163

4-15 Pasteurized (3C) 350 ppb month 6 chromatogram ...... 164

4-16 Pasteurized (3°C) 350 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28 ...... 164

4-17 Pasteurized (20°C) 350 ppb month 6 chromatogram ...... 165

4-18 Pasteurized (20°C) 350 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28 ...... 165

4-19 Structures of compounds formed during fumonisin B2 degradation ...... 166

5-1 Mean fumonisin B2 contamination concentrations in wine by grape cultivar for each sampled state (n=68) ...... 180

6-1 Mean fumonisin B2 toxin concentrations throughout Carlos wine production after artificial inoculation at 200 and 1500ppb FB2 ...... 200

6-2 Mean fumonisin B2 concentrations in wine yeast sediment during Carlos wine production after artificial inoculation at 200 and 1500ppb FB2 ...... 200

6-3 Evolution of fumonisin B2 in two artificially inoculated samples during the Carlos wine-making process...... 201

6-4 Mean fumonisin B2 toxin concentrations throughout Noble wine production after artificial inoculation at 200 and 1500ppb FB2 ...... 203

6-5 Mean fumonisin B2 concentrations in wine yeast sediment during Noble wine production after artificial inoculation with 200 and 1500ppb FB2 ...... 203

6-6 Evolution of fumonisin B2 in two artificially inoculated samples during the Noble wine-making process ...... 204

6-7 Noble wine production process and sampling protocol ...... 205

6-8 Carlos wine production process and sampling protocol ...... 206

LIST OF ABBREVIATIONS

ADI acceptable daily intake

AEPC annual eatings per capita

AFB1 aflatoxin B1

AML acute myeloid leukemia

AP aminopentol

ATCC American Type Culture Collection

ATM atmosphere

Aw water activity

BCRP breast cancer resistance protein

B.W. body weight

CBS Centraalbureau voor Schimmelcultures

CV cultivar

CYAS Czapek yeast agar with 5% salt

CYA20S Czapek yeast agar with 20% sucrose

DA daltons

DON deoxynivalenol

ELEM equine leukoencephalomalacia

E.U. European Union

FB fumonisin

FDA United States Food and Drug Administration

FFQ food frequency questionnaire

FRAC fungicide resistance action committee

GDDA45A growth arrest and DNA-damage-inducible protein GADD45 alpha

GPI glycosylphosphatidylinositol

HPLC-FD high performance liquid chromatography/ fluorescence detection

HUVEC Human Umbilical Vein Endothelial Cells

IAC immounoaffinity column

IARC International Agency for Research on Cancer

IC inhibitory concentration

IΚB-Α nuclear factor of kappa light polypeptide gene enhancer in B- cells inhibitor, alpha

JEFCA Joint FAO/WHO Expert Committee on Food Additives

LD50 lethal dose of 50% of population

LOQ limit of quantification

M/Z mass to charge ratio

MEA malt extract agar

MEOH methanol

MDR multidrug resistance phenotype

NaCL sodium chloride

NDA naphthalene-2, 3-dicarboxaldehyde

NFKAPPAB nuclear factor kappa-light-chain-enhancer of activated B cells

NOAEL no observed adverse effects level

NRRL Northern Regional Research Laboratory

NTD neural tube defects

OTA Ochratoxin A

OIV International Organisation of Vine and Wine

PANC-1 Human pancreatic carcinoma, epithelial-like cell line

PAP N-palmitoyl-AP

PPE porcine pulmonary edema

PCR-RFLP polymerase chain reaction-restriction fragment length polymorphism

PDI probable daily intake

PMTDI provisional maximum tolerable daily intake

RH relative humidity

RP-HPLC/ESI-ITMS reversed phase- high performance liquid chromatography/ electrospray ionization –tandem mass spectrometry

RT retention time

SA sphinganine

SPE solid phase extraction

SPP. species

SO sphingosine

TDI tolerable daily intake

WHO World Health Organization

WTO World Trade Organization

ZEN zearalenone

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

ASSESSMENT OF FUMONISIN B2 CONTAMINATION IN MUSCADINE WINE AND GRAPE JUICE AS A MAJOR RISK FACTOR TO HUMAN HEALTH

Devin C. Lewis

August 2016

Chair: Reneé Goodrich-Schneider Major: Food Science

Mycotoxins are the secondary metabolites of filamentous fungi. These fungi are ubiquitous in nature and many are known to cause spoilage of agricultural commodities.

Fumonisins are mycotoxins that were once thought to only be associated with the

Fusarium fungal genera. Currently, Aspergillus niger has also been reported to produce fumonisin B2 (FB2), via the shared fum8 gene. A. niger is a fungal species that thrives in warmer climates and readily produces the FB2 mycotoxin. The environmental conditions that facilitate fumonisin B2 production are also the same conditions required for the growth and maturation of muscadine grapes. The occurrence of mycotoxin contamination of wine and grape beverages within the U.S. has been studied very little.

Government guidelines do not address beverages, alcoholic or not, with regard to fumonisin contamination. These published guidelines are only valid for maize and corn products. This study determined that muscadine grapes can serve as a viable substrate for colonization, growth, and subsequent mycotoxin production by A. niger. The stability of this toxin within the white muscadine grape juice matrix was found to be high when stored at three different temperatures (-18, 4, 20°C) over a six month period. Unlike the toxin patulin FB2 did not degrade to concentration safe for human consumption. When

surveying the FB2 contamination of commercially available muscadine wines from across the Southeastern U.S., all the sampled wines were found to be contaminated with the toxin. However, the observed toxin levels of these wines were below the provisionary maximum daily tolerable intake of 2µg/kg b.w./day for a 70kg adult set by the FAO/WHO. An assessment of the fate of fumonisin B2 during the production of

Noble and Carlos muscadine wine found that juice fermentation played a secondary role in the removal of toxins from artificially contaminated wine. Toxin decontamination was found to be carried out primarily via physical means from the adsorption of the mycotoxin to spent yeast cells. Although this mechanism removed measurable quantities of the toxin, concentration levels still remained beyond the levels for safe consumption.

CHAPTER 1 INTRODUCTION

Typically inconspicuous to most, fungi are ubiquitous in nature. To the naked and untrained eye these complex organisms can go unnoticed for months and even years until furnished with the proper environment to flourish. Although vast amounts of research has been initiated to fully understand these unassuming creatures, there is still much to be elucidated about their interactions within the ecosystem.

Classified as neither plant nor animal, fungi serve as consummate decomposers, playing a strategic role in nutrient cycling, degrading organic matter into inorganic matter. Due to their lack of chlorophyll or photosynthetic function, fungi gain their energy and nutrients via this mechanism. After this degradation occurs, inorganic molecules can reenter the anabolic pathways of other organisms (Lindahl et al., 2007).

Fungi do not grow in isolation, and have evolved to coexist in varying relationships with other organisms for their benefit and survival. These symbiotic relationships can be described as mutualistic, antagonistic, or commensal, having no detrimental effects on either the fungus or host (Paszkowski, 2006).

Within agriculture, plant fungal diseases are one of the major adversaries to a profitable harvest. For people in developing countries that live by subsistence farming for not only food stuff, but clothing and furniture, a low yielding harvest may have ramifications that go as far as death from starvation. Crop loss quantities may range from slight to 100%, and have serious effects on product quality thereby hindering marketability. In the case of losses for farmers, the burden of higher prices is passed on to the consuming public. These diseases have been noted to not only attack crops while in the fields but also in storage facilities as well. To control this devastation, an

entire industry was created, and devoted to the production of fungicides and fungistats, followed by another to regulate the production and use of these products. Historically, a large portion of the interactions between humans and fungi have been detrimental to humans. One of the earliest incidences (857 A.D.) was burning sensations within the extremities, leading to blackened toes, feet and fingers, of those effected by Hell Fire, later renamed St. Anthony’s fire. These symptoms and others stemmed from the ergot poisoning of rye, a staple grain along the Rhine River and later in Eastern Europe and western Russia. This poisoning was a direct result of infection by Claviceps purpurea, a fungus that causes the plant disease ergot, to replace healthy rye grains with fungal fruiting bodies. High mortality rates were associated with this episode due to population reinfection from a lack of knowledge pertaining to fungal diseases at the time. It was not until 1850 that the nature of ergot plant infection was understood. However in the 20th century, extracts from ergot have been used to produce pharmaceuticals that aid in migraine headache relief and uteral contractions (Matossian, 1989).

The effects of fungal diseases have also been demonstrated in domesticated animals. During the summer of 1960, a shipment of Brazilian ground peanut meal was exported to Great Britain for use as a protein supplement for animal feed. Unbeknownst to anyone this meal was highly contaminated with Aspergillus flavus and A. parasiticus.

Once the meal was fed to animals, they began to die by the thousands, with the majority being turkeys. Infected birds displayed symptoms of hemorrhagic gastroenteritis with abnormally swollen kidneys and liver lesions. While the deaths were being investigated, the “virus” was dubbed “Turkey X” syndrome (Cole, 1986). The common element between the Turkey X syndrome and St. Anthony’s fire incidents was the fungi that

contaminated the foodstuffs elicited a secondary metabolite that caused the malaise in the human and animal victims. These secondary metabolites are defined as

“mycotoxins”, and are classified as secondary because they are not needed for growth and survival of the fungus. Mycotoxins may cause diseases known as mycotoxicoses via all natural routes of exposure, including inhalation, dietary and dermal contact.

Mycoses, on the other hand, are diseases that stem from the direct growth of fungi on the host. These fungi are generally either opportunistic, infecting immunocompromised/debilitated hosts, or primary pathogens that infect healthy hosts.

Several factors determine the severity of mycotoxicoses including: the type of toxin, toxin concentration, duration of exposure, age and health of the exposed, and dietary status. Mycotoxicoses have the potential to enhance the susceptibility of the infected to microbial diseases, compound interactions with other toxins, and worsen already present malnutrition (Bennett and Klich, 2003). The distinction that all mycotoxins are of fungal origin, but not all toxic compounds produced by fungi are mycotoxins must be kept in mind. This distinction is validated by the importance of the metabolite target and concentration. Fungal products such as penicillin that are toxic to bacteria are classified as antibiotics, while fungal products that are toxic to plants are called phytotoxins. With regard to concentration, mycotoxins are typically toxic at very low concentrations, whereas a fungal product such as ethanol is not deemed to be a mycotoxin because it is harmful primarily in higher concentrations (Bennett, 1987). The mycotoxin associated with C. purpurea during the St. Anthony’s Fire epidemic was an ergot alkaloid, a derivative of lysergic acid, as for the toxin of the Turkey X syndrome, aflatoxin was

extracted from contaminated groundnut meal, with the distinct possibility that cyclopiazonic acid may have also been a constituent (Hanson, 2008).

It has been known for some time that Aspergillus flavus produces the highly pathogenic mycotoxin aflatoxin. However, the dangers of a pathogenic secondary metabolite from another species of the Aspergillus genus, Aspergillus niger, went undetected until the work of Frisvad et al., in 2007. A. niger has been known to demonstrate many beneficial attributes including its production and/or over-production of citric acid (Moyer, 1953), fermentation of Puer tea (Mo et al., 2005), and extracellular enzyme production (Wosten et al., 2007). Aspergillus is currently considered one of the most economically important genera of micro fungi due to its collection of value-added traits. On the other hand, this fungal genus has been linked to very harmful and toxic instances to human health. Filamentous fungi within the Aspergillus genera are well- known for being linked to the spoilage of agricultural commodities. This spoilage is generally proportional to the growth of the fungus. During this growth, some Aspergillus species not only contaminated commodities via growth, but also through the production of harmful mycotoxins. Aspergillus niger is one of these such species, secreting the mycotoxin Fumonisin B2 (FB2). This mycotoxin has been linked to cases of esophageal cancer in South Africa (Marasas et al., 1988), leukoencephalomalacia in horses

(Marasas et al., 1988), and pulmonary edema in pigs (Haschek et al., 2001). Although are there hundreds of mycotoxins, few of them are detected in food and are considered to have little impact on human health. The toxicity of mycotoxins is generally associated with low doses over long periods of time (Serra et al., 2005). Until recently the fumonisin toxin has been solely associated with the Fusarium genera, but multiple studies have

been published verifying that the mycotoxin is also produced by A. niger. A. niger, being ubiquitous in vineyards and causing black rot of grapes, may also pose a risk to human health via FB2 and /or ochratoxin A (OTA) contamination. At this time there are no published reports on the fate of this mycotoxin in wine/ grape juice production or if there is an occurrence of this type of contamination from grapes grown in the southeastern United States.

From the study of this agricultural product, there are several objectives we hoped to achieve. Although various grape varietals can be sourced from all over the world, the focus of this study was grapes and grape products (juice) sourced from the southeastern United States. Within this grape growing region the Vitis rotundifolia grape species, also known as muscadines are quite popular due to their resistance to the area’s harsh weather extremes. The environmental conditions these grapes are grown under are very similar to those in the southern regions of affected European countries.

Since these growing conditions are equivalent, there is a possibility that the black mold seen in vineyards of the southeastern U.S. may be isolates of Aspergillus niger that produce fumonisin B2. The threat of FB2 contamination in grape products is not one that has been thoroughly investigated within the United States, although all the risk factors including moisture, temperature, and high soluble solids content are prevalent. With regard to food products contaminated by fumonisins, the recommendations and guidelines handed down from the FDA/USDA only pertain to corn and corn products, with no mention of beverages. The first objective of this study was to demonstrate the toxin production of A. niger on Noble and Carlos muscadine grape varietals. These varietals were chosen because these grapes are highly suitable for wine and grape

beverage production. This study serves as a foundation for further studies because if it can be shown that this fungus will use muscadine grapes as a substrate to produce this toxin, then the possibility for contamination in other grape products is relevant. It is common place for juice products made available for commercial sale to be pasteurized per the U.S. FDA Juice HACCP Hazards and Controls (2004) guidelines. These processing guidelines allow for a five log reduction of pathogenic bacteria. Mycotoxins, however, have the ability to survive thermal processing. The second objective was to ascertain whether various storage conditions after thermal processing have any effect on FB2 concentrations in contaminated grape juice over a designated time period and under various storage conditions. There have been instances of contaminated juices being stored over long periods of time under refrigeration leading to the reduction in toxin concentrations such as in the case of Patulin contaminated apple juice (Koca and

Ekşi, 2005). Although contamination risk factors persist, Aspergillus secondary metabolite contamination has yet to be quantified during the production of the aforementioned products. The third objective was to conduct a survey of commercially available wines and grape juice beverages produced in the southeastern United States for fumonisin contamination. This objective provided a clear indication of whether there was a natural occurrence of fumonisin contamination of grape-based beverages for public consumption within the region. The fourth and final objective of the study was focused on the fate of the fumonisin B2 toxin during the wine making process. From these observations, we determined if any of the vinification steps had an effect on the reduction of toxin concentrations. This objective served to ascertain whether the toxin concentration is reduced via fermentation or by other means, the possible mechanisms

behind this reduction and if the toxin is recoverable or structurally converted to another compound.

CHAPTER 2 LITERATURE REVIEW

The term mycotoxin is derived from the Greek word “mycos” meaning mold, and the Latin word “toxicum” meaning poison. Harmful to humans and animals that may ingest them, mycotoxins are low molecular weight secondary metabolites of various fungi. Mycotoxins are considered secondary metabolites because they are not needed for fungal growth, and are produced by primary metabolic processes (Bellí, 2006).

Although they have been studied since the late 1960’s, the exact function of mycotoxins remains a mystery. It is hypothesized that mycotoxins act as a defense mechanism, protecting the fungus from plants, animals and other competing fungi (Smith and Moss,

1985). The infectious dose of mycotoxins necessary to produce adverse health effects varies between fungi and the infected animal or human. Toxicological effects on humans and animals can be either acute or chronic. Acute toxicity is the rapid onset of an adverse effect from a single exposure, whereas chronic toxicity is a slow or delayed onset and is typically due to multiple or long term exposure. Most often, ingestion of mycotoxins and the toxicity they confer to humans is through chronic exposure over long periods. Chronic exposure, being the primary mechanism of infection, creates difficulties in risk assessment and mitigation across populations, especially those in developing countries.

Major Genera of Mycotoxigenic Fungi

Most mycotoxigenic fungi tend to be classified in one of the following genera:

Aspergillus, Penicillium, or Fusarium. Members of the genus Aspergillus are very diverse and found throughout the world, but largely occupy subtropical and warm climates. They are generally regarded as saprophytes with great importance to the

nutrient cycle. Their growth at high temperatures and low water activity (aw) allows for their involvement in the colonization of a variety of crops, at times with limited parasitism under the favorable conditions. Many economically viable toxigenic species belong to this genus. The genus Penicillium generally grow and can produce mycotoxins over a wider range of temperatures than those of other genera (Ominski et al., 1992).

Members of this genus are most commonly associated with storage than with preharvest contamination of grain and are more abundant in temperate climates (Sauer et al., 1992). Fusarium is a large and complex genus whose species have adapted to a wide range of habitats. They are world-wide in distribution and serves as plant pathogens, living in a saprophytic lifestyle degrading plant residues. Some species are prevalent toxin producers and contaminate crops during preharvest. Stachybotrys and

Claviceps also produce toxins but their pathogenicity with regard to humans is of a lesser prevalence. However, historically, the genus Claviceps is one of the oldest known mycotoxigenic genera due to its causation of ergotism. The genus Stachybotrys is characterized by its saprophytic species that produce toxins contained in the conidia and other particulates that can become airborne resulting in illness via respiratory or digestive routes of exposure (CAST, 2003).

Aspergillus niger: Taxonomic Classification and Life Cycle

Aptly known as an opportunistic pathogen, the haploid filamentous ascomycete fungi, Aspergillus niger (Fig. 2.1) is classified within the subgenus Circumdati, section

Nigri, (Samson, 1992) with other associated synonyms including: Aspergillus niger var. niger, Aspergillopsis nigra (Tiegh.) Speg., Rhopalcystis nigra (Tiegh.) Grove,

Sterigmatocystis nigra (Tiegh) Sacc. 1877 (Raper and Fennell, 1965). Aspergillus received its name from Micheli in 1729, viewing the fungus under the microscope, its

spore-bearing structures reminded him of a device used by the Roman Catholic clergy to sprinkle holy water during a portion of the liturgy called the asperges (Ainsworth,

1976). Its ubiquitous nature stems from its ability to grow over a wide range of temperatures from 6-47°C with an optimum of 35-37°C. A. niger has also been noted to grow successfully at the maximum water activity of 0.88 and at pH ranges of 1.4-9.8

(Schuster et al., 2002). Possessing characteristics such as these lend themselves to a lifecycle inherent to survival. The lifecycle of A. niger begins as over-seasoning spores, mycelium, or sclerotia (compact mass of mycelium) living on dead leaf/plant debris as a saprophyte. Mycelium consists of an interwoven system of branching tubes classified as hyphae. The walls of hyphae enclose multinucleated protoplasm that produce new cell walls at the growing tip. These extensions are divided by cross walls known as septa, which contain central pores that allow the movement of cytoplasm and nuclei. Since the hyphae are not closed mononucleated cells they are classified as coenocytic. When warm and humid conditions persist, the mycelium produce conidiophores, hyphal branches developing from foot cells. These conidiophores enlarge at their apex to form a rounded or elliptical-shaped vesicle. A layer of cells known as “phialides” emerge from the fertile area of the vesicle and produce long chains of spores or asexual fungal fruiting bodies known as conidia or condiospores. Fully formed conidia are detached from the conidiophore by various elements including wind, rain etc., many of these conidia will land on substrate that are inhospitable for growth and will begin to over- season until an acceptable host/ substrate is found. For those spores that do find acceptable hosts, a water-insoluble polysaccharide mucilage is secreted to aide in the adherence to plant surfaces (Petrini and Ouellette, 1994). This is followed by a cascade

of stimuli that include: contact with the host surface, hydration, an availability of nutrients, and absorption of low molecular weight ionic material that culminate in the germination of spores. If this stimuli threshold is not met or the spore senses too many like spores in the vicinity, germination will be delayed by an action known as quorum sensing. Host invasion and colonization commence via the mobilization of food stores and carbohydrates, funneling these resources to form the germ tube and its extension

(Whitehead and Salmond, 2000). The growth and differentiation of the germ tube into an appresorium leads to either direct penetration, penetration through wounds, or natural openings by the narrow penetration peg at the appresorial tip. The penetration of plant cuticle is predicated on the accumulation of glycerol, which draws water into the penetration peg generating the needed hydrostatic pressure to overcome plant cuticle turgor (Emmett and Parbery, 1975). Once the conidia penetrates the host cuticle layer or gains access via openings (wound or natural), A. niger begins to infect the host, coming in contact with susceptible cells and tissues. As the fungus robs the host of its vital nutrients, infected cells collapse and disintegrate. At this point, the infection is successful and colonization, growth and reproduction, begins showing symptoms such as softened tissues and rot (Tucker and Talbot, 2001). However, there are some instances when A. niger demonstrates an endophytic association with some agricultural products such as maize and peanuts. Species of black aspergilli were isolated from these surface disinfected items, with no signs or symptoms of fungal colonization

(Palencia et al., 2009). A similar endophytic relationship was thought to have occurred as well with garlic and onions, this proved to be a symptomless infection, metabolically active, producing toxic secondary metabolites (Hayden and Maude, 1992). Although all

four of these agricultural products (maize, onions, garlic, and peanuts) are known substrates for A. niger colonization, it is yet to be fully understood why there are instances of non- infection in these plants.

Fungal Metabolism

Primary Metabolism

Prior to the production of secondary fungal metabolites, primary metabolism must occur as in all living organisms. Primary metabolism is the culmination of a series of degradative and/or synthetic enzyme catalyzed reactions, yielding an organism’s energy and macromolecules, i.e. protein and DNA. The synthesis and activity of enzymes of primary metabolism are controlled by various regulatory mechanisms, some of these same mechanisms govern enzymatic activity during secondary metabolism as well.

Properly functioning primary metabolism depends on the coordination of catabolic, anabolic, and amphibolic pathways. Enzymes and intermediates of primary metabolism serve to secure the growth and reproduction of various organisms. The aforementioned regulatory mechanisms are controlled at the physiological and genetic levels. Several physiological factors are known to play regulatory roles in primary metabolism such as: induction of enzyme synthesis, feedback/end product repression of enzyme synthesis, feedback inhibition of enzyme activity, activation of enzymes, catabolic inhibition, nitrogen metabolite regulation, and phosphate regulation (Betina, 1984). The major energy source for heterotrophic organisms, glucose, may be used by some fungal species directly to produce secondary metabolites, however most secondary metabolites are formed as intermediates in a series of energy-releasing reactions.

Without these intermediates being used for such purposes the reaction would oxidize glucose to CO2, water, and adenosine triphosphate (ATP). During this conversion, the

production of shikimic acid yields an intermediate for aromatic 2° metabolites, whereas further down the carbon pathway the carboxylation of acetyl CoA from pyruvate produces malonyl CoA, a substrate for a linear condensation with acetyl CoA creating polyketide or fatty acid 2° metabolites. Finally, the condensation of acetyl CoA with oxaloacetate during the tricarboxylic acid (TCA) cycle, completes the oxidation of glucose, but is also a source for other 2° metabolites to be produced such as itaconitin from Aspergillus itaconicus (Turner, 1971). As efficient as it maybe, primary metabolism is a controlled process, not over producing one intermediate at the expense of another. This control is exerted via the production of two classes of enzymes, inducible and constitutive. Constitutive enzymes are typically produced in the same quantities under all circumstances, whereas inducible enzymes are only synthesized in the presence of inducers, their substrates. If for any reason those controls fail, organisms also have end product repression of enzyme synthesis and allosteric inhibition of enzyme activity to regulate primary metabolism. End product repression is denoted by the final product of the biosynthetic sequence repressing all catalytic enzymes of the sequence, while allosteric inhibition inhibits the activity of a single enzyme via absorption of the end product on an enzymatic receptor site causing a conformational change that will no longer accept the substrate (Lehninger et al., 2005).

Secondary Metabolism

Although primary metabolism is virtually the same amongst the majority of living organisms, secondary metabolism is restricted to lower life forms and is quite species specific. It involves primarily synthetic processes that conclude with products that demonstrate no true role for the organism. However, many of these secondary metabolic products have proved useful to humans. Citric acid, a secondary metabolite

derived from Aspergillus is one of the most widely used food ingredients, and is known for its properties as a buffer for pH adjustment and antioxidant use in the cosmetic and toiletries industries (Schuster et al., 2002). This metabolite from a fungal source is so widely used that it is estimated at production rates of 1.6 kg per year (Dodds and Gross,

2007). Another A. niger metabolite, gluconic acid, also has a wide range of uses spanning from a key component in metal cleaning applications, to therapies focused on the elimination of calcium and iron deficiencies (Ruijter et al., 2002). Enzymes such as pectin esterase, protease, pectin lyase and glucoamylase have been commercially produced by A. niger for the degradative purposes of pectin and carbohydrates (Frost and Moss, 1987) (Grassin and Fauguenbergue, 1999). Hemicellulases sourced from A. niger have been used to improve the baking process through its addition to doughs. The enzymes modify the rheological properties of the dough, yielding a higher loaf volume and better crumb structure for breads and pastries (Schuster et al., 2002). One of the earliest approved cholesterol lowering statin drugs for human use, lovastatin, was a secondary metabolite isolated A. terreus, and was successfully sold by Merck under the brand Mevacor™. Other biologically and pharmacologically-active metabolites isolated from this genus include: cholecystokinin, ion channel ligands, and neurokinin antagonists (Alberts, 1998).

Secondary metabolites are typically associated with sporulation processes and placed into broad categories including: metabolites that activate sporulation, pigments required for sporulation structures, and toxic metabolites secreted by growing colonies during the time of sporulation (Calvo et al., 2002). The production of secondary metabolites typically commences later in the growth of fungi, around the stationary or

resting phase. The environmental conditions necessary for sporulation and secondary metabolism have been observed to be much more stringent than those required for vegetative growth, i.e. primary metabolism. However, it was once conceived that secondary metabolites were essential for fungal sporulation, but this has been disproven by several fungi that sporulate and are deficient in 2° metabolite production.

Even with this observation it cannot be discounted that some 2° metabolites may have subtle effects on sporulation (Bu’lock, 1961). It has been noted that there are some 2° metabolites that affect fungal development in various was. Fusarium graminearum’s production of zearalenone, an estrogenic mycotoxin, enhances the perithecial production within the fungus. Effects such as these are also exhibited by butyrolactone

I, an inhibitor of eukaryotic cyclin-dependent kinases produced by A. terreus, which increases the hyphal branching, sporulation, and lovastatin production by this fungus

(Schimmel et al., 1998).

The most common 2° metabolite associated with developmental structures (as there are many others) is melanin. Melanin are dark brown pigments form by oxidation polymerization of phenolic compounds and synthesized during spore formation for deposition into cell walls. In pathogenic fungi, this 2° metabolite acts within the fungal spore as a barrier against damaging UV light and is an important virulence factor also.

In some fungal species, it has been demonstrated that melanin biosynthesis is associated with appresorial formation and spore development. The appresoria, being an infection structure, is required for host penetration. Impairment of this structure or its formation can greatly reduce fungal virulence. Reduced melanin concentrations have

also contributed to smaller, less viable spores that were more sensitive to UV light than wild types (Kawamura et al., 1999).

A positive correlation has been observed between mycotoxin production and sporulation within several mycotoxigenic genera. In Aspergillus parasiticus, compounds that inhibit sporulation have also been shown to inhibit aflatoxin production as well. The chemical inhibition of polyamine biosynthesis inhibits the production of sterigmatocystin, aflatoxin, and sporulation in A. nidulans and A. parasiticus. Furthermore, it has been noted that Aspergillus mutants deficient in sporulation did not produce their associated toxins (Guzman de Peña et al., 1998). Pazoutova et al., (1977) reported that Claviceps purpurea mutants lacking the ability to form conidia on agar, also produced a less toxic alkaloid, the lack of similar activity was also identified in the production of FB (fumonisin

B) biosynthesis from Fusarium verticillioides.

The “workhorses” of secondary metabolism are the enzymes that have exhausted their primary metabolism functionality. These enzymes are of low specificity, which results in the production of chemical families of structurally related metabolites.

This lowered specificity lends itself to the “plasticity” of these enzymes during secondary metabolic regulation. An example of this is the enzyme system isolated from Penicillium urticae and is responsible for the synthesis of 6-methylsalicylic acid, a multi-enzyme complex involved in fatty acid synthesis. However, other fungal species have the ability to transform 6-methylsalicylic acid into a series of other secondary metabolites, including mycotoxins (Bettina, 1983). This phenomenon was described by Bu’ Lock

(1975) as a metabolic reaction grid, which demonstrated how both genotypic and phenotypic selectivity may be evident, due to the production of some enzymes for

specific types of transformations and the manipulation of cultivation conditions.

Characteristics such as these make it clear that in spite of the wide array of chemical structures, secondary metabolites are formed by only a few biosynthetic pathways.

These pathways are related to the primary metabolic pathways and tend to use the same intermediates. With regard to fungal secondary metabolism, the most important pathways are the polyketide, terpenoid route and processes that utilize essential amino acids. It has been noted by Borrow et al. (1961) that fungal secondary metabolism passes through distinct phases during fungal growth. The initial or “balanced” phase is characterized by the exponential growth of the fungi and the uptake of essential nutrients in a constant ratio. Secondary metabolites are generally not produced during this phase and ends with the exhaustion of nitrogen or phosphorus. This nutrient exhaustion also precipitates a decline in cell replication. The following phase for nitrogen-limited fungal growth is the “storage phase” which is noted via cell weight increases due to the accumulation of fat and carbohydrates, and secondary metabolite production begins. At this point, dry weight remains constant beginning the maintenance phase, further continuing the uptake of glucose and production of secondary metabolites, which is triggered by either the induction of enzymes required by secondary metabolism or activation of enzymes formed during the storage phase.

For those organisms that are not nitrogen-limited but have exhausted other nutrients,

Borrow et al. (1961) suggests a “transition” phase between the balanced and storage phases, where the rate of cell proliferation decreases. Although these phases of secondary biosynthesis have been determined, there are still some mechanisms that remain obscure, and still does not give rhyme or reason as to why these metabolites are

produced to begin with. With regard to secondary metabolism and growth, most microorganisms produce high levels of secondary metabolites after most of the cellular growth has occurred. This observation led Bu’Lock (1961), to distinguish the growth phase as “trophophase” and the production phase as “idiophase”. This phase separation is clearly observed in antibiotic-producing unicellular bacterial cultures, but is not plainly observed in fungi. Cultures of fungi, after a period of unlimited growth accompanied by the progressive consumption of nutrients begin to slow their growth.

This usually controls the onset of mycelial differentiation and the biosynthesis of secondary metabolites. It has been observed that there are three distinct variations of growth and 2° metabolite synthesis. The first was noted during the culturing of

Penicillium urticae, in the trophophase, nitrogen, phosphorus, and RNA reach maximal level with rapid glucose oxidation. The phase transition was marked by minimal acetate utilization and respiratory activity. While the idiophase was distinguished by decreased levels of RNA and thiol compounds with slower glucose oxidation, but with patulin accumulation. The second growth and metabolite scenario was that of P. islandicum. It was noted that during the synthesis of pigments, a secondary metabolic process, proceeded at a maximum rate in the middle of the growth phase. The final scenario is that of P. janthinellum, which yielded a diphasic production of citrinin and other secondary metabolites. Citrinin and related phenolic compounds were produce in two separate phases with two maxima in the exponential and the stationary phases (Betina et al., 1979). In a review of these metabolic processes and growth, Bu’Lock (1975) stated, “The two activities, secondary biosynthesis and growth, are often complementary alternatives which may be said to compete for key intermediates.

However it cannot be asserted that these activities are mutually exclusive and incompatible.” It has been suggested by Bu’Lock (1967) that it is not the secondary metabolite that is important to the fungus, but the metabolism itself. It is postulated that secondary metabolism is a means to remove intermediates that would otherwise accumulate, which in turn enables the primary processes to remain functional during times of high stress. Although there is a vast array of secondary fungal metabolites, we will only focus on polyketides since our mycotoxin of focus is of polyketide origin.

Secondary Metabolite Biosynthesis- Polyketides. Fungal polyketides are metabolites derived by the formation of a β-polyketide chain through the repetitive condensation of acetate units or other short carboxylic acids via enzymatic mechanisms similar to those of fatty acid synthesis (Hopwood, 1997). A group of enzymes classified as polyketide synthases (PSKs) catalyze these reactions, and contain several catalytic subunits or “domains” that function to extend the polyketide chain. These domains include: β-ketoacyl synthase(KS), acyltransferase (AT), acyl carrier protein (ACP), β- ketoacyl reductase (KR), dehydratase (DH), C-methyltransferase (C-MeT) and enoylreductase (ER). Polyketides tend to be reduced based on the type and number of domains present in said polyketide synthase (Huffman et al., 2010). Polyketide synthases are classified into three types based on their protein structure. Type I PKSs are large modular proteins consisting of numerous domains, type II PKSs consist of multiple separate protein subunits, each with an independent catalytic site, and finally type III PSKs are a distinctive group of synthases that lack an ACP domain. During synthesis, type I PKSs act only once whereas type II PKSs act iteratively in chain establishment and elongation. Fungal PKSs in particular tend to have a modular

organization like type I PKSs with each domain acting iteratively, hence they are termed iterative type I PSKs (Stauton and Weissman, 2001). Fungal PKSs have the capacity to produce polyketides with one of three reductive states classified into three distinct groups, non-reducing PKSs, partially reducing PKSs, and highly reducing PKSs. Each of these various reductive states has its own distinct domain organization from the N- terminus to its C-terminus which dictates the produced polyketide. Fungal PSKs are further organized on the basis of whether their produced polyketide will have an aromatic functional group or not. The aromatic-type PKSs which typically have a domain organization of KS-AT- ACP-ACP-C-MeT, in which C-MeT is a thioester enzyme(TE)- like domain that contributes to the chain length determination in fungal aromatic polyketide biosynthesis (Fujii et al., 2001). On the other hand, fungal nonaromatic-type

PKSs have additional domains such as DH, ER, KR, and MT. Fungal highly reducing

PKSs such as those that synthesize fumonisins do not include a thiesterase/Claisen-like cyclase for the condensation reaction that is typically associated with other polyketide formation (Cox, 2007). A pyridoxal 5´- phosphate dependent mechanism is responsible for the offloading and elongation of the polyketide carbon chain, and the introduction of the characteristic amine group (Gerber et al., 2009) which we will further discuss later.

Mycotoxin Classes

Major

The vast majority of fungal toxins are not pathogenic and do not cause disease in humans or animals. However, those that do pose the greatest potential risk to humans and animals via food and feed are: aflatoxins, tricothecenes, zearalenone, ochratoxin A, ergot alkaloids, and patulin (Bryden, 2007). The following is a brief overview of the

major fungal secondary metabolites with the exclusion of fumonisin which will be discussed in depth later.

Aflatoxins

This toxin can be produced by four species of Aspergillus: A. flavus, A. parasiticus, A. nomius, and A. pseudotamarii. The four major aflatoxins produced by these fungi are B1, B2, G1, and G2, the “B” and “G” designations indicate blue and green fluorescence under UV illumination on thin-layer chromatography, respectively, with subscripts as indicators of chromatographic mobility. Two minor aflatoxin metabolites also exist, M1 and M2, which are the hydroxylated forms of B1 and B2 with lower toxicity, and were initially isolated from the milk of lactating animals fed aflatoxin preparations

(Ito et al., 2001). This toxin has also been detected in cheese, corn, peanuts, cottonseed, nuts, almonds, figs, and spices. Aflatoxins receive greater attention than some other toxins due to their demonstrated carcinogenic effects in susceptible laboratory animals, also their acute and chronic toxicological effects in humans (Eaton and Groopman, 1994).

Trichothecene

Constituted by a family of nearly 150 related compounds produced by several genera including: Fusarium, Cephalosporium, Myrothecium, Stachybotrys, and

Trichoderma, this toxin’s most important structural feature is the 12, 13-epoxy ring and the hydroxyl group’s position around the nucleus. Trichothecene, DON, or T-2 toxin is characterized as powerful protein synthesis inhibitors, reacting with components of cellular ribosomes. Regardless of the route of exposure, once tricothecene toxins enter systemic circulation, it affects rapidly proliferating tissues and is cytotoxic to most eukaryotic cells. One of the most unique aspects of this toxin is its use in biological

warfare. From 1974 to 1981, the former Soviet Union is alleged to have used this toxin in Cold War countries such as Afghanistan, Laos, and Cambodia. The attacks were administered in aerosol and droplet cloud delivery systems commonly called “Yellow

Rain” (Wannemacher and Weiner, 1997).

Zearalenone

Produced by multiple Fusarium spp. and Gibberlla zeae, this xenobiotic is a non- steroidal estrogenic toxin biosynthesized via polyketide pathways. Fungi producing this toxin frequently colonize corn and to a lesser extent, barley, oats, wheat sorghum, millet, and rice. Typically characterized as a preharvest toxin, postharvest occurrences have been reported due to storage mishandling and improper crop desiccation. After oral administration, ZEA is rapidly absorbed, but is difficult to quantify due to extensive biliary excretion. ZEA contamination has been linked to scabby grain toxicoses in China,

Japan, the U.S.A., and Australia, with associated symptoms if nausea, vomiting, and diarrhea. Although ZEA has a low acute toxicity when orally administered, intraperitoneal injection has been reported to yield a route of exposure with an even lower LD50 in some species (Zinedine et al., 2007).

Ochratoxin A

OTA is produced mainly by Aspergillus ochraceus, A. carbonarius and

Penicillium verrucosum in temperate and sub-tropical climates. This toxin has been found to contaminate various commodities including: coffee, grapes, beer, spices, cacao, dried fruit, cereals, and grape juice. OTA, like most mycotoxins is relatively stable within the range of conventional processing methods and has been detected in manufactured products as well. This toxin is a well-known nephrotoxic agent, associated with the fatal human kidney disease known as Balkan Endemic Nephopathy.

It has also been implicated as being carcinogenic, teratogenic, immunotoxic, genotoxic and possibly neurotoxic in some species. The International Agency for Research on

Cancer (IARC) has classified OTA into group 2b (possible human carcinogen) (Wu et al., 2011).

Ergot alkaloids

Cool damp conditions promote the proliferation of this toxin by Claviceps purpurea, which invades grain kernels to form its characteristic sclerotium. This colonization transforms the grain into an enlarged, hard, dark spur-like structure. Ergot produces numerous alkaloids including: ergotamine, ergocristine, and ergonovine, of these ergonovine is more readily absorbed. Over forty alkaloids have been identified, and all are derivatives of lysergic acid, some of which are inactive. One of the unique traits of the ergot toxicoses, ergotism, is its tendency to manifest in one of two ways in human. This toxin may exhibit via gangrene in the extremities characterized by severe pain, inflammation and burning sensations, or convulsive ergotism which is displayed via blindness, hallucinations, and temporary psychosis (Schardl et al., 2006).

Patulin

This mycotoxin is produced by Aspergillus, Penicillium, and Byssochlamys spp. and is most commonly associated with apples an apple juice contamination. However, it has also been isolated from grains, cherries, plums, bananas, grapes, and vegetables.

Studies have shown this toxin to be geno- and immunotoxic, and have antimicrobial properties against some microorganisms. Although it is not as potent as other mycotoxins, its rampant contamination of apples and apple products was great cause for concern due to the volume of these products consumed by children, a particularly vulnerable population. Its heat stable nature enables it to endure thermal denaturation

and pasteurization, but its stability is lessened after fermentation. The PTDI for patulin was set at 0.43µg/kg b.w. by the FDA, based on an NOAEL of 0.3 mg/kg b.w. per week.

The regulatory limit for apple juice is 50µg/kg worldwide, 25µg/kg for apple products, and 10µg/kg in products intended for infants and young children in the E.U. (Puel et al.,

2010).

Minor

Although the following mycotoxins are considered to be minor, that is by no means an indication of their toxicity. These toxins are classified within this class because they contaminate a sole commodity, are less studied due to a low contamination frequency or under unusual circumstances.

Cyclopiazonic acid

Cyclopiazonic acid is a calcium uptake inhibitor via the deregulation of the calcium pump in the sacroplasmic and endoplasmic recticulum. This toxin is produced by Penicillium and Aspergillus spp, that are instrumental in cheese and sausage manufacturing and is related to the ergoline alkaloids. There is some evidence to implicate this toxin in the Turkey X syndrome in England during the 1960’s (Bradburn et al., 1994).

Sterigmatocystin

Structurally similar to aflatoxins, sterimatocystin is produce by A. nidulans and A. versicolor. This toxin has been found to contaminate grains, green coffee beans, and is naturally occurring in cheese. It is considered to be a potent liver carcinogen, mutagen and teratogen but its effects are not widespread. The IARC classifies this toxin in Group

2B, possibly carcinogenic to human, however a definitive link has not been proven. At this time no country has regulatory guidance for sterigmatocystin in foods (Horn, 1985).

Citrinin

Citrinin acts as a nephrotoxin causing nephropathy in livestock and has been implicated as a cause of Balkan nephropathy. Used as a reagent in biological research, this toxin induces mitochondrial permeability leading to an inhibition in respiration and readily permeates through human skin. It is produced by Peniciliium, Aspergillus, and

Monascus spp. and has been found to contaminate grain, cheese, sake and red pigments, including red yeast rice supplements (Bennett and Klitch, 2003)

Gliotoxin

One of the few toxins with sulfur as a component of its structure, gliotoxin is a member of the epipolythiopiperazines, precursors to therapeutic drugs. This toxin in produced by A. fumigatus, Trichoderma, and Penicillium spp. It is highly immunosuppressive, accelerating apoptosis in neutrophils, granulocytes, and macrophages. Camels have been known to become intoxicated after ingesting hay contaminated with gliotoxin (Gareis and Werney, 1994).

Citreoviridin

Citreoviridin causes paralysis, dyspnea, cardiovascular disturbances and loss of eyesight in experimental animals. Initially isolated from cultures of moldy rice associated with cardiac beriberi. This toxin contaminates corn and other feeds, at times simultaneously with aflatoxin. Its mode of action is binding to and inhibiting mitochondrial ATPase and uncoupling oxidative phosphorylation. Several species of

Penicillium and one Aspergillus have been reported to produce this toxin (Ueno, 1974).

Toxicology-Biological Effects of Mycotoxins

Acute and Chronic Toxicity

Mycotoxin toxicity can vary greatly, and is dependent on the toxin’s structure and the animal species infected. In most cases, the larger the animal species the greater the LD50 value for the species. This is exemplified when comparing an LD 50 of 0.3-

0.6mg/kg body weight in ducklings versus 9.0 mg/kg in mice dosed with aflatoxin B1

(Heathcoate, 1978). With regard to structure-activity relationships, the toxic effects of various mycotoxins are dictated by the organs they affect. Citrinin has been known to cause nephrotoxicity, aflatoxins cause hepatotoxicity, penicillic acid has cardiotoxic effects and vomitoxin causes vomiting in subjected species (Vesonder et al., 1976).

Although chronic intake is the most widespread form of mycotoxin exposure, there are instances of acute mycotoxicoses, many of which can be directly related to floods, famine, or war (Matossian, 1998). Acute liver disease has been reported in Malaysia, and Kenya following aflatoxin consumption (Bryden, 2007). In India, gastrointestinal pain and diarrhea from a foodborne outbreak associated with high fumonisin intake was reported by Bhat et al (1997). Local villagers in 27 villages, also in India, experienced mycotoxin exposure after consuming maize and sorghum, once harvested, were left in the fields to be damaged by unseasonal rains. In this incident, patients also experienced transient abdominal pain and diarrhea, which began half an hour to one hour following the consumption of unleavened bread prepared from the moldy crops. The inflicted fully recovered when the exposure ceased with no fatalities (Bhat et al., 1989).

Dietary staples in many regions of the world such as cereal grains and corn contain low concentrations of mycotoxins. The impact of regular consumption at these low levels on human health is highly significant with a number of consequences

including, but not limited to impaired growth and development, immune dysfunction, and alterations in DNA metabolism. Gong et al. (2002) conducted an epidemiological study in West Africa to determine aflatoxin exposure in children 9 months to 5 years versus a

WHO reference population. A strong association between exposure to aflatoxin in the children and both stunting and being underweight were revealed. These conditions demonstrated malnutrition and exposure to the toxin in utero and subsequently after birth. The children were also co-exposed to a number of infectious diseases which compromise growth and development via reduced food intake, leading to the repartitioning of nutrients to maintain an upregulated immune system, depressing development.

Cytotoxicity

Mycotoxins form adducts with various molecular receptors such as DNA, RNA, functional proteins, enzymatic cofactors and membrane constituents. The adverse effects are mainly related to genotoxicity, carcinogenicity, mutagenicity, teratogenicity, and immunotoxicity. AflatoxinB1 has been shown to be both genotoxic and one of the most powerful hepatocarcinogens. According to the International Agency for Research on Cancer (IARC), it has been classified in group IA (adequate evidence for carcinogenicity for humans). Ochratoxin A is a nephrotoxin, and is correlated to concentrations in food and Balkan endemic nephropathy, and urinary tract tumors.

Ochratoxin A has also been included on the list of carcinogens as “possible carcinogen to humans.”

Carcinogenicity

Many of the common mycotoxins have modes of action that promote tumors and lead to metabolic events that display carcinogenic effects. Aflatoxin has demonstrated

an ability to inhibit the synthesis of both DNA and RNA when fed to rats over a six week period. The activated aflatoxin B1 (AFB1) metabolite, AFB1-8,9-epxide formed a covalent bond with N7 of guanine, subsequently forming adducts within target cells

(Bailey, 1994). Wang and Groopman (1999) concluded that these cellular adducts led to guanine → thymine transversions, DNA repair, lesions, mutations, and tumor formations. These transversions have also been linked to human hepatocellular carcinomas at codon 249 of the p53 tumor suppressor gene. Although this epoxide has been shown to be short-lived due to hydrolysis to form AFB1-8,9-dihydrodiol, it has also been associated with reducing the synthesis of vitamin K causing a lack of blood coagulation, and other clotting factors. AFB1 also has cytotoxic effects that induce lipid peroxidation in rat livers leading to hepatocyte oxidative damage (Shen et al., 1995).

Competitively binding substrate due to the structural similarities it has with mitochondrial enzymes, OTA’s open lactone moiety enhances its mode of action. This mycotoxin competitively inhibits ATPase, succinate dehydrogenase and cytochrome C oxidase in rat liver mitochondria (Xiao et al., 1996). Within the spleen, OTA is reported to disrupt protein synthesis through the inhibition of phenylalonyl-tRNA synthase, and the promotion of further cellular damage due to hydroxyl radical formation and lipid peroxidation (Cheeke, 1998). After exposing human kidney cells to OTA (100nmol/l), a stimulatory effect on extracellular protein kinase and caspase was reported to lead to apoptosis. Upon exposing human urothelial cells to OTA (50-500nmol/l), there was an induction of unscheduled DNA synthesis with subsequent repair and lesions (Schwerdt et al., 1999) (Dorrenhaus et al., 2000).

The 12, 13 epoxytricothecene nucleus of tricothecene has been experimentally linked to a cytotoxic nature, contributing to the inhibition of protein, RNA, and DNA synthesis (Liao et al., 1976). This inhibition has demonstrated itself to be the precursor of tricothecene binding to active polysomes and ribosomes, interrupting peptide linkages and diminishing initiation and termination sequences (Ueno, 1977). T-2 toxin and DON have been shown to disrupt membrane transport and function, suppressing immune responses, and yielding abnormal blood function. This leads to the reduction of granulocyte-macrophage colony forming cells in the bone marrow of mice, also the inhibition of granulo-monocytic and erythrobalstic progenitors, two stem cells that effect cellular leukemia expression (Rio et al., 1997).

The estrogen binding effect of zearalenone (ZEN) has been well–studied, and is reported to influence estrogen dependent transcription in the nucleus (Kolb, 1984). The receptor binding by ZEN inhibits the estrogenic hormones in rat mammary tissues.

Furthermore, this toxin has displayed the potential to stimulate growth in human breast cancer cells which contain estrogen response receptors (Withanage et al., 2001).

Although fumonisin is cytotoxic and carcinogenic, its complete mode of action has yet to be fully elucidate. However, much of these effects are attributed to the disrupting sphingolipid metabolism via the inhibition of sphingosine N-acyltransferase or ceramide synthase in rat liver microsomes. The accumulation of sphingoid bases has been linked to unscheduled DNA synthesis, the alteration of signaling by cAMP (Huang et al., 1995), and protein kinase C (Yeung et al., 1996), and disruption of normal cell cycling (Ramljak et al., 2000). This fungal toxin also inhibits protein phosphatases and

arginosuccinate synthetase, which further hinders the urea cycle leading to cytotoxic effects.

Antitumor Properties

As counter-intuitive as it may seem, researchers have uncovered bioactive mechanisms from unlikely sources such as snake venom, bee venom, and mycotoxins that have demonstrated a propensity to inhibit tumors. The antitumor activity of these biotoxins has been displayed in several respects including: killing tumor cells directly, inhibiting angiogenesis and inhibiting tumor growth (Orsolic, 2012).

Some of the more prolific fungal secondary metabolites with anti-carcinogenic properties belong to the statin family. This family is most known for their therapeutic use to treat hypercholesterolemia and cardiovascular disease. Statin structure may be the key behind their mode of action, consisting of a dicyclohexene ring system connected to another dicyclohexene ring system linked with a closed lactone ring or open acid form (Wong, 2002). Compactins, a statin-metabolite produced by P.solitum and P. hirsutum has been shown to inhibit acute myeloid leukemia (AML) cells at an inhibitory concentration (IC100) of 2.6µm, with its analogs, lovastatin and simvastatin

(synthetic) being even more potent. The mode of action for lovastatin is to selectively inhibit the growth of primary AML cell colonies. This is further demonstrated in its reported reduction of proliferation in four lung cancer cell lines with median inhibitory concentration (IC50) values between 1.5 and 30µm by lovastatin (Maksimova et al.,

2008). Martirosyan et al. (2010) noted that lovastatin induced apoptosis in ten ovarian cancer cell lines, and was found to inhibit breast cancer MCF-7, liver cancer HepG2, and cervical cancer HeLa cell lines. Simavastatin, the synthetic statin analog, has

inhibited three melanoma, two lung, and four breast cancer cell lines, induced apoptosis with reduced tumor growth in hepatic cancer cells, and has entered clinical trials as an anticancer drug. The alkaloid fungal metabolites isolated from A. fumigatus known as tryprostatins A and B, are known to inhibit the mitogen activated protein MAP-kinase- dependent microtubule assembly. This occurs via the disruption of the microtuble spindle to inhibit cell cycle progression at the mitotic phase. The cytotoxic activity of the tryptostatin B stereoisomer mimics the actions of etoposide in human carcinoma cell lines, which is typically used to treat cancer of the testicles and small cell lung cancer

(Zhao et al., 2002). Furthermore, these two toxins act against the multidrug resistance phenotype (MDR), one of the major causes behind chemotherapy failure in patients.

The breast cancer resistance protein (BCRP) was shown to confer the MDR phenotype to tumor cells, and tryprostatin A acts as a direct inhibitor (Woehlecke, 2003).

At this point, most of the discussed secondary metabolites produced by

Aspergillus spp. have been pathogenic to human health, but A. terreus and A. nidulans produce metabolites that are contrary to this. The small polyketides terrein and asperlin are derived from the two aforementioned fungal species, respectively, and have anticancer activities. Almost 80 years after its discovery, terrein has been found to inhibit breast cancer via the induction of apoptosis at IC50 values of 1.1nM in MCF-7 cell lines. Activity such as this makes terrein100 times more potent than the commonly prescribed cancer medicine Taxol against this line of cells. This metabolite is also active against pancreatic and liver cancer cell lines PANC-1 (IC50 9.8 µM) and HepG2 (IC50

66.8 µM) (Liao et al., 2012). The secondary metabolite asperlin, has been credited with the reduction of cellular proliferation and the induction of G2/M cell cycle arrest in the

human cervical carcinoma HeLa cell line (He et al., 2011). It was also found to impede the phosphorylation and degradation of the IκB-α protein, a compound that does not allow NF-κB to bind with DNA, keeping it sequestered in the cytoplasm and diminishing its activity in lymphoma tumor cells. Asperlin has been noted to reduce the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. (Lee et al., 2011). A. nidulans also produces a tricyclic metabolite, norsolorinic acid, which also induces cell cycle arrest selectively in the G0/G1 phase of the cell cycle. This induces apoptosis in human bladder cancer T-24 and human breast cancer MCF-7 at IC50 values of 10.5 and

12.7µM, respectively (Yamada et al., 2011). Isolation techniques performed on A. pseudoustus as reported by Bennett et al. (1971), yielded the secondary metabolite austocystin D. Austocystin D selectively inhibits the growth of tumor cell lines that overexpress the multidrug resistance-associated protein and human colon carcinoma

LS174T cells in mice. Although it inhibits a number of cancer cell lines in the colon, brain, uterus, breast, bone marrow, and prostate, this metabolite has never entered clinical trial due to a low therapeutic index (Wang et al., 2008). Similar activity was seen in brefeldin A, a polyketide metabolite isolated from P. brefeldianum. Reports indicated the induction of apoptosis in leukemia (HL-60 and K-582), colon (HT-29), prostate (DU-

145), cervical (KB and HeLa), breast (MCF-7 and BC-1) and lung (SPC-A-1 and NCI-

H187) cancer cell lines (Bladt et al., 2013).

The isolation of gliotoxin and methylthioglitoxin from liquid culture filtrates of fungal strains Y90086 and Y80805 proved to have inhibitory capabilities against the proliferation of human umbilical vein endothelial cells (HUVEC) cells. In addition, the toxins reduced the migration of HUVEC cell and the formation of HUVEC-related tubes.

The activity of both compounds displayed growth-inhibitory actions highlighting their potential antiangiogenic effects. Hur et al. (2008) reported gliotoxin-mediated enhancements of radiotherapy efficiency through the activation of NFkappaB (controls

DNA transcription) and the inhibition of radiation-induced GADD45a, the induction of apoptosis of cancer cells in vitro, and in vivo anticancer activity in human cancer xenographs.

One of the more unique sources for acquiring anti-carcinogenic agents is Aplysia kurodai, also known as the sea hare. From this marine organism, Periconia bissoides is isolated and creates the secondary metabolites, perybisin and macrosphelide. These metabolites are cell adhesion inhibitors and act as antimetastatic compounds, inhibiting the adhesion of leukemia HL-60 cells to HUVEC cells. From these isolates, synthetic macrosphelides were prepared and noted to exert highly potent apoptosis-inducing activity in human lymphoma cells, which can be enhanced via hypothermia (Ahmed et al., 2009).

Under the proper conditions, Fusarium spp. produce the fusarisetin A metabolite.

FN080326 was isolated from soil samples collected in the Daejeon region of Korea, and found to be a morphogenesis inhibitor. This compound has been reported to be a cancer migration inhibitor, and ex vivo studies revealed that has the capability to inhibit various types of cellular migration (Xu et al., 2012)

Embryogenicity / Teratogenicity

A large number of mycotoxins have been recognized as teratogens or embryonic agents including but not limited to: aflatoxin, DON, fumonisin, patulin, and ochratoxin A.

Embryogenesis is a complex process that merges cell proliferation, differentiation, migration, organogenesis, and apoptosis to generate species offspring. A toxin that

disrupts any of the aforementioned processes is a potential teratogen or embryonic agent. The most dramatic manifestation of teratogenesis is the birth of grossly deformed offspring, but it must be recognized that any toxin that causes teratogenesis has the potential to also cause embryotoxicity and fetotoxicity (Caldwell-Smith et al., 2007).

Due to the varied structure of most fungal toxins, determining their teratogenic nature from chemical structures alone is virtually impossible. The mutagenic activity of mycotoxins can be unpredictable due to a multitude of factors involved such as: dosage, period of exposure, species, nutrients, feed additives, environmental stressors, and other co-occurring toxins (Bryden, 1982). Several different species have been used as the animal model for developmental and mycotoxin study which has led to variation between species, also, recognized teratogens have displayed interspecies variation.

LeBreton et al. (1964) demonstrated mammalian teratogenicity by inducing fetal growth retardation following administration of repeated small doses of aflatoxin to rats. The administration of small doses at early stages of development was shown to cause much more significant abnormalities than larger doses at later stages of development. Similar studies using hamsters as the model showed that the major factor in retarding development was a dependency on the period of gestation during which the hamsters were treated with aflatoxin. Abnormalities ranged from fetal death and resorption to malformed, but live fetuses when the toxin was administered during organogenesis. It was noted that when the same toxin concentration was administered after the completion of organogenesis, no malformed fetuses were present with a decrease in dead fetuses (Elis and DiPaolo, 1967). Administration of OTA in CD-1 mice by Hayes et al. (1974) was found to cause similar malformations in conjunction with increased

prenatal mortality, craniofacial defects, and skeletal malformations. A wide spectrum of lesions, fetal death, and resorptions were induced under the same conditions for hamsters. From these studies, it was found that dietary protein levels and protein composition are factors that significantly alter OTA-induced teratogenesis (Mayura et al.

1984).

Empirical risk figures, along with numerous clinical and experimental studies, suggest that NTDs are multifactorial in origin, having genetic, environmental, and nutritional components that contribute to their prevalence (Campbell et al., 1986).

Epidemiological evidence points to neural tube defect (NTD) causation by fumonisin.

NTDs are a congenital malformations that occur when the embryonic neural tube, which ultimately forms the brain and spinal cord, fails to properly close during the first weeks of development. A cluster of cases of anencephaly (developmental absence of brain, skull, or scalp) and increased neural tube defects in South Africa (Viljoen et al., 1995),

Guatemala (Voss et al., 2006), and among Mexican-American women who conceived in the Lower Rio Grande Valley during the 1990’s (Kendricks, 1999) were of significance. The prevalence of tube defects in these areas was 6-10 times higher than the world average (1 per 1000 live births), folate deficiency and genetic susceptibility, such as Merkel syndrome, were two compounding factors in many of these cases

(Marasas et al., 2004). In NTDs occurring without other syndromes, the recurrence risk for siblings is approx. 2-5%, which is up to a 50-fold increase over that observed in the general population. Khoury et al. (1988) demonstrated that for the recurrence risk to be this high, an environmental teratogen would have to increase the risk at least 100-fold to exhibit the same degree of familial aggregation, making a genetic component required.

Although efforts have been made to induce teratogenicity and embryotoxicity to study this phenomenon with regard to fumonisin, experimental evidence is not as clear. Voss et al. (1996) suggested that the developmental effects of fumonisin are mediated through maternal toxicity, but this was later refuted by Voss (2006) after inducing NTDs in rat embryos of mothers exposed to fumonisin. Furthermore, impaired folate metabolism was noted in this study as well since fumonisin inhibits folate transport contributing to teratogenic activity and the disruption of apoptosis.

Another factor to consider is that some fungi produce more than one toxin, and multiple toxic secondary metabolites may accumulate in feed from one or several different species. In this case, animals may be subjected to low concentrations of a

“cocktail” of toxins manifesting in toxin interactions. Interaction studies performed by

Wangikar et al. (2004) between aflatoxin and OTA demonstrated a reduction in teratogenicity, but an increase in embryotoxicity. However, when OTA was paired with zearalenone the number of fetotoxic and teratogenic effects were reduced (Arora et al.,

1983). Co-injection of fusaric acid and fumonisin by Bacon et al. (1995) resulted in a synergistic toxic response in chicken embryos. The injections caused reduced reproductive efficiency by increasing teratogenicity and embryotoxicity. Instances such as these serve as examples of the difficulty created when attempting to extrapolate single toxin versus field intoxications.

After the ingestion of mycotoxin contaminated foods, humans eliminate the toxin via bodily fluids or they accumulate in tissues. This accumulation raises concerns for the contamination of human breast milk as a serious potential hazard. Occurrences in human tissues and fluids have been documented in tropical and subtropical countries,

with detection in breast milk, cord blood, and maternal blood on the African continent

(Galvano et al., 1996), United Arab Emirates ( Abdulrazzaq et al., 2003), Australia and

Thailand (el Nezami et al., 1995). Aflatoxin contamination poses the greatest prenatal health risk because it can cross the human placental membrane, with possible concentration in the developing feto-placental unit. In more temperate or colder countries, the risk of OTA residues in human milk have been uncovered in Italy (Micco et al., 1991), Switzerland (Zimmerli and Dick, 1995), and Germany (Gareis et al., 1988).

In Norway, Skaug et al. (2001) examined the relationship between OTA contamination of breast milk and dietary intake, finding that the risk was related to the consumption of breakfast cereals, processed meat products, juice and other foods. They also concluded that the risk of toxin ingestion from multiple sources by infants is increased in the first months of life due to receiving breast and dry milk sources concurrently.

Immunosuppressive Effects

Many fungal toxins including fumonisin, ochratoxin A, patulin, and aflatoxin have been shown to cause immunosuppression and increase the susceptibility of animals to infectious disease (CAST, 2003). Mycotoxins have been proven to be immunotoxic and exert effects on cellular responses, humoral factors, and cytokine mediator of the immune system (Oswald et al., 2005). The effects of immunity and resistance are more difficult to separate in field settings because signs of the disease are typically associated with the infection instead of the toxin that caused said infection through decreased resistance and/or reduced vaccine or drug efficacy. In animal models, immunosuppressant effects of toxin occur at lower levels of intake than do other toxin effects such as feed intake or growth rate and can go unnoticed for substantial periods.

Studies by Peska et al. (2004) revealed that DON can both stimulate and suppress the

immune system. This activity was demonstrated via the toxin’s effects on dysregulation of IgA, which led to the development of kidney disease in animal models that closely resemble human glomerulo-nephritis IgA nephropathy. Immune suppression can interfere with vaccination and can predispose to cancer induction. The effects of low level, long term exposure on the immune system is highly relevant due to the increase in susceptibility to opportunistic microbes or severe infections related to zoonotic disease or food safety.

Mycotoxin Exposure

Knowing the occurrence and distribution of toxins in foods and feeds is important because exposure to unknown bioactive agents is an important factor in attempts to explain the etiology of chronic disease in animal and humans. However, it is uncertain exactly how many toxic secondary metabolites or mycotoxins exist in nature.

Throughout the years several estimates have been made. In 1978, Turner cataloged

1,200 secondary fungal metabolites, produced by approximately 500 fungal species.

Four years later, Alderidge and Turner (1983) cataloged 2,000 more metabolites which were believed to be produced by 1,100 species. From this work, the conclusion that on average there are two unique secondary metabolites per fungal species arose. Based on this assumption, and the estimation that there are 69,000 known fungal species, which represents 5% of the world’s total fungal species which is further estimated to be around 1.5 million, there are 3 million unique secondary fungal metabolites

(Hawksworth, 1991). The usual route of mycotoxin exposure is ingestion as food or contaminated feed. Dermal contact and inhalation are also important routes for toxin exposure also. Direct effects of mycotoxins range from acute disease where severe conditions are most likely followed by exposure to high levels of a mycotoxin. On the

other hand, chronic disease such as tumor formation or conditions such as growth retardation, impaired immunity, decreased disease resistance, or decreased milk/egg production may result from long term exposure to small toxin quantities. The low level of exposure is of considerable concern where food and feeds are of better quality and would typically contain lower concentrations of mycotoxins. Populations that depend on a single staple diet are of great concern also. Toxin contamination of said staple items would yield greater exposure to consumers over a given time period, as opposed to populations that have diets based on a wide variety of foods. Indirect exposure of humans to mycotoxins is likely to occur when toxin residues or metabolites persist in milk, eggs, or edible tissues are consumed.

Preharvest and Postharvest Occurrence of Mycotoxins

The contamination of foodstuffs via mycotoxins has the tendency to be an additive process, beginning in the field and increasing during harvest, drying, and storage (Wilson and Abramson, 1992). Fungi that are known to colonize agricultural products have been divided into two groups, field fungi and storage fungi. Storage fungi are those that grow at moisture levels in equilibrium with relative humidity at 70-75%, with damage and growth intensifying at higher humidity levels (Christensen, 1974).

Species that fit this classification includes: Aspergillus and Penicillium, leaving

Alternaria, Cladosporium, Fusarium, and Helminthosporium, noted as field fungi. If crops contaminated with field fungi are stored in conditions with moisture contents below 22%, they tend to die off after a few months due to inhospitable conditions and their need for environments with higher relative humidity. This identification was based on studies performed in temperate climates, however it must be noted that under warm, humid subtropical/ tropical climates or temperate areas with hot and dry growing

seasons, Aspergillus and Penicillium spp. can cause infection in the field (Wilson and

Abramson, 1992). Aspergillus flavus is an example of a species that infects during temperate conditions while in storage, but colonizes crops such as corn during preharvest in the southern United States. The importance of environmental conditions cannot be understated in the contamination of food crops by mycotoxigenic fungi during preharvest. Temperature and moisture are two growing conditions that highly influence plant growth and health, as well as the competitiveness of toxin producing fungi. Due to the diversity of mycotoxigenic fungi and the substrates they have the ability to colonize, no sole set of conditions can be described as conducive to mycotoxin contamination

(CAST, 2003). This is demonstrated by the contamination of wheat. Aflatoxin infection is predicated by years with above average temperature and below average rainfall; whereas head scab infection of wheat is favored by warm and wet spring conditions.

Damage from insects, rodents, and birds also plays a role in exacerbating fungal colonization within the field.

With regard to the infection of stored agricultural commodities, several factors influence the severity of contamination, including water activity, substrate aeration, substrate temperature, inoculum concentrations, microbial interactions, mechanical damage, and insect infestation (Ominski et al.,1994). Of these factors, temperature and the distribution and availability of water remain key to fungal colony growth. The lack of water and/or lower water activity greatly prohibits fungal toxin production, although fungi may still colonize the substrate (Wilson and Abramson, 1992). The humidity requirements of fungi are not static across the kingdom, thus there are three classifications to describe this attribute. Hydrophytes require high humidity between 90-

100% relative humidity (RH) to enable them to fully develop. Mesophytes are fungal species that require intermediate moisture in the range of 80-90% RH. This category contains some of the most toxic species of the genus Aspergillus and Penicillium. The final class is known as the Xerophytes, which are capable of developing under fairly dry conditions of between 70-80% RH. Few fungi grow at Aw values below 0.70 and, the increase of this value is proportional to the fungal life a substrate can support. However, two Aspergillus species, A. ehinulatus and A. restrictus have been noted to develop at humidities as low as 65% RH (Navarro et al., 2002).The production of water by metabolic processes and the respiration of xerophytic fungi yields conditions that can support other fungal species. When conditions are favorable for fungal contamination, it is not uncommon for more than one fungus to be present. In storage, substrates such as grains are often colonized by a succession of fungi. In many cases, substrates such as seeds can support the growth of several fungal species at a time. Initially, xerophytic species such as A. restrictus colonize the substrate by producing moisture to create a hospitable environment, then a mesophilic species such as P. aurantiogriseum will colonize the same substrate, a seed in this case, and propagate. Some species have been found to colonize various substrates at conditions that are suboptimal for growth because they can outcompete other species under those conditions (CAST, 2003). The aforementioned P. aurantiogriseum is most abundant at 0°C and 1.0 aw, although it grows best in pure culture at 25°C and 0.7 aw (Lacey et al., 1991).

Harvest Timing Effects

Adapting to colonize substrates with a low moisture content essentially increases the amount of varying substrates that mycotoxigenic fungi can contaminate. Due to this fact, delayed harvesting can result in increased contamination concentrations.

According to Payne et al. (1981), aflatoxin concentrations increase in corn left in the fields until the kernel moisture is reduced to 16-18%, a similar increase is observed in late harvested grains also. Thus, the early harvesting of crops, followed by drying, may help avoid increased toxin contamination. However, for some agricultural products, a drying strategy such as this may not be economically feasible due to the added cost of drying. Late season rains after crop maturation have also been associated with increased toxin levels in various commodities. Peanut harvests are a prime example of this type of toxin contamination. If peanuts grown under drought stress are left in the field and subjected to precipitation, the chances for toxin contamination increases two- fold (Payne et al.,1988). During harvesting it is important to limit excess damage that maybe caused by harvesting equipment such as combines that may predispose crops to infection. The highest mycotoxin levels are often associated with broken and insect damaged crops (Malone et al., 1998). It must be pointed out that the careful adjustment of agricultural equipment may eliminate contaminated pieces with minimal loss of sound crop portions. However, sound crop portions have also demonstrated concentrations of fumonisins (Munkvold and Desjardins, 1997) and aflatoxin (Jones et al., 1980), so removal of only damaged portions will not fully eliminate contamination. Malone (1998) observed an overall decrease of total fumonisins B1-2 of 60% was obtained by screening and gravity separating corn being discharged form a storage silo. Sieving techniques have been successful in eliminating larger ergots from rye and wheat seeds whose strains are approximately the same size and shape as the seeds (Tudzinsky et al.,

1995). Along with the aforementioned techniques, care should be taken in the cleaning of storage bins, auger pits, and to maintain clean trucks, trailers and combines that

come in contact with agricultural products, minimizing future contamination (Widstrom,

1996).

Fungicide Treatments

The association between fungal toxins and fungicides is quite simple. Kill the fungus that produces the toxin, then there is no chance for food commodities to be contaminated by said toxin. Although a strong statement, this line of thinking does not consider several confounding factors. Fungicide use is targeted to eradicate fungi, but the improper or over use of these treatments lead to fungal resistance. At the moment,

Aspergillus niger is not a targeted fungal invader in vineyards; they are therefore not generally treated for this particular organism.

Fungicides are classified in only a few ways: mobility, biochemical modes of action, and fungicide resistance action committee (FRAC) code. The FRAC code is a number and/or letter combination assigned by the committee to group together active ingredients which demonstrate a potential for cross resistance. Fungicides with the same FRAC code are at risk for cross resistance due to having the same fungal targeting site. When a certain plant pathogen is not controlled by fungicides affecting the same growth process, it is said to be cross-resistant. More problematic than this is multiple resistance. This resistance is characterized by a plant pathogen not being controlled by fungicides that affect different processes associated with fungal growth

(Fischel and Dewdney, 2006).

Fungicide mobility is an indication of how the fungicide moves after plant application. There are two general schemes involved in plant and fungicide interaction via adsorption or absorption. Fungicides that are taken up by the plant through absorption are described as penetrants, while those that adhere in very thin layers on

the plant surface via adsorption are classified as contact fungicides. Despite the mobility that a fungicide may possess, none are effective after the development of visible disease symptoms. Hence the timely application of fungicides is rather imperative with regard to optimal disease management (Beckerman, 2011).

Contact fungicides are highly susceptible to being washed away by rain or irrigation, and have a tendency not to protect plant parts that grow and develop after application. This type of fungicide must be applied before fungal spores come in contact with plants as they inhibit spore germination, but have no effect after infection is established. On the other hand, penetrant fungicides move into plant tissues, beyond the plant cuticle. Localized penetrants remain in the initial area of contact undergoing minimal plant movement, whereas xylem-mobile, amphimobole, and translaminar fungicides translocate throughout the treated plant. Xylem-mobile fungicides move upward from the point of entry through the plant’s xylem. Amphimobiles move via the xylem and phloem, while translaminars are absorbed by the leaves and move solely thorough leaf tissues. These systemic fungicides can stop or slow infections with 72 hours of exposure and must be applied soon after the initial infection. However, they have limited curative activity, and are ineffective once the fungus begins sporulation

(Beckerman, 2011). Furthermore, the misuse or over use of fungicides leads to resistance that leaves crops vulnerable to fungal colonization. Fungicidal resistance is usually manifested in one of four ways. A) Target site alteration- fungi evolve to alter the target site of the pesticide while maintaining its normal metabolic activity. B)

Detoxification- fungi use metabolic pathways within the fungal cell to rapidly degrade fungicides and inactivate them before reaching its site of action. C) Removal- Fungal

cells quickly export the fungicide before attacking the target site, and D) Uptake reduction- the fungus absorbs the fungicide at a rate so low that it has no degradative effects (Buhler, 2015).

Of the targeted fungal contaminants in vineyards, Aspergillus niger is generally not listed. This could be because it is considered a postharvest contaminant, but in the southeastern portion of the United States conditions for this fungus to infect in the field are prevalent. Common fungal diseases that are treated for and the fungi that are associated with them are listed below:

Angular Leaf Spot- Mycosphaerella angulate

Black Rot- Guignardia bidwellii

Ripe rot-Glomerella cingulate

Bitter rot- Melanconium fuligineum

Macrophoma Rot- Botryosphaeria dothidea

Powdery Mildew-Uncinula necator

Eutypa Dieback- Eutypa lata

With regard to compounds that may have adverse effects on A. niger, researchers have confirmed that carbendazim (30 μg/mL), hexoconazole (75 μg/mL), and nanosulfur are deleterious to this fungus. However, this is not without some drawbacks. According to reports by Choudhury et al., 2010, sulfur nanoparticles were demonstrated to be very effective against A. niger colonization, this study was not conducted in the field under actual growing conditions, only in vitro. As for the other two compounds, studies conducted by Anand et al., 2010, were carried out in the field under normal growing conditions with groundnuts as the substrate for fungal colonization. Both hexoconazole

and carbendazim were found to inhibit fungal colonization via uptake through plant roots when compared to four other possible fungicide treatments. Bioassay techniques determined that the mode of action for these fungicides was the inhibition of spindle formation during fungal mitosis, a key element for fungal growth and development.

The use of pesticides as an agricultural practice has always raised questions pertaining to residue concentrations left behind in the finished product. Wine, being an agricultural product has not escaped such scrutiny. With regard to the aforementioned fungicides that act against A. niger, studies have confirmed that these fungicides can be effectively removed from wines. The fining agent bentonite, at concentrations of 2.5g/L was demonstrated to completely remove carbendazim from semillon wine samples and reduced residue concentrations far below the maximum allowable limit for the reporting country for shiraz samples (Ruediger et al., 2004). As for hexoconazole, Cabras and

Conte (2001) included attempts to detect this fungicide among others in a survey of 259 wine samples, but were unable to detect concentrations of the pesticide in wines available on the Italian market. Although the wines used in the pesticide analyses from the previously mentioned studies were Vitis vinifera, it is believed that under similar conditions Vitis rotundifolia would demonstrate identical results i.e. pesticide concentrations below allowable levels.

Human Risk Assessment

Foodborne risks to human health can arise from hazards that are of a biological, chemical or of a physical nature. Akkerman et al. (2010) defines food safety as the prevention of illness resulting from the consumption of contaminated food. Risk assessment, risk management, and risk communication have been incorporated under the term food safety risk analysis. These measures have become the preferred

methodologies in the assessment of possible links between hazards in the food chain and actual risks to human health, and takes into account a wide range of inputs to decision-making on control measures. For the purpose of establishing food standards and other food control measures, risk analysis relies on comprehensive scientific evaluation, process transparency, consistent treatment of different hazards and systematic decision-making. The application of harmonized methodologies and risk analysis principles within various countries aides in the facilitation of food trade

(FAO/WHO, 2006).

Food related chemical hazards include: food additives, environmental contaminants such as mercury and dioxins, natural toxicants in food such as glycoalkaloids in potatoes and aflatoxins in peanuts, acrylamide, and pesticide residues in veterinary drugs. Differing substantially from microbiological hazards, chemical hazards usually enter food in the raw state or ingredients, or via processing steps, and typically the level of hazard present in the food after introduction does not significantly change. With this sort of contamination, toxic effects are similar from person to person, with differentiation exhibited in individual sensitivities, health risks may be acute, but are largely chronic (FAO/WHO, 2006). With regard to mycotoxins as chemical hazards, there is the potential to increase in concentration via processing steps, if conditions are optimal for fungal growth. Fungal toxins are natural contaminants of food, feed, and raw materials. According to CAST, (2003), some mycotoxins can cause autoimmune illnesses, have allergenic properties, and be teratogens, carcinogens, or have mutagenic properties. The conditions needed for mycotoxin production vary as much as the actual toxin structures themselves. It is dependent upon pH, temperature, moisture

levels, strain, and, nutrient availability. With this in mind, the presence of toxigenic fungi does not always imply that mycotoxins are present. Furthermore, finding mycotoxin contamination is not definitive proof that a fungal species is or was present (Fung and

Clark, 2004).

Hazard Characterization

To characterize hazards, risk assessors describe the nature and extent of the adverse health effects known to be associated with the specific hazard. Mechanistic aspects of the hazard are considered, along with the establishment of a dose-response relationship between different levels of exposure at the point of consumption and the probability of differing health effects. Typically, adverse health effects are predicted for long term exposures only, but in the case of mycotoxins, both acute and chronic health effects are given close consideration. According to the FAO/WHO (2006), in scenarios where the toxic effect is the result of a mechanism that has a threshold, hazard characterization results in the establishment of a safe level of intake, an acceptable daily intake(ADI) or tolerable daily intake (TDI) for contaminants. It is commonly understood that the majority of fungal toxins do not act through a genotoxic mechanism.

This attribute, or the lack thereof, allows for the derivation of a practical, biological threshold of effect, and a tolerable intake level from the determination of a no observed adverse effect level (NOAEL) for biological endpoints and the application of factors to ensure safety. The estimate of the amount of a contaminant that can be ingested over a lifetime without appreciable risk is known as a tolerable intake and can subsequently be expressed on a daily, weekly, or monthly basis (Garcia-Cela et al., 2012). When extrapolating data from an animal model to humans and to account for inter-individual variability, estimations of the ADI or TDI known as the provisional tolerable weekly

intake (PTWI), include the application of default “uncertainty factors” to a no-effect-level observed in experimental or epidemiological studies. It must be noted with regard to toxicological reference values used by different authorities that genotoxic or carcinogenic chemicals vary. This is due to some of the values being based on a combination of epidemiological and animal data, some based on animal data alone, and the use of different mathematical models to extrapolate risk estimates and low doses.

These differences have the potential to lead to significant variability in cancer risk estimates.

Exposure Assessment

One of the key points in a risk assessment is the exposure assessment. This assessment typically describes the pathway of exposure and estimates total intake.

Depending on the scenario, toxin intake may be associated with a single food, and for others the toxin may be present in multiple foods. Characterizing the amount of toxin consumed by the various members of the exposed population(s) is the primary role of the exposure assessment. This analysis makes use of the concentrations of toxin in raw materials, the food ingredients added to the primary food and the general food environment to track changes in levels throughout the food production chain..

Estimating the degree of human exposure is an essential step in the risk evaluation or exposure assessment of chemical substances in food products. The exposure to mycotoxins may be measured by using food consumption data from contaminated foodstuffs, and the average occurrence of the mycotoxin among the studied population during a particular timeframe. From this estimation, the degree of exposure is measured in terms of probable daily intake (PDI) per unit of body weight, and is generally expressed in ng/kg b.w. (Jager et al., 2013). In risk analysis, PDI can be compared with

TDI determined in toxicological studies. The WHO (2002) has established a maximum tolerable daily intake of 2 µg/kg b.w. for fumonisin B1, B2, B3, alone or in combination.

Risk Characterization

During risk characterization, an estimate of risk is generated via the integration of hazard identification, hazard characterization, and the exposure assessment. These estimations are scenario-dependent and must also take uncertainty and variability into account. Risk characterizations typically include a narrative on other aspects of the risk assessment, which include: a comparative ranking with risks from other relevant foods, the impact on risk of various “what if’ situations and the reduction of scientific gaps through research. It is difficult during the characterization process to address estimates for chronic exposure that speak to the likelihood and severity of adverse health effects associated with various levels of exposure due to its inherently unpredictable complexity. A “notional zero risk” approach is generally taken, where the goal is to limit exposure to levels deemed unlikely to have an adverse effect on human health.

Methodologies that support quantitative risk assessment have not been associated with hazards that demonstrated no appreciable risk below certain very low levels of exposure, mechanisms of toxic action believed to exhibit a threshold. This has been because it was widely recognized that this approach was considered to provide an adequate margin of safety without the need for further risk characterization. However, quantitative risk assessment models have been applied by the Joint FAO/WHO Expert

Committee on Food Additives (JEFCA) for effects that have no apparent threshold, genotoxic carcinogens. Biologically-based mathematical extrapolation models from observed animal cancer study data derived from tests using high dosages have been use to estimate the expected cancer incidence at lower levels generally experienced by

humans. If available, epidemiological cancer data can also be used in these models

(Weed, 2005).

Risk Management

After the risk assessment process, maximum levels are set under the consideration of the concentration of a contaminant in the food, socio-economic impacts, and the technical feasibility of the derived values. Food safety measures based on risk assessments are designed to reduce risk to a targeted level, achieving a predetermined degree of health protection. Those assessing risk must examine the relative impacts of different controls on reducing risks, through the use of objective data that supports decisions on the most appropriate controls. The overriding objective of risk management is to maximize risk reduction while ensuring that the measures employed are efficient and effective and not overly restrictive (FAO/WHO, 2006).

Exposure guidelines can provide a reference point for maximum safe intake, and risk management measures can be put into place that are aimed to prevent consumers form exceeding that safe upper limit of exposure. When managing the risks to vulnerable commodities, the Acceptable Level of Protection (ALOP) concept is one that is adaptable across a wide range of products and initiated by the World Trade

Organization (WTO). An ALOP is defined as “The level of protection deemed appropriate by the Member [of the WTO] establishing a sanitary or phytosanitary measure to protect human, animal or plant life or health within its territory”. According to de Swarte & Donker (2005), an ALOP can be expressed in a range of terms that include broad public health goals to quantitative expressions of the probability of an adverse public health consequence or incidence of disease. There are two primary approaches to setting an ALOP in risk management. The first is the “notional zero risk approach”,

where hazards are kept at levels that are on par with pre-determined “negligible” risk based on assessments indicating that low exposure levels will not cause harm to humans. For the majority of mycotoxins, no acute effects in humans are observed, therefore it is difficult to derive a dose- response relationship. Furthermore, this approach does not produce precise estimates of risk versus dose and cannot model the impact of various interventions in terms of the reduction of risk. Another methodology to setting ALOPs is the threshold approach. This method is based on the idea that risk must be kept below a specific numerical level as predetermined by public policy, this approach is used in particular with possibly carcinogenic toxins. Levels of risk that are deemed acceptable are defined within public policy, and risk management measures are then chosen to keep the risk below that threshold, which may also be referred to as a “virtually safe dose”.

The Food Safety Objective (FSO) concept is another tool used in risk management. The FSO is the maximum frequency and/ or concentration of the hazard in a food at the time of consumption and is preceded by the performance objective

(PO), which is defined as the maximum frequency and/or concentration of a hazard in a food at a specified step in the food chain before the time of consumption (ICMSF,

2002). Generally, FSOs are used with regard to microbiological hazards, however, this methodology has worked effectively with other hazards also. In the context of fungal toxin contamination, FSOs serve as a means to coordinate risk management in the production process throughout the farm to fork production chain (de Swarte & Donker,

2005). After the FSO is set, management systems are put into place to deliver a level of food safety in compliance with said FSOs. “Chain reversal” is used to derive

performance criteria (PC) and other metrics to demonstrate appropriate food safety standards for individual links in the chain. To fully implement the FSO concept, a quantitative FSO is relied upon so that performance criteria and objectives are specified, leading to the development of an appropriate HACCP plan. The combination of this HACCP plan and good hygiene practices (GHP) are ground level tools needed to keep toxin contamination low. After these come the PO which serves as a guidance to meet the FSO in place, and PC which is the effect of one or more control measures need to meet or contributes to meeting a PO. POs are not intended to be enforced per say, but should lead to designing the correct operational control measures at the step in the production chain that the specific PO pertains to. Throughout the food production process it is imperative to understand the effects of every treatment and process parameter to ensure that hazard levels do not exceed safety levels before processing or consumption (Stringer, 2005). The development of PCs must consider the initial level of a hazard and the changes occurring during production, distribution, storage, preparation, and use of a product. Performance criteria must account for hazard increases and reductions which can be expressed by the following equation:

Ho –ΣR+ΣI ≤ FSO (2-1)

where

Ho= Initial level of the hazard

ΣR= Total (cumulative) reduction of hazard

ΣI= Total (cumulative) increase of hazard

FSO, Ho, R, and I are expressed in µg/kg

PCs are an indication of the change in hazard levels required at specific steps in order to reduce the hazard levels at the beginning of the step (Ho) to a level that is compliant with the performance objective or FSO. PCs are orchestrated by food safety managers as essential points in the design of food production in a supply chain. It should be noted that the above parameters in the equation are point estimates that rely on numerical point values at each step in the risk assessment (typically the 95th percentile value of measured data), in production scenarios a distribution of values would be relevant

(Cole, 2004). The aforementioned control measures are actions or activities that can be used to prevent or eliminate a food safety hazard or reduce it to an acceptable level.

With regard to fungal toxins, some control measures may include: ensuring control of initial levels of toxins, prevention of unacceptable increases of toxins, preventing fungal growth during transport, storage and processing, and the if possible the reduction or elimination of the overall toxin (Garcia-Cela, 2012).

Fumonisins

Structural Characteristics and Biosynthesis

Typically not associated with fruit or fruit products, fumonisin (C34H59NO15) (Fig.

2.2) MW=705.39, has recently become a topic of interest in the contamination of these agricultural products. The basic structure of the fumonisin molecule is a nonaromatic diester of propane-1, 2, 3-tricarboxylic acid and a pentahydroxicosane which contains an amino group. Fumonisins are classified into three analog states, A, B, C, or P which all have slight variations that yield differing biological activities and characteristics. The

Fumonisin A-series is acetylated on the amino group, but are produced at low levels and has minimal biological activity (Gelderblom et al., 1988 and Abbas et al., 1993).

The B-series analogs, the most abundant and toxic due to their capacity to induce

resistant hepatocytes causing cancer initiation in rodents (Gelderblom et al., 1993), are characterized by a terminal 2-amino-3-hydroxy motif on an eicosane backbone and two hydroxy groups esterified with tricarboxylic acids. These fumonisin analogues differ structurally with 2-3 hydroxyl groups located at different positions on the toxin molecule

(Rheeder et al., 2002). According to Stockmann-Juvala and Savolainen (2008), despite minor structural variations, the magnitudes of toxicity of FB1-3 are at a similar level, with

FB2 being slightly more cytotoxic (Gutleb et al., 2002). There are several structural similarities between the B and C series, but the C-series lacks the C-1 terminal methyl group (Seo et al., 1996). Fumonisin P1-3 are characterized by a 3-hydroxypridinium functional group on the C-2 amine group (Musser et al., 1996). Although rarely quantified or extracted from food sources, long chain pyridiniums such as the FPs have been cited as causing long term neurotoxic effects (Tian and Griffiths, 1993). Originally, the sole source of fumonisin was thought to be Fusarium and Gibberella molds, such as

Fusarium verticillioides and Gibberella fujikuroi (Proctor et al., 2003). Recent studies

D confirm similar overall stereochemistry, optical rotation ([α] 20 -11.36 (c 1 mg/mL,

MeOH)) and unique structural folding characteristic of the globular FB2 molecule verify this to no longer be the case with production of fumonisin B2 (FB2) by Aspergillus section Nigri isolates (Frisvad et al., 2011; Mansson et al., 2010). Biochemical analyses of F. verticillioides identified the fumonisin gene cluster, FUM, which is composed of 17 genes, and serves as a biomarker for fumonisin production (Brown et al., 2007). This cluster encodes a toxin transcription factor, enzymes, and transport proteins, along with highly reducing polyketide synthases that play a vital role in backbone structure (Gerber et al., 2009). Genes FUM1 and FUM8 encode the polyketide synthase consisting of KS,

AT, DH, C-MeT, ER, KR and ACP domains; Fum1p & Fum 8p genes, and an α- oxoamine synthase which are both essential in the formation of the 20 carbon backbone chain of FBs 1-3 (Proctor et al., 2006). It has been noted that FUM1 disruption in gene engineered mutants produced no measurable quantities of fumonisins, demonstrating that this gene is also required for fumonisin production. Oxoamine synthases, a small family of enzymes involved in important primary biosynthetic pathways including L- serine palmitoyltransferas in ceramide-sphingolipid biosynthesis (Gerber et al., 2009), play a role in the mode of toxicity for fumonisins which will be discussed later. According to Alexander et al. (2009), and Susca et al. (2010), Fum1p catalyzes the synthesis of the linear polyketide that is associated with carbon atoms 3 through 20 of the backbone and the methyl groups at carbons 12 and 16. Fum8p catalyzes the condensation of alanine and acetate to form carbons 1 and 2 of the backbone, as well as the amine group at carbon 2. The hydroxyl groups at carbons 3 and 5 are derived from molecular oxygen, while the methyl groups at C-12 and 16 are derived from methionine. This further illustrates that FB2 is a highly reduced type I polyketide. FUMs 2, 3, 6, &14 are not dedicated to backbone synthesis, but are essential in the catalyzation of functional groups along the backbone. There are four genes involved in the formation of the two tricarballylic esters located at C-14 and C-15, FUMs 7,10,11, and 14. It is presumed that FUM 11 is encoded with a tricarboxylate transporter that could be involved in vivo substrate shuttling across cellular compartments (Butchko et al., 2006). Genome sequencing of A. niger has uncovered a biosynthetic gene cluster, which includes orthologues of 10 genes also found in the Fusarium FUM cluster and three genes that are distinctly characteristic of A. niger. The prevailing distinction between the two

clusters is the FUM cluster of Aspergillus lacks the FUM 12 gene which is directly related to the production of FB1 and FB3 via the encoding of P450 monooxygenase that catalyzes hydroxylation at the fumonisin backbone at carbon 10. Since the A. niger

FUM cluster lacks the FUM 12 gene it can only produce FB2, FB4, and FB6. Mutants with a deleted FUM 12 also produced only FB2 and FB4 suggesting that this gene is also required for C-10 hydroxylation found in FB1 and FB3 (Proctor et al., 2006). Three strains of Aspergillus have been verified as producers of FB2 through transcriptomic and metabolomic examination: ATCC 1015, NRRL 3, NRRL 567, and CBS 513.88. All three strains contain a fumonisin gene cluster similar to the cluster associated with fumonisin production by Fusarium spp. (Frisvad et al., 2011).

Mechanism of Toxicity

Fumonisin’s structure is closely related to that of sphingoid bases sphinganine

(SA) and sphingosine (SO) (Figure 2-3), a characteristic that aides in the action of its toxicity via an inhibition of sphingolipid biosynthesis which consequently disrupts multiple cellular functions (Gutleb et al., 2002). Sphingolipids are primarily located in cellular membranes, lipoproteins, and other lipid-rich structures, and identified by their polar head group ranging from simple phosphodiesters to complex carbohydrates, in conjunction with a fatty acid backbone attached in an amide linkage (Merrill et al.,

1996). The backbones vary in length, degree of unsaturation with most being unsaturated, and the presence or absence of a hydroxyl group attached at the α or ω carbon. Mammalian sphingolipids include: sphingomyelin, cerebrosides, globosides, gangliosides, and sulfatides (Hakomori and Igarashi, 1995). These lipids are critical to the maintenance of membrane structures, serving as binding sites for extracellular matrix proteins, and modulating the behavior of growth factor receptors. The first event

in the disruption of sphingolipid metabolism is the binding of fumonisin to the catalytic site of ceramide synthase, the enzyme that acylates sphingoid bases. Acylation produces sphinganine from de novo synthesis and sphingosine from sphingolipid turnover. This bond is easily facilitated due to the recognition of both the amino group and the tricarboxylic side chains of fumonisin. Ceramide synthase catalyzes the formation of ceramide from the condensation of fatty-acyl CoA and Sa or So. Inhibition of ceramide synthase by fumonisin is competitive, and involves the tricarboxylic acid and amine groups, which occupy the enzyme’s respective fatty-acyl CoA and sphingoid base binding sites (Merrill et al., 2001). The inhibition of ceramide synthase causes Sa to accumulate, and at times increases sphingosine, which occurs much later when advanced cell injury prompts membrane degradation (Riley, 1994). Experimental evidence indicates that the primary amine is essential for the fumonisin molecule’s ceramide synthase inhibitory activity, which is believed to be the mechanistic “trigger” for toxicity (Lemke et al., 2001). The inhibition of ceramide synthase results in an accumulation of upstream sphingoid bases and sphingoid base-1-phosphates (which may be transported to other organs, augmenting concentrations), and a depletion of downstream complex glycolipids (Riley et al., 2001). Several biochemical modes of action have been postulated to explain the fumonisin-induced toxicity and carcinogenicity in animals, but the primary hypothesis involves the perturbation of sphingolipid metabolism. It has been noted that the removal of the tricarballylic acid side chains of FB yielding AP (aminopentol), decreases the potency of ceramide synthase inhibition 10-fold in vitro. The side chain cleavage converts the inhibitor into substrate.

However, this only occurs under the condition that AP occupies the sphingoid-base

binding site and the fatty acyl-CoA site remains open, which yields PAP (N-palmitoyl-

AP) and is more toxic than FB or AP at similar concentrations. The formation of AP is most readily demonstrated in the nixtamalization of contaminated corn products making that of PAP less prevalent due to the specific orientation needed for formation (Humpf et al., 1998). These lipids are not only important structural components of the plasma membrane as mentioned previously, but also function as intra- and intercellular signaling molecules (Chalfant and Spiegel, 2005). Fumonisin’s toxic effects depend on the disruption of various aspects of lipid metabolism, membrane structure, and signal transduction pathways mediated by lipid second messengers. These demonstrated effects include altered rates of cell proliferation vs. apoptosis, altered cell-cell communication and cell adhesion, oxidative stress, and the modulation of gene expression (Abel and Gelderblom, 1998).

Apoptosis and mitosis in liver hepatocytes and the kidney outer medulla, are early consequences of fumonisin exposure. They occur simultaneously and it is proposed that an imbalance between cell death and increased regenerative pressure through continuous mitosis is a critical component of fumonisin’s carcinogenic mode of action (Dragan et al., 2001). Stevens and Tang (1997), reported that fumonisin-induced depletion of glycosphingolipids inhibited vitamin uptake via the glycosylphosphatidylinositol (GPI)-anchored folate receptor and suggested that dietary exposure to fumonisin could therefore adversely affect folate uptake, and potentially compromised cellular processes dependent on this vitamin. The high-affinity folate receptor is a GPI-anchored protein associated with membrane micro-domains rich in cholesterol and sphingolipids known as “lipid rafts” (Elortza et al., 2003). The role of lipid

rafts in the regulation of the folate receptor was validated during a study in which the depletion of cellular cholesterol was found to impair uptake of 5-methyltetrahydrofolate into cells (Chang et al., 1992).

In vitro assays have demonstrated the reversibility of the inhibition of ceramide synthase via the dilution of the inhibitor and removal of FB from the medium of cells in culture. According to Riley et al. (2001), FB appears to inhibit ceramide synthase via a noncovalent interaction, and its reversibility is predicated on FB reductions in the cytosol, with other interacting factors being: cellular concentration of the substrates and rate of removal of accumulated So and Sa. In vivo reversibility was determine by Wang et al., (1999), when feeding rats 10µg FB/g feed and then changed their diet to feed containing 0,1, or 10µg FB/g feed. It was observed that urinary Sa returned to their initial concentrations within 10 days when the rat’s diet was changed to 0µg FB, but if changed to 1µg FB (an amount alone that did not elevate Sa levels) urinary Sa concentrations remained elevated. This illustrates the possibility that amounts of FB that are below TC when consumed alone could be more damaging if there are occasional increases in the diet.

Biodistribution and Pharmacokinetics

Within rodents and nonhuman primates results of biodistribution studies indicated that FB1 and FB2 are poorly absorbed from the gastrointestinal tract, cleared rapidly from the blood and low concentrations are accumulated in tissues, however low amounts are found in the liver and kidney (Norred et al., 1993). The absorption of rats that were orally administered FB1 (10mg/kg b.w.) was low (3.5% dose) with a maximum plasma concentration of 0.18 µg/ml. The elimination half-life was 3.15h for plasm, 4.07h for liver, and 7.07h for kidney. It was noted from the AUC tissue/AUC plasma ratios

(2.03/29.9) that FB accumulates in the kidney to a greater extent than in the liver. This was further exemplified by Riley and Voss (2006), where LC-MS quantifications of fumonisin in the liver and kidney of rats given diets containing the toxin for three consecutive weeks, demonstrated kidney concentrations that were 10- fold higher than the liver.

Prelusky et al., (1994, 1996), found similar results when using pigs as the test animal to evaluate fumonisin distribution and kinetics. Intravenous administration of

14 0.4mg/kg b.w. [ C] FB1 displayed a half-life of 10.5-182 min. After 72 h, the recovery form the urine and plasma ranged from 0.76-0.83 of the dose. A large majority of the radiolabeled toxin was recovered from the bile urine and feces within 72h. Tissue recovery averaged about 0.12 of the dose, with the highest activity in the liver and kidneys. Similar results were observed in pigs that were dosed in an intragastic fashion.

The radiolabeled toxin appeared in the blood after 30 mins, but was eliminated in the feces (0.87-0.95 of the dose) within 72h. Less than 0.005 of the dose was excreted via urine on average, with 0.86-0.95 of the dose found in the feces. The liver and kidneys consistently contained the highest specific activities at all time periods. This activity rose continuously and the respective tissue burden was calculated to be approx. 160 and 65ng/g at the end of the 24-day exposure period. There was a decline in activity when the pigs were returned to control feeds, with small amounts of labeled-toxin in the kidneys and liver. Overall, it was concluded that toxin absorption from feed was low, but the toxin remained in tissues over extended periods. Absorbed fumonisin was shown to preferentially accumulate in the liver and kidneys with enterohepatic recirculation contributing to long biological a half-life.

Fumonisin was found to be minimally absorbed by ruminants, explaining their tolerance for ingestion of the toxin. In steers, more than 0.80 of ingested toxin went unmetabolized and excreted in the feces, with trace amounts in urine (Smith and

Thakur, 1996). The clearance of FB in i.v. dosed lactating dairy cows was 15.1 min (low dose) and 18.7 min (high dose), with no detection in the plasma after 2 h and a low volume distribution (0.251-0.278), which suggests that there was no uptake by the tissues. No toxin was detected in the plasma of orally dosed cows, which is attributed to the first-pass through the liver reducing toxin concentration in the blood to undetectable levels (Prelusky et al., 1995). No detectable levels of FB or hydrolyzed FB were found in the milk of dairy cows dosed orally or intravenously with an LOD of 3 and

20ng/ml, respectively. Similar results were reported from dairy cows that were fed diets containing approx. 75 ppm FB (daily intake 3 mg/kg b.w.) for 14 days (Richard,

1996)These studies all indicated that the carryover of FB from feed into milk does not pose a potential threat to human health.

It must be noted that the aforementioned studies and results were investigating the acute effects of fumonisin ingestion of domesticated animals. The damage conferred on animals from this toxin is typically seen under chronic exposure.

Fumonisin is known to induce spontaneous disease in pigs and horses, with horses being the most susceptible. Equine leukoencephalomalacia (ELEM) and porcine pulmonary edema (PPE) are diseases that are species and target organ specific, the brains of horses and the lung in pigs, with liver and kidney targeting also.

Cardiotoxicity, a more recently recognized lethal toxicity, has been reported in pigs and horses and is believed to be the precursor to PPE in pigs (Smith et al., 1999). The

equine central nervous is also susceptible to FB attack, causing necrotic softening of the brain tissue. Onset of the disease has been reported to occur as early as seven days of consuming contaminated feed, but typically needs 14-21 days for full manifestation, with rare cases of 90 days of more. It is common for outbreaks to effect several horses on the same farm (Morgavi and Riley, 2007). Outbreaks have been reported since 1901, with large numbers of outbreaks in the USA during the 1980’s. The overall morbidity of an outbreak is low, less than 0.25, but mortality usually approaches

1.00 in effected animals. Classic symptoms include: decreased feed intake, depression, ataxia, blindness, hysteria, and anorexia. Those equine that do survive have permanent neurological disease. Contamination levels required to induce ELEM is low, as opposed to the higher concentrations required for PPE manifestation. The first sign of

PPE following exposure is a decline in feed consumption. Within 4-7 days, pigs show respiratory distress and cyanosis that is rapidly followed by death due to acute pulmonary edema and hydothorax. Symptoms typically decline within 48 h of withdrawal of contaminated feed (Haschek et al., 1992). Cattle and poultry are much more resistant to FB contamination. To achieve toxicity levels, diets containing at minimum 100g/kg fumonisin were fed to poultry, with the possibility that other toxins may have caused the observed malaise in these animals (Webking et al., 1993a). There has been no reporting of natural toxicosis in ruminants, and no fumonisin is excreted in the milk once ingested by ruminants. Furthermore, cattle and ruminants have been observed avoiding fumonisin contaminated feed and continue to consume clean feed when offered (Voss et al., 2007).

Historical Relevance and Discovery

Although the isolation, chemical characterization, mode of action and biological activity of fumonisins has been elucidated to a degree this has not always been the case. Initially, the causation behind the equine leukoencephalomalacia (ELEM) outbreak in 1970 within South Africa was unknown until the isolation of Fusarium verticillioides from moldy corn. This outbreak was characterized by liquid-filled necrotic lesions in the cerebral hemispheres of effected horses. Further investigation revealed bile duct proliferation, multinucleated hepatocytes, and large hyperchromatic nuclei caused by the infection of horse livers, indicating the possibility F. verticiliodies may be a carcinogenic fungus. Since this fungus was found on moldy corn fed to animals, it was hypothesized that there may have been a connection between the outbreak and the high incidence of esophageal cancer in the southern former Transkei region of South

Africa. The corn for humans and domesticated animals was all home grown within the region, with F. verticillioides as the fungus most prevalent in the crop consumed by the population (Marasas, 1984). The conclusion that the fungus in both the outbreak and human cases were of the same etiology was confirmed when fungal isolates from both occurrences caused ELEM experimentally in horses, porcine pulmonary edema in pigs, and hepatoxicity and cardiotoxicity in rats, finally being designated as F. verticilioides

MRC 826 (Kriek et al.,1981). Although this confirmation was made in 1984, the chemical nature of the carcinogenic metabolite still had not been identified. The motivation to determine the pathogen’s chemical nature was increased when researchers in the United States reported that corn from field outbreaks of ELEM and ears naturally infected by F. verticillioides were not only hepatocarcinogenic in rats, but also exhibited identical pathologic changes in the rats fed MRC 826 culture material and

corn from affected U.S. harvests.(Marasas, 2001). To characterize the toxin behind these incidences, researchers used the Ames test to narrow the scope of possible pathogens because most mutagens are also carcinogenic as well. During this period, it was known that a chloroform /isopropanol extract of MRC 826 was highly mutagenic to

Salomonella typhimurium in the Ames test. This knowledge revealed a group of structurally related compounds, the fusarins, was not only mutagenic, but fusarin C occurred naturally in corn samples from the former Transkei region and the U.S. To this end, short term carcinogenicity assays and long term trials in rats fed cultures containing high concentrations of fusarin C did not confirm the metabolite’s carcinogenicity. Due to its heat and light sensitivity, fusarin C was found not to be a threat to human health (Jaskiewicî et al., 1987). Through the development of these short term initiation/promotion assays to investigate fusarin C, other assays using diethylnitrosamine (DEN) as an initiator and phenobarbital as a promotor began to demonstrate promise in elucidating the offending toxin. Following the aforementioned initiation/promotion pathway, the addition of culture material fractions from MRC 826 displayed early lesions on treated rat livers resulting in cancer-promoting activity. This promotion led to selective stimulation of gamma glutamyltranspeptidase (GGT)-altered foci (disease biomarker) in rat hepatocytes, which was consequently used to isolate fumonisin B1 and B2 as the active toxins in the infection incidences (Bezuidenhout et al.,

1988). After purifying this newly discovered toxin in 1988, confirmation of its carcinogenic nature were demonstrated by intravenously injected horses contracting

ELEM, pigs displaying PPE and liver cancer in male BD IX rats when fed a dietary level of 50µg/g (Harrison et al.,1990).

Government Guidelines and Recommendations

Mycotoxins, from a regulatory stand point are considered to be unavoidable contaminants i.e. those that cannot be avoided by good manufacturing practices. With this in mind, small amounts of these contaminants may be legally permitted, provided that the contamination levels do not negatively impact human or animal health. In the

U.S., the U.S. Food and Drug Administration (FDA) regulates mycotoxins under

Sections 402 and 406 of the Food, Drug, and Cosmetic Act. This act states that a food is considered adulterated if it bears or contains any poisonous or deleterious substance that may render it harmful to health. By enforcing statues of this nature, the FDA can prohibit the entry of, and remove from interstate commerce, any food or feed that is thought to be adulterated. The objective is to balance the benefits of food availability against the risk that a contaminant in that food might pose to the public health (Bolger et al., 1996). Based on the adverse health effects observed in animal, the Center for Food

Safety and Nutrition (CFSAN), FDA issued industry guidance levels for fumonisin in maize in 2001 (“Guidance for Industry on Fumonisin Levels in Human Foods and

Animal Feeds”) stating that human health risks associated with exposure to fumonisin were possible. Recommended levels for foods range from 2 to 4 ppm total fumonisin

(fumonisin B1 plus fumonisin B2 plus fumonisin B3) depending on the intended use of the maize and whether or not the kernels are whole or degermed. Currently, within the

United States, guidance levels for total fumonisins in human foods are as follows:

2mg/kg corn grits, meal, and flour, 3 mg/kg popcorn grain, and 4 mg/kg maize products

(US Food and Drug Administration, 2001). Proposed limits (fumonisin B1 plus fumonisin

B2) in Europe, (effective October 2007), range from 0.2 to 2ppm depending upon the commodity and the targeted consumer (i.e., adults or infants) (Commission of European

Communities, 2005). The International Agency for Research on Cancer (IARC, 2002) classified fumonisin B1 as “possibly carcinogenic to humans (class 2B)” and in 2001 the

Joint FAO/WHO Expert Committee of Food Additives (JECFA) recommended a PMTDI of 2µg/kg body weight for fumonisin B1, fumonisin B2 and fumonisin B3, “alone or in combination (Bolger et al., 2001). This “dose” is based on a no-observable-adverse- effect level (NOAEL) in the male rat kidney and incorporates a 100-fold safety factor

(FAO/WHO, 2002). At this time there is no total fumonisin guideline for beverage contamination.

Dietary Exposure

Ideally, the determination of exposure and diagnosis of a mycotoxicosis should depend upon the absence of other diagnosable diseases and finding the particular mycotoxin within the suspected food (Richard and Thruston, 1986). Simply isolating the fungus is insufficient, there must be a presence of biologically effective concentrations of the toxin. Thus, it is difficult to rely on the chemical analysis of food because the representative consumed sample must contain the toxin as well. It is evident from research studies that ingesting fumonisin via the diet has harmful effects on animals, due to the compound’s carcinogenic nature, studies such as these are unconscionable for human subjects. To this end the employment of epidemiological studies are used to ascertain any correlation between the known physiological effects of consuming fumonisin contaminated foods and any damage done to humans. The estimation of the degree of human exposure is an essential part of the risk evaluation of chemical substance in food products. Human exposure to mycotoxins may be measured using data on the consumption of contaminated foodstuffs, and the average occurrence of the toxin. From this estimation, the degree of exposure is measured in terms of probable

daily intake (PDI) per unit of body weight, and is expressed in ng/kg of body weight

(b.w.) per day (Jager et al., 2013). Studies such as these focus on geographical areas where corn and cereals play a pivotal role in the diet since they are the most highly adulterated commodities with regard to fumonisin.

In Italy, Cirilio et al. (2003), performed a survey to ascertain the frequency and level of fumonisin contamination that may lead to dietary exposure from Italian marketed foods. The group collected 202 samples that were classified into six classes: raw cereals (maize, wheat, barley, farro, rice, etc.), breads, durum wheat pasta, breakfast cereals, biscuits, and baby/infant foods. Samples were purchased from area supermarkets, bread shops, and department stores over a 12-month period. For each product, two different packages of the same quantity lot were collected for analysis.

From LC-MS analysis, FB1 and FB2 occurred in 26 and 35% respectively of the tested samples. No FB1 was detected in durum wheat pasta, biscuits or baby/infant foods, with its highest concentrations found in raw cereals ranging from 0.010-2.870 µg/g. FB2 was detected in all food classes except baby/infant foods, with its highest levels ranging from

0.010-0.790 in durum wheat pasta. In the traditional Italian diet, bread, pasta and similar products are very common with high consumption levels. The Italian Statistical Study

Centre evaluated bread and pasta consumption as 150 and 51.6g/day, respectively for

90% of the Italian population. From this study the calculated exposure to fumonisin from pasta and bread consumption were well below the TDI level of 2 µg/g b.w./day.

One area where maize is of dietary importance is Brazil, a region that ranks corn as the second most cultivated crop (Rocha et al., 2009). Pirassununga, São Paulo,

Brazil was the site of a study conducted by Bordin et al. (2014), to determine the

fumonisin consumption of the residents within the city. The research group collected

100g of samples of corn meal, corn flour, corn flakes, polenta, canned corn, and popcorn four times every three months from the resident volunteers from June 2011 to

March 2012. All of the collected samples totaled approx. 120 corn-based food products.

Collecting samples from subject homes yielded a more well-rounded perspective of contaminated food that consumers actually encounter, as opposed to sampling only commercial products. A food frequency questionnaire (FFQ) was also given to all volunteers (21 women and 18 men) to determine consumption habit of foods susceptible to fumonisin contamination. The aim of the questionnaire was to capture as many consumed foods as possible, including those that are ingredients in other foods such as cornmeal which is used in processed foods and homemade foods i.e. couscous, corn bread, and corn cake. Subject consumption frequency was measured using a range from less than a month to twice every day. Portion sizes were chosen for each food according to standard measures used in the preparation of homemade foods.

The FFQ was filled out by all the subjects that submitted samples for analysis. The mean consumption of each corn product by each volunteer was estimated from the portion sizes reported in the FFQ and was expressed in g/month. The body weight of each individual was recorded and used to calculate the PDI value. Food ingestion habits and food intake were calculated on a monthly basis due to the fact that some foods were not consumed on a daily basis by subjects. The most frequently consumed corn products were popcorn (716.0± 1178.5g/person/month) and couscous

(661.5±1255.3g/person/month). Other foods such as polenta, cake or cornbread, cornflakes and canned corn were determined to be a small part of the overall diet of the

sample population. According to the “Household Budget Survey 2008/2009” conducted by the Instituto Brasileiro de Geografia e Estatística (2013), corn flour and corn meal were the most consumed corn-based products in Brazil, with daily intake levels of

51g/person/month and 189.3g/person/month, respectively. This difference could be due to the fact that all the subjects in this study were from the local university and not a vertically diverse population that would have captured subjects of various income levels and thus varied consumption choices. The mean PDI of FB1 within the population of

Pirassununga resulted in 63.3ng/kg b.w./day representing 3.1% of the PMTDI

(provisional maximum tolerable daily intake-2µg/kg b.w.) , which is similar to the estimated intake in the Federal District of Brazil at 9.0% of the PMTDI as reported by

Caldas and Silva (2007). It must be noted that the fumonisin consumption in the study was lower than previous studies in Brazil. Machinski and Soares (2000), assessed fumonisin consumption in the same region and found a rate of 1276 ng/kg b.w./day which coincides with 63.8% of PTMDI . In the state of Paraná, southwest of the current study location, mean PDI was estimated at 950 ng/kg b.w./day, which represents 47.5% of PMTDI (Moreno et al., 2009). In the state of Santa Catarina, the mean PDI was estimated to be 1600 ng/kg b.w./day, 80% of PMTDI , according to Westhuizen et al.

(2003). When comparing the data from the previous reports to the current study, one may assume that corn product manufacturers have become more conscientious to the fumonisin content within in their products. However, this is a conclusion that cannot be drawn because the estimated mean PDI values were derived from the analyses of commercial corn products and not from those obtain from consumer homes.

Leblanc et al. (2005), performed a Total Diet Study to assess toxin dietary exposure in France, also focusing on analyzing food “as consumed by the eater” over raw food materials. In this study, 456 composite samples were prepared from 2280 individual samples and analyzed for aflatoxin, OTA, patulin, trichothecene, zearalenone and fumonisin contamination to estimate intake by the French population, but we will focus solely on FB data. The list of food to be analyzed was based on data from individual food consumption surveys representative of the diet of the French population collected over 11 months using 7-day dietary records. Portion sizes and food descriptions were standardized using a photographic food atlas. To capture a true representation of the population, stratification by region of residence, town size and application quotas that included age, sex, individual professional/cultural category, and household size. A separate food list was compiled for the vegetarian population based on results from a consumption survey using dietary records over 5 consecutive days, covering 138 subjects. This survey identified three vegetarian population sub groups: ovolactovegetarians, lactovegetarians, and vegans, representing 2-3% of the overall

French population. The foods from the list could be raw or used as an ingredient in a prepared dish as long as it was “as consumed” before analysis.

The results of the study with regard to fumonisin intake showed that 12 of 34 composite samples exhibited levels above the LOD for FB1 and one sample for FB2 (35 and 3% respectively). Of the twelve samples five were breakfast cereals (42%) with concentrations between 60-120ppb, with the remainder being three poultry liver samples containing concentrations between 90-120ppb. One canned sweet corn and canned soybean sprout sample were detected to have levels at 80ppb and 100ppb

respectively. Two beer samples were also found to be contaminated at 11-14pb. The rationale given for the contamination was that maize is feed to poultry and used as a raw material in beer, which may have led to their presence in these commodities. The estimated average total intake of fumonisin in the French population was 14ng/kg b.w./day in adults and 46ng/kg b.w./day in children. From this study, the 95th percentile of exposure is 64ng/kg b.w./ day in adults (3% of TDI) and 175 ng/kg b.w./day in children (9% of TDI). These results were on average lower by a factor of 10-20 than those estimated during previous dietary evaluations. In the vegetarian population, the estimated average intake of fumonisin is between 40-100ng’kg b.w./ day, depending on the group studied. The 95th percentile exposure is between 120 and290 ng/kg b.w./day

(1-15% of TDI). No proportion of the population was found to intake fumonisin levels that exceeded the TDI.

Measurement of Fumonisin Exposure Difficulties

One of the major difficulties in studying environmental components of fumonisin- related birth defects is that the time of injury occurs far ahead of the condition by weeks or even months. In humans, neural tube closure is complete approximately 28 days after conception, frequently before the women knows she is pregnant. Studies of exposure would require extensive population screening to identify the effected cases and years to develop a sample size large enough to analyze gene-environment interactions. Krapels et al. (2004), suggests the use of biomarkers at time points after pregnancy may be a useful approach for characterizing environmental influences.

Fumonisin is rapidly cleared from the system and can only be detected in human urine and feces for up to 3-5 days after ingestion (Sewram et al., 2003). In some developing countries, without the aid of modern prenatal monitoring such as ultrasound, fetal

malformations may not be detected until the child is born. At this point, it is too late to accurately determine the level of maternal fumonisin that occurred during the critical gestational window spanning, 3-4 weeks post conception. The sphinganine

/sphingosine ratio has been suggested as an alternative exposure biomarker, but this measurement is limited to short term detection unless the exposure is fairly constant

(Sewram et al., 2003). In several rodent studies, it was noted that once elevated by a high dose, the levels of sphinganine in either urine or kidney will remain significantly elevated by exposure to a low dose of fumonisin that would not ordinarily elicit an increase in sphinganine (Enongene et al., 2002). Rats were fed diets containing 10ppm of fumonisin b1 for 10 days and then changed to either diets containing 0 or 1 ppm fumonin. In the rats consuming the 0 ppm diet, the urinary sphinganine returned to control values (0.2 nmol/ml) by day 20, whereas in the rats changed to the 1ppm diet, sphinganine was still significantly elevated on day 20 (2.5 nmol/ml) (van Waes et al.,

2005). Chemical analysis of hair samples has shown promise as a method for examining chronic mycotoxin exposure. In 2003, Sewram et al. reported that human hair testing could be used to detect fumonisin. After extraction and clean up, HPLC-ESI-

MS was able to detect fumonisin B1, B2, and B3 in hair samples. However, the samples were composites taken from barbershops in various regions of South Africa.

Nonetheless, hair analysis has advantages when compared to urine and blood testing, which are only detect able to detect exposures from 2 to 4 days, hair analysis provides a larger window of xenophobic detection. Detection can range from weeks to months depending on the length of the hair, and evaluation of hair segments can be performed so that the segment most closely reflecting time-at-exposure is selected for analysis

(Kintz, 2004). The method for measuring fumonisin in hair has yet to be validated so that hair concentrations can be used to predict exposure.

Fumonisin Effected Commodity Occurrences

Due to the ubiquitous nature of A. niger, and Fusarium’s field hardiness and spore-forming capacity, corn is the major commodity effected by fumonisin.

Coffee

From studies performed by Noomin et al. (2009), FB2 and FB4 were found to have contaminated Thai Arabica and Robusta coffee beans and cherries. Both commodities were incubated on 18% glycerol agar and malt extract agar plates with and without surface sterilization for seven days at 25°C, then inspected for fungal growth. From this incubation, isolates of Aspergillus section Nigri were isolated and identified to species level using morphology and molecular characteristics. Within Arabica coffee samples, sections Nigri and Circumdati were found to account for 75 and 77% infestation respectively, whereas in the Robusta samples Nigri was the predominate species with 100% infestation. No Fusarium species were detected from coffee bean samples. Representative samples of each Aspergillus species was isolated and incubated at 25°C for 7 days on CYAS. All ten tested A. niger isolates from Arabica bean and cherry samples produced FB2 from 0.4 to 2 µg/cm, and 3 out of 7 of the

Robusta coffee bean/cherry isolates from 0.3 to 2 µg/cm. Coffee bean samples, after

LC-MS/MS analysis were found to be contaminated between 1.3 and 9.7ng/g. It was noted that a high percentage of infection was determined after the surface disinfection of the green coffee beans indicating that A. niger actually grows actively inside the coffee bean itself. However, the lack of an abundant source of carbohydrates needed for substantial FB2 contamination was missing from these samples. Coffee cherries did

provide a carbohydrate source, but not enough to represent a high risk for widespread contamination.

Dried Vine Fruit

After Frisvad et al. (2007), reported that A. niger had the capacity to produce fumonisins in high quantities on agar media with low water activity, food matricies that met this criterion were investigated as well. Vargas et al. (2010), investigated the role that black Aspergilli played in the contamination of raisins, dates, sultanas, figs, and currants from various countries including Turkey, South Africa, Greece, Iran, USA,

China, and India. The vine fruit were surface sterilized with 96% ethanol by immersion for 5 min, then placed on malt extract and dichloran-rose bengal medium. The samples were incubated under standard conditions, purified and identified by classical taxonomic methods. Following this, the purified fungal isolates were cultivated on CYA20S agar plates for seven days at 25°C. After removing agar plugs of the cultivated isolates, the fumonisins were extracted with MeOH/H2O 3:1 (v/v) and analyzed using RP-HPLC/ESI-

ITMS. From a morphological analysis of the fungal cultures grown from the dried vine fruit samples demonstrated that all except two raisin samples were contaminated with black Aspergilli. Using a PCR-RFLP approach, two types of Aspergilli were identified, those with the RFLP pattern N i.e. A. niger and A. awamori and those with the RFLP pattern T such as A. tubingenisis, A. vadensis, A. costaricaensis, A.acidus and A. piperis. Due to this finding only isolates displaying A. niger contamination were considered since the latter fungal species did not have a history of fumonisin production. The average total fumonisin content of the dried vine fruit samples was

7.22 mg/kg which ranged from 4.55-35.49 mg/kg. Furthermore 66% of the raisin-derived

A. niger and A. awamori strains were found to produce fumonisins in detectable

quantities. Cultivated isolates of A. niger produced 6.39mg/kg of fumonisins on average.

Similar values were observed by Mongensen et al. (2010), for A. niger isolates from raisins (5-784 µg/kg and 12-672 µg/kg for FB2 and FB4, respectively). Raisins taken directly from retail markets in U.S.A., Greece, South Africa, Chile, and Turkey were also analyzed for fumonisin contamination by Knudsen et al. 2011. The group found that

48% of the 21 tested raisin brands contained FB2 and 43% contained FB4, with concentrations of 0.3-13 µg/kg and 0.26-1 µg/kg respectively. The highest concentrations of contamination were observed in samples from California, but the reasoning behind this are unknown and may be attributed to several factors including: climate, preharvest weather conditions, differing grape cultivars, or different drying or sorting methods. There was also no ability to correlate fumonisin content and the type of cultivation strategy (conventional or organic). It was surmised by researchers (Vargas et al., 2010 and Knudsen et al., 2011) that the drying process may have led to the accumulation of these toxins in dried vine fruit, which is an indication that the fumonisins could have been present before drying.

Maize and Corn-Based Foods

Maize is arguably one of the most important crops worldwide. Many countries of varying socioeconomic levels use this grain for subsistence in the daily lives of their populations. A.niger has been found to colonize maize in the pre-harvest period, during harvest, or post-harvest during storage, and fumonisin contamination may occur at any of these stages. Lorieco et al. (2013), demonstrated the role of A.niger in the contamination of maize kernels by isolating this fungus from kernels grown in central

Italy. While in the United States, Palencia et al. (2010), reported 19 isolates of

Aspergillus sec. Nigri from maize using rep-PCR, however no fumonisin data was

presented. To entertain this deficit, Susca et al. (2014), collected 199 Italian and 101

U.S. derived maize isolated strains to evaluate the fumonisin production potential of these strains. To encourage mycelium production, a spore suspension from each strain was grown in Wickerham medium (40 g glucose, 5g peptone, 3 g yeast extract, 3g malt extract and 1 L water) for 48 hrs. After mycelial filtration, DNA was isolated with quality determinations performed using electrophoresis and spectrophotometry. PCR amplification and purification led to sequencing for species identification and comparison. Following this, isolated fungal strains harvested from corn samples were cultivated on CYA20S under standard conditions and analyzed via HPLC-FD.

Identification of 148 strains based on the beta-tubulin locus were characterized as

A.niger and showed a 99.1-100% similarity with previously identified strains (accession number AY585536, A.niger type strain). Of the strains evaluated for FB2 production,

72% of the strains from the U.S. and Italy were FB2 producers with concentrations up to

890 µg/kg in culture, and a detection limit of 10 µg/kg. The relative frequency of A.niger infected corn for both countries was quite different, in Italy the species composition was approx. 60% A.niger and 40% A. tubingensis. Within the U.S. the frequency was observed at 73% A.niger and 27% A. tubingensis. A tubingensis has not been characterized as a FB2 producer, noting a higher risk of FB2 contamination from black

Aspergilli in the U.S. Several agronomic practices for the reduction of fumonisin in corn were offered from a study performed by Ariño et al. (2009). The group found that type of crop rotation, varietal resistance, fungicide application, corn borer infection, and cultivation techniques tend to affect the infection levels within crop growing areas.

Fumonisin infection in conventional corn (729 ± 429 µg/kg) was found to be higher when

compared to transgenic corn,Monsanto-810, (395± 121 µg/kg) grown under the same conditions, indicating that plants protected against borer insects are less susceptible to fungal infection and subsequent mycotoxin contamination. Statistically, however there was no significant difference in the contamination levels. The lack of significant differences between corn kernels grown from conventional and transgenic seed may be explained by the abnormally low incidence of borer insect infestation during the 2007 crop year. Work within the United States has revealed that the control of insects on maize via the use of genetically modified insect-resistant maize lines can reduce fungal colonization and the resulting contamination of corn indirectly, by reducing insect damage. It was also observed that maize fields subjected to dry planting contained significantly higher fumonisin concentrations (1004±379 µg/kg), when compared to those sown by wet planting (230±88 µg/kg). This result was attributed to the crop undergoing hydric stress which has been shown to increase mycotoxin concentrations in plants (Barrier-Guillot, 2006). Although tillage methods (plowing or minimal tillage) were found to have no direct effect on fumonisin contamination, the removal of residue from the previous crop harvests showed decreases in fumonisin levels (246±216 µg/kg) versus the practice of incorporating leftover residue into the soil (683±239 µg/kg). The removal of the residues removed detritus that spore forming fungi use in their saprophytic life cycle during the over seasoning period, thus alleviating any chance of toxin production. Field fertilization with nitrogen was also found to increase fumonisin levels in maize, with a weak correlation of r=0.46 between kilograms of N per hectare and fumonisin level. Concentrations in fertilized and unfertilized fields were 687 and 345

µg/kg respectively (Marocco et al., 2008).

Silva et al. (2007), evaluated commercially available products in Portugal for the occurrence of fumonisin contamination. The evaluated products included: yellow maize, white maize, maize flour, maize semolina, maize starch, sweet maize, maize snacks, corn flakes, and mixed breakfast cereals, all purchased from shops, health food stores, and supermarkets. Samples were extracted three times with methanol and water

(80:20 v/v). The extract was then cleaned up via glass microfiber filtration and added to an immunoaffinity column (IAC). Final analysis was performed after pre-column derivatization with NDA, via HPLC-FD. Of the 67 samples tested, 22.4% were verified to be contaminated with fumonisin. Samples revealed high contamination levels, between not detected and 1569 µg/kg for FB1 and not detected and 457 µg/kg for FB2. In yellow and white maize, FB1 concentrations were 332 and 363 µg/kg respectively, and 99 and

275 µg/kg respectively. However these values are much lower than those observed by

Doko et al. (1995), in 1992, where levels for the same commodity were 1031 µg/kg-FB1 and 1077 µg/kg FB2. Due to these findings, surveillance efforts in other countries of that consume corn products identified high incidence and contamination levels. In Spain, the incidence rate for yellow maize was 87.3% with a mean contamination level of 4800

µg/kg for FB1 (Castella et al., 1999). In Morocco, the percentage of positive samples for

FB1 was 50% with a maximum of 5960 µg/kg (Zinedine et al., 2006). Similar levels were also found in maize imported into the U.K. (3406 µg/kg -FB1, 1268 µg/kg-FB2)

(Scudamore and Patel, 2000), Nigeria, and Brazil. With regard to maize flour, mean

FB1 values were 822 µg/kg with a maximum contamination level of 1569 µg/kg. Maize flour is used in the production of breakfast cereals and similar foodstuffs, an evaluation of these items in other locales demonstrated proportional contamination. Molinie et al.

(2005), found 1113 µg/kg FB1 in breakfast cereals from French markets made exclusively of maize flour. In Italy, Cirillo et al. (2003), found in flour and whole meal cereals ranging between 10 and 2870 µg/kg for FB1 and 10-420 µg/kg for FB2. All samples from Sao Paulo, Brazil, were positive for both toxins as well. Mean values were 2100 and 700 µg/kg, respectively, for FB1 and FB2 (Bittencourt et al., 2005).

However, fumonisins were not detected in maize starch. It is thought that the wet milling process leads to losses that explain these results. Analysis of fractions from pilot-scale wet milling of corn tainted with fumonisin revealed the starch fraction did not contain measureable amounts of FB1 or FB2 due to the migration of the toxin into aqueous solutions (JEFCA, 2001). From the Silva et al. (2007) study, none of the cornflake cereals were found to be contaminated with the fumonsin toxin, which is in agreement with studies by Sydenham et al. (1991), and Pittet et al. (1992). In direct juxtaposition to these findings, Park et al. (2004), found that 14 out of 15 cornflake samples and other maize-based breakfast cereals contained detectable levels of FB1 with a range from13-

237µg/kg and 21-23 µg/kg FB2. In 1997, Tseng and Liu reported an FB1 incidence rate of 3.5% (140-1281 µg/kg), whereas the incidence rate for FB2 in the same cornflake samples was 17.6% (120-466 µg/kg). It has been reasoned that one of the reasons the fumonisin levels in cornflakes in particular tends to be lower than other products is due to the fine nature of the corn grit during commercial dry milling of maize. Free fumonisin are lost during extrusion cooking of maize flour, flaking, and roasting of maize grits and the overall processing of maize flour to cornflakes. The addition of glucose has also resulted in fumonisin reductions during the extrusion of maize grits, coupled with the protein binding displayed during the heat processing (Kim et al., 2003; Girolamo et al.,

2001). Of all the commercially available products evaluated, maize-based snacks had the lowest incidence rate of approx. 16.6% or less in the U.S., Spain, the U.K., and several Nordic countries. Incidences were highest in Taiwan at 33% for FB1 (0-2395

µg/kg) and 21% for FB2 (0-715 µg/kg). Jackson et al. (1997), attributed the minimal contamination of maize snacks as a result of the non-enzymatic browning involving the primary amine in fumonisin and the reducing sugars in the food matrices.

Beer

A beverage that enjoys worldwide sales and consumption, beer is not typically associated with fungal contamination. The labor-intensive preparation of this fermented alcoholic beverage has reportedly not completely escaped contamination by mycotoxins. During instances of contamination, the source has been molded barley or moldy corn, used for adjunct purposes. Moldy corn has also been implicated as a contaminant during fermentation activities for commercial ethanol production.

According to Bothast et al. (1992), small concentrations of fumonisin degradation were noted during the 3-day fermentation process to produce ethanol. Measurable quantities of the toxin were extracted from the distillers’ grain, thin sillage, and distillers’ soluble fractions. Furthermore, the addition of FB1 and FB2 to wort that was fermented over an

8- day period using three different strains of Saccharomyces cerevisiae had minimal effect on toxin reduction with losses of 3-28% and 9-17% respectively (Scott et al.,

1995). This contamination issue is not isolated to one portion of the beer-drinking population, but has been observed in varying countries including: Canada, Spain,

Kenya, and the U.S. A survey of 46 Canadian beers found that 22 (48%) were contaminated at levels of 0.2-24.7 ng/mL. However, 21 of the tested beers were found to have fumonisin concentrations of less than 4.8 ng/mL (Scott et al., 1997). Indirect

competitive ELISA was the preferred method of beer analysis during a toxin evaluation study in Spain. The evaluation consisted of a total of 32 beers, alcoholic and non- alcoholic. 14 of these beer samples contained quantifiable concentrations between 4.76 and 85.53ng/mL (Torres et al., 1998). A similar study was conducted in Kenya by

Mbugua and Gathumbi (2004), and included two brands of lager-style beer with contamination rates of 72% with toxin concentrations up to 0.78 ng/mL. Hlywka and

Bullerman (1999), employed the use of HPLC for FB quantification in beers purchased in Lincoln, NE. Of the extracted samples, 25 (86%) were found to be positive for FB1 contamination, and 41% had concentrations of FB2. Total FB content ranged from 0.3-

12.7ng/mL, with the sample population averaging toxin levels of 4.0 ng/mL. None of the aforementioned countries has any regulations pertaining beverage contamination levels of alcoholic beverages. The vast majority of the beers sampled in these studies were clear, lager-style beers. There was no explanation given as to why this style was chosen over other darker or ale style beer varieties even though both styles use similar raw ingredients.

The fate of several mycotoxins including fumonisins during the brewing and fermentation process was assessed by Inoue et al. (2013). After artificially spiking malt with the various toxins, it was used as an ingredient in the production of beer on a lab scale. During this brewing process, samples were taken from unhoped wort, spent grain, cooled wort, and beer. By the conclusion of the study it was observed that fumonisin concentrations decreased to less than 20% of their initial levels. The mashing step saw the largest decrease in toxin levels with smaller decreases in other portions of the process. Residual ratio percentages of the spiked toxin levels were used to

calculate toxin loss. For fumonisins B1 and B2, the residual ratios were as follows: unhoped wort-47.2 and 19.3, cool wort-32.4 and 17.0%, beer-46.1 and 10.6%, spent grain- 16.4 and 38.2%, respectively. It was concluded that, toxin decomposition was possible during the mashing and boiling processes because not all of the spiked malt was recovered from the wort or spent grain fractions. The adsorption of toxins to spent grains also contributed to the decrease in toxin levels.

Wine and Grapes

Initially, wine and wine grapes were analyzed to identify contamination levels from ochratoxin A, a well-known and well-characterized mycotoxin also derived from various Aspergillus spp. However, reports by Frisvad et al. (2007), uncovered the production of FB2 and FB4 by A.niger, and the potential mycotoxicological risks posed created a shift in the attention given to this species of Aspergilli. To make this declaration, the Frisvad group cultured several different strains of A. niger and F. verticilliodes as a reference, on thirteen different agars. Using an agar plug method for mycotoxin extraction and a selective ion monitoring electrospray LC-MS, the group was able to compare the similarities in toxin production of the two different fungi. It was noted that the A. niger strains produced higher amounts of toxin on media with greater contents of sugar, glycerol, and NaCl. Based on these findings, an evaluation of musts naturally contaminated by black Aspergilli in southeastern Italy was undertaken by

Logrieco et al. (2009). The group use thirty-one strains, each derived from four species of Aspergillus section Nigri (A. niger, A. carbonarius, A. tubingensis, and A. uvarum), which are commonly isolated from Italian grapes. These species were used to artificially infect Vitis vinifera cv. Negroamaro grapes found in the region. Upon extraction and analysis, it was established that only the A. niger inoculated grapes

produced detectable concentrations of FB2 in the range of 0.2-293g/g. To further investigate toxin contamination in wines within the region, must samples were obtained from freshly pressed grape juice containing solids, such as skins, stems, and seeds that would be used for the production of “Primitivo” red wine. Samples were analyzed via

LC/MS/MS, with chemical identification of FB2 from the samples based on three parameters: molecular weight, fragmentation pattern, and comparison of the retention time (RT) of sample peaks with fumonisin standards. Of the twelve collected must samples, two were contaminated with FB2 at levels of 0.01 and 0.4µg/mL.

To further examine the depth of FB contamination, in 2010, Lorieco et al. investigated the natural occurrence of FB2 in commercially available wines from Italy.

Fifty-one wines, 45-red,5-white, and 1-rose were randomly purchased, originating from the wine making regions of Piedmont, Trentino Alto Adige, Veneto, Apulia, Tuscany,

Lombardy, Campania, Siciliy, Friuli Venezia Giulia, Lazio, and Umbria, with years of production from 2004-2008. Samples were prepared and loaded onto a SPE cartridges for extraction and final analysis was performed by HPLC-MS/MS. Recoveries for the toxin were measured by spiking 7.5 mL of wine with FB2 and FB4 at three different concentrations (1,5, & 10 ng/mL). Nine red wine samples (17%) were found to be contaminated by FB2 at levels ranging from 0.4-2.4 ng/mL, with no FB4 contamination.

No measureable concentrations of FB2 or FB4 were found in the analyzed white and rose wine samples. Mogensen et al. (2010), performed a contemporary study that evaluated 77 wines (red-56, white-13, port-6, rose-1, champagne-1, madeira-1) purchased in Denmark with vintages from 1991-2009. Of the 77 wines, 13 countries were represented including Argentina, Australia, Chile, China, France, Germany, Italy,

100

New Zealand, Portugal, Romania, Spain, South Africa, and the U.S.A. These samples were also extracted via SPE cartridge and analyzed with LC-MS/MS. Analysis of the 77 wines demonstrated the occurrence of FB2 in 23% (18 of 77) of the samples. A high number of the samples were close to the LOQ, so the results were further confirmed via a secondary extraction using immunoaffinity purification which mimicked early results with contamination ranging from 1-25ng/mL. It was noted that in both studies the rate of contamination of red wine samples was much higher than for other varieties.

As with many other grape growing countries, the predominate black Aspergillus found in Portuguese vineyards are strains that belong to the A.niger aggregate. To evaluate the propensity of these strains to produce FB2, Abrunhosa et al. (2011), cultured 700 black Aspergilli isolated from wine grapes that were collected to ascertain the occurrence and distribution of OTA in the five major wine-making regions of

Portugal (Douro, Ribatejo, Alentejo, Vinho Verde, and Madeira). All strains used in this study were collected between 2001 and 2003, and were preserved in glycerol and stored at -80°C, which would have minimal effect on the culturing or secondary metabolism of the isolates. The represented strains were A. niger (597), A. carbonarius (75) and A. ibericus (9). The strains were revived in MEA for 7 days in the dark at 25°C, and then subcultured into CYA and incubated at 25°C for 8 days in the dark. FB2 determination was made from the extraction of five agar plugs of each culture, then analysis via LC-FLD with immunoaffinity confirmation. FB2 was not detected in the A. carbonarius or A. ibericus isolates, but was detected in 176(29%) of the strains from the A.niger aggregate. The amount of FB2 produced by Portuguese grapes ranged from 0.003-6.0mg/kg, with a mean of 0.66mg/kg. The potential risk of

101

FB2 contamination of grapes and wines is a viable one when consideration is given to the fact that A. niger aggregates are consistently present in vineyards at higher levels than other black Aspergilli, and warm, humid weather conditions abound in grape- growing regions.

Thermal Degradation from Processing

The majority of mycotoxins are heat resistant within the range of conventional cooking methods and food processing temperatures (80-121°C). Thus minimal toxin reduction is achieved during boiling, frying, or pasteurization (Scott, 1984). There are many factors that play vital roles in the degradation of fungal toxins in food including: toxin concentration, moisture level, heating temperature and time, pH, food ionic strength, degree of heat penetration, and the type of mycotoxin (Samarajeewa et al.,

1990). Fumonisin is heat stable up to101-120°C, which renders it undisturbed by many processing applications. Alberts et al. (1990), demonstrated that boiling F. verticillioides culture for 30 min did not result in a reduction in the overall levels of fumonisin present.

The pasteurization of milk spiked with 50ng/mL FB1 and FB2 at 62°C for 30 min led to no significant reductions of toxin contamination in the milk (Maragos and Richard, 1994).

Fumonisin was still found to be present at toxic levels in maize products that were nixtamalized which produced FB1 and hydrolyzed FB1 (HFB1) (Humpf and Voss, 2004).

However, progress has been made in determining the proper conditions for FB degradation. Within an aqueous buffered solution at pH 4, 7, and 10, the effects of processing time (10-60min) and temperature (100-235°C) displayed that FB1 and FB2 are least stable at pH 4, followed by 10 then 7. Decomposition began at or above

150°C, and at 175°C more than 90% both fumonisin compounds was lost after a 60 min treatment, regardless of pH (Kabak, 2009). It was observed by Scott and Lawrence

102

(1994), that the heating of dry and moist corn meals at 190°C for 60 min caused a 60-

80% reduction in fumonisin levels, while close to 100% of the toxin was degraded following baking at 220°C for 25 min. Baking corn muffins spiked at concentrations of 5 mg/kg FB1 at 175° and 225°C resulted in 16 and 28% toxin reductions respectively

(Jackson, 1997). It must be noted that no organoleptic attributes were evaluated during the processing of the aforementioned products.

During processing, fumonisin is known to bind to components within the food matrix or react with reducing sugars. The reaction of FB with D-glucose or D-fructose at

65°C for 48 hours appeared to block the primary amino group, thereby preventing FB- induced toxicity in rat and pig cell culture tissues (Fernandez-Surumay et al. 2005).

There is also the phenomenon of “hidden fumonisin” as well. These fumonisin are described as being bound to sugars or other matrix components during cooking/processing and are present throughout the raw material all the way to processing, but cannot be detected by conventional methods. Evidence of this phenomenon was presented by Kim et al. (2003), when evaluating cornflake samples.

When placed under the conditions associated with extrusion processing, high temperature, high heat, and excessive shear forces, toxins levels may be reduced. This reduction is contingent upon the extruder temperature, screw speed, moisture content of the food product, and residence time within the extruder. Castelo et al (1998), reported 31-95% reductions of fumonisin in maize grits(18-26% moisture), spiked at 5

µg/g when using twin screw extruders with screw speeds of 142 &160 rpm and barrel temps ranging from 14°-200°C .

103

Muscadine Grapes

For the majority of the consuming public, when considering grape varietals associated with beverages, well-known varietals such as chardonnay, pinot grigio, cabernet sauvignon, merlot, and syrah come to mind. However, in the southeastern

United States muscadine grapes have been cultivated for decades and were the first native grape species to be cultivated in North America (Andersen et al., 2003). With regard to botanical classifications, the Vitis genus includes two subgenera: Euvitis

(European, Vitis vinifera L. grapes and American bunch grapes, Vitis labrusca L.) and the Muscadinia grapes (muscadine grapes). The three species within the Muscadinia subgenera include: Vitis munsoniana, Vitis popenoei, and Vitis rotundifolia. The Euvitis and Muscadinia sub genera have been reported to have somatic chromosome numbers of 38 and 40, respectively. Our focus is solely on Vitis rotundifolia, which are known to have thick skins, large seeds, and a soft, translucent, musky-flavored pulp. Berries vary in size ranging from ¼- 1 ½ inches in diameter and 4-15 grams in weight, containing five hard, oblong seeds. Grape cultivars vary over a range of colors from white, also known as bronze, to pink, red, blue, nearly black, and purple (Olein, 2001). Muscadine grapes are a rich source or polyphenols such as resveratrol (3, 4, 5-trihdroxystilbene), ellagic acid, catechins, tannins, quercetin, delphinidin, petunidin, gallic acid,myricetin, kaempferol and ellagitannins (Talcott and Lee, 2002). The resveratrol content in muscadines is noted as being one of the highest in most crops, and has been shown to demonstrate anticancer properties against blood, colon, and prostate cancers (Mertens-

Talcott and Percival, 2005). Overall phenolic concentrations in muscadines was found to be higher when compared to bunch and Florida hybrid bunch grapes (Basha et al.,

2004). The native growing area of these grapes extends from Delaware to central

104

Florida, and includes all the states along the Gulf of Mexico reaching northward along the Mississippi River into Missouri. The history of these grapes in the region are not clear, but there is a muscadine vine on Roanoke Island that has been in continuous cultivation for nearly 200 years, with a trunk over two feet thick covering half an acre. It is believed that from this vine is where others were taken.

Muscadine grapevines are capable of growing wild in forests, uncleared lots, on fences, and along highways. Known for their vigorous growth, they can pose a vegetational threat to unused land by becoming invasive and stymieing the growth of other native plant species. Muscadines found in the wild are described as being functionally dioecious because of female vines with incomplete stamen formation and incomplete pistil formations in male vines. The majority of the wild muscadine grape population is composed of male vines. These vines are known to grow best in deep, moderately fertile sandy loams and alluvial soils (pH 5.5-6.5), with poor growth performance in calcareous, heavy or poor draining soils. Vines have been known to grow from 60-100 ft. in the wild, with tight, non-shedding bark, warty shoots, and unbranched tendrils. The foliage is described as slightly lobed with yellow-tinged green leaves ranging from 2.5-5.0 in., and round to broadly ovate with serrated edges

(Vasanthaiah et al., 2011). In nature, muscadine grapes are propagated sexually from seed and asexually from shoot formation after making contact with the ground. Asexual propagation typically produces a plant that is genetically identical to the mother plant, whereas seed propagation produces plants with genetic variation. Planting areas where the temperature does not drop below 0°F is best, however the minimum temperature vines can withstand is dependent upon their overall condition. Temperature fluctuations

105

from high to low can be substantially damaging to vines due to a loss of hardiness.

Typically, muscadine grape clusters are quite small with 6-24 berries, and are late in breaking bud in the spring requiring 100-120 days to mature fruit. Mature berries characteristically do not adhere to the stem. Berries that are fully matured when harvested have a dry stem scar, whereas grapes harvested too early will tend to have a wet stem scar due to tearing at the point of pedicel attachment. Stem scarring can be used as an indication of berry quality and storage life. Wet stem scars appear when the cap stem does not separate from the berry, and yield a site for spoilage organism invasion or colonization, leading to premature spoilage (Ahmedullah and Himelrick,

1989).

Harvest and Post-Harvest

The harvesting period for muscadines begins as early as late July, and extends into late September for some cultivars. For most cultivars, two harvests will be necessary, but for Fry or Welder as many as five maybe needed. As with most agricultural commodities, harvesting muscadines at peak maturity ensures a high quality final product. These grapes are typically harvested around a pH value of 3.2 and the soluble solids range from 15-19% (Ahmedullah and Himelrick, 1989). Walker et al.,

2001, reported that berry color, sugar and acid contents were indicators of an increase in fruit ripeness correlating to higher fruit densities. Due to the increase in sugars and the decrease in acids during ripening, percent soluble solids and tartaric acid are parameters used to determine grape maturity. A drawback to muscadines is that some cultivars do not ripen uniformly with a range of ripeness during harvest periods. Once harvested, the management of temperature and humidity play a pivotal role in the final quality of muscadine grapes (Morris and Brady, 2004). Slowing the respiration process

106

protects grapes from premature deterioration. Lowering storage temperatures does just this, for every 18°F the temperature is reduced, the respiration rate is lowered by a factor of two to four. It is recommended to ensure high quality grapes, pre-cooling with forced air to 36°F or lower within twelve hours of harvest is the best practice (Perkins-

Veazie, 2002). The average brix content for selected cultivars at full maturity is listed in

Table 2-1.

Juice and Wine Considerations

The juice of muscadine grape is described as having a unique flavor and bouquet. In the past, juice for local consumption has been produced from cultivars including: Hunt, Scuppernong, Creek, Dulcet, Yuga, Noble and Carlos (Murphy et al.,

1938, Sistrunk and Morris, 1982). According to Bates, 2001, changes that occur in muscadine grapes during growth and maturation determine the quality of the juice. The development of flavor and aroma volatiles occurs during the ripening process. As the fruit matures, sugar concentrations and color intensity increase, as pH and titrable acidity decreases. The composition of muscadine grape juice differs very little from that of the whole grape with the exception of a decrease in fiber from the skins and lack of oils which are contained in the seeds. Glucose and fructose are the major sugars found in muscadine juice, in combination with other flavor components such as: acids, volatile esters, and aldehydes. Anthocyanin pigments near grape skins are responsible for juice color. Juice quality is highly dependent upon sugar concentrations, acid content and the aforementioned flavor components. Juice composition from a particular cultivar will vary from year to year and changes from one growing area to another due to soil compositions and vineyard management practices. During juice production juice yields tend to be low, with approx. 130 gallons juice/ ton of muscadines, while other grapes

107

yield 180 gallons juice/ton (Ahmedullah and Himelrick, 1989). Juice from muscadines may be extracted with hot or cold pressing techniques. However, Threlfall (2002), reported that hot pressed juice had higher color density and yielded more juice when pressing methods were compared across three cultivars. Sistrunk and Morris (1982) noted that juice extracted at higher temperatures increased the occurrence of browning, overall acidity and total phenols in samples.

During processing and storage protocols for wine making, muscadine grapes are highly susceptible to color loss and browning (Sims and Morris, 1985). Storage conditions with increased temperatures were associated with visual browning which were attributed to the chemical changes of anthocyanin pigments. Muscadine wines stored at 104°F experienced unacceptable browning after three months of storage, whereas at 86°F similar color degradation was demonstrated after nine months (Sims and Morris, 1984). Color changes are also effected by increases in pH, which displayed muscadine wines with lighter hues than initially observed. In comparison to V. vinifera berries (24-26%), muscadines have a lower sugar content (14-16%). Therefore, muscadines tend to require the addition of sucrose to promote alcohol conversion during fermentation to create a desirable final product (Vasanthaiah et al., 2011).

Cultivars

There are over 100 improved cultivars of muscadine grapes, with the oldest being found in the Scuppernong River and being named so. Cultivars meant for the fresh market are typically large, sweet, and attractive with thin skins. They have yields that are consistent, and moderate to high with vine vigor at the same rate and some disease resistance. Many fresh market cultivars have imperfect flowers, requiring a pollinizer and are female cultivars. These cultivars classified as pistillate, and have

108

stamens that are not completely developed and are usually non-functional. Self- fertilizing cultivars tend to average higher yields that are 40-50% higher than female cultivars. The production of cultivars destined for processing must have relatively high yields, with berries at least 14° Brix and have a favorable sugar-acid ratio. Uniform berry ripening is also an important characteristic to limit the need for multiple harvests.

Vine berry appearance is not very critical, however, these cultivars must have a high degree of color stability, especially darker cultivars.

Fresh market

African Queen- patented, pistillate cultivar; large black berry, mid-season harvest

Big Red- patented, pistillate cultivar; large red berry, low vigor, mid to late season harvest

Black Beauty- patented, pistillate cultivar; mid-season harvest, large black berries, high quality

Black Fry- patented, pistillate cultivar; large black berry, moderate vine vigor and yield

Creek- not patented; small purple berry, high acid, moderately high vigor and yield, late harvest

Darlene- patented, pistillate cultivar; large pink berry, high vigor, early to mid- season harvest

Doreen- not patented, self-fertile; small to medium-sized berry, high yield, late season harvest

Early Fry- patented, pistillate cultivar; dark bronze berry, vine vigor and yield low, early harvest

Fry- not patented, pistillate cultivar; large bronze berry, moderate vine vigor and yield, 6 week ripening period

Golden Isles- not patented, self-fertile cultivar; small to medium-sized berry, non- muscadine flavor, mid-season harvest

Granny Val- patented, self-fertile; bronze berry, heavy yield extended harvest into late September and October, may overproduce and stress vine

109

Farrer- patented, pistillate cultivar; large elongated purple berry

Ison- patented, self-fertile; moderate to large black berry, high yields, good pollinizer, early harvest season, high vine vigor

Pam- patented, pistillate cultivar; large yellowish-bronze grape, mild flavor, moderate yield, high vine vigor, mid-season harvest

Pineapple- patented, self-fertile; medium-large bronze berry, moderate vine vigor and yield, mid to late harvest season, tendency to over crop

Polyanna- not patented self-fertile, potential as pollenizer; medium-large dark purple berry, moderate to high yield, sweet berry, medium-high vigor, mid to late harvest

Southern Home- patented, V. rotundifolia x V. vinifera hybrid; black, medium berry, moderate to high yield, ripens August to November, flavor uncharacteristic of muscadine

Summit- not patented, pistillate cultivar; very sweet medium-large, bronze berry, high vigor, medium to high yields, mid-season harvest

Supreme- patented, pistillate cultivar; sweet large dark purple berry, high yields in southern Ga., low vigor and vine mortrality if allowed to over produce, mid to late season harvest

Sweet Jenny- patented, pistillate cultivar; large bronze berry, moderate vigor and yield, early season harvest, may blemish easier than other cultivars

Tara- not patented, self-fertile, potential pollenizer; large bronze berry, moderate vine vigor and yield, early to mid-season harvest

Wine, juice, jelly processing

Alachua- not patented, self-fertile; medium-sized black berry, medium high vigor and yield, uniform ripening, mid-season harvest

Carlos- not patented, self-fertile; medium-sized bronze berry, high vigor and yield

Nesbitt- not patented, self-fertile; medium to large black berry, high yield, late to mid-season harvest

Noble- not patented, self-fertile; medium-sized red berry, high vigor and yield, high disease resistance, uniform berry ripening

Welder- not patented; bronze berry moderate yield, extended harvesting throughout season

110

Hardiness- insect control and disease

One of the well-noted advantages to cultivating muscadine grape is their tolerance against most insect pests and diseases (Olien and Hegwood, 1990).

Persisting for no more than a few weeks, aphid outbreaks may occur on shoot terminals. However, the grape root borer (Vitacea polistiformis) is a significant pest of muscadine vines and have contributed to vine mortality. Wasp-like adult moths emerge and lay eggs on trunks and leaves. The eggs fall to the ground and hatch within 2-3 weeks. Newly hatched larvae burrow into the ground to feed on roots, moving to larger rootstock as they grow. Following one-two years of this activity, the larvae travels to the surface to enter the pupal stage finally becoming a moth. Infected vines have small yellowish leaves lacking vigor and looking weak, with underdeveloped berries. Upon sighting symptoms of this pest, it is usually too late to save the vine (Ducher, 2001).

Control of this pest can be maintained via the use of soil insecticides and keeping in row strips weed-free through herbicide use minimizes borer density in the roots (Andersen et al, 2003). It must be noted that other animal pest also consume muscadine grapes including raccoon, squirrels, crows, opossums, coyote and deer. This should be kept in mind when planning vineyards near thriving wildlife habitats, larger vineyards can out produce losses from these pests, but smaller vineyards may have more substantial losses.

As a sustainable fruit crop, there are some muscadine cultivars that can be grown with minimal use of pesticides. However, there are some more recent cultivars that are not as disease-resistant. Following some basic cultural practices have been demonstrated to aid in disease minimization. One such practice is using training systems that confer good circulation of air through the canopy, such as bilateral cordon

111

systems, and debris removal below vines to promote air flow. The use of drip or microjet irrigation systems, replacing systems that wet the foliage aid in reducing fungal incidence. Furthermore, harvesting muscadines at the proper time and removing mummified berries, pruned and dead wood from the vineyard reduces place for fungal spores to over season. The most common disease on muscadine grapes and leaves are angular leaf spot, black rot, ripe rot, macrophoma rot and powdery mildew. All above ground tissues can be infected by bitter rot, while black rot and powdery mildew are cool-weather disease and easily controlled for. The closer the berry is to full maturation, bitter rot, macrophoma rot and ripe rot can be much more problematic

(Andersen et al., 1985). Instances of “dieback” or “dead arm” have also been associated with muscadine grape vines. This disease, caused by Botryosphaeria, manifests itself in the spring when new shoots appear. Related symptoms include necrosis, dying back from new shoot tips, stunted shoots with shortened internodes, and small misshaped leaves. Further disease development is identified via dark, wedge-shaped cankers in the woody vascular tissue. If left untreated, the fungus will travel down the trunk, killing the entire vine. Infection of muscadine vines is attributed to pruning wounds and dead infected wood carrying the pathogen. Dipping pruning equipment in 10% bleach solutions during winter pruning, wound protection, cross infection prevention, complete removal of infected wood, and spraying with a suitable fungicide after pruning are all viable controls of the disease (Ren and Lu, 2003).

Breeding and Cultivation

One of the major challenges to gaining wider acceptance for muscadine grapes is the lack of fruit quality when compared to V. vinifera grapes. Attempts have been made to combine the high fruit quality of V. vinifera with the disease and pest resistance

112

of V. rotundifolia through hybridization by breeders with some success. This was demonstrated when V. vinifera was used as the parent seed. The challenge in this hybridization is the difference in the chromosome number of the two grape varieties, V. vinifera (2n=38) and V. rotundifolia (2n=40) (Dermen, 1964). This chromosomal inconsistency led to the few hybrids that have been either highly or completely sterile.

However, through the use of colchicine and chromosome doubling, two hybrids “N.C. 6-

15” and “N.C. 6-16” were made fully fertile. From these colchicine-induced amphiploids, a small population of tetraploid seedlings were raised. The seedlings displayed enough variation in percentage of good pollen and in vegetative and fruit characters to indicate that segregation of character acquired from the two parental species had occurred.

Furthermore, breeders have obtained seedlings from berries set on the 2x hybrid “N.C.

6-15”. The fruit produce by these plants exhibited large fruit bunches, a characteristic of

V. vinifera, and other shared characteristics of both parental species. According to

Dermen et al., 1970, previous hybridization trials resulted in diploid hybrids with 2n=39 chromosomes, 19 contributed from the haploid gamete of V. vinifera and 20 chromosomes from the haploid gamete of V. rotundifolia, all of which were self-sterile.

Since 1993 over 50 cross combinations of the aforementioned varieties have been made, with limited success only two hybrids were produced from the crosses when muscadine grapes were used as the female parent. Many of the morphological and biological characteristics of these hybrids including, leaves, stems, tendrils, time of bud break, bloom date, and ripen date were intermediate between the parents, with some of them being partially fertile. It was found that the hybrids did exhibit a disease resistant capacity to Pierce’s disease, downy mildew, and anthracnose disease. Hybrids such as

113

these can be used as a bridge to carry important genes and improve characteristics of

Muscadinia and V. vinifera grapevines through breeding. With regard to seedlessness, this breeding discovery has not been associated with V. rotundifolia. Lu et al., 1993, reported that it is difficult to use muscadines as the female parent when crossing with bunch grapes due to pre-fertilization barriers. However, researchers have observed genetic transformations and discovered grape-derived genetic elements that could be used to regulate transgene functions in embryos and muscadine seeds (Li et al., 2007).

One of the most powerful tools being utilized to advance muscadine breeding is the use of DNA markers for new cultivar creation (Dalbo et al., 2000). According to Riaz and

Meredith, 2000, microsatellite markers provide unique genetic profiles for each cultivar, leading to unambiguous identification, with no relation to environment, disease or farming practices. Molecular marker-based mapping also aides in the early selection of promising seedling. The combination genetic and physical mapping may lead to the isolation of genes that regulate specific traits with unknown mechanisms. This may lead to a reduction in disease losses and pesticide usage in classical breeding without changing quality attributes (Grando et al., 2000). These genetic maps enable breeders to detect traits that are linked and are subsequently likely to be inherited together, determining the number and location of genetic factors controlling quantitative traits.

Dalbo et al. 2000, has mapped genes controlling flower gender, quantitative trait loci analysis has been used to identify several genetic regions that control fungal disease resistance in an interspecific cross.

114

Figure 2-1.Fully formed Aspergillus niger with conidia (spores) for dissemination

Figure 2-2. Fumonisin B2 structure

115

Figure 2-3. Molecular structure of “fumonisin-like” sphingolipids

Adapted from Stockmann-Juvala and Savolainen, 2008

116

Table 2-1. Summary of vegetative characteristics of select muscadine Cultivar pH Brix Mass (g) Yield* Vigor* Alachua 3.7 17.5 6.5 7.3 7.6 Black Beauty 3.3 17.3 11.9 6.9 7 Black Fry 3.3 17.7 10.6 5.7 5.8 Carlos 3.5 15 5.4 8.7 8.8 Creek 3.2 15.1 3 7.9 7.6 Darlene 3.4 22 15 5.6 8.7 Early Fry 3.5 17.4 10.2 4.2 4.4 Farrer 3.5 18 12.5 5.2 8.8 Fry 4 16 12.5 5.7 4.3 Golden Isles 3.1 15 6.5 5.6 9.1 Granny Bal 3.3 17 11 7.4 6.9 Ison 3.4 19 11.5 9.2 9.5 Nesbitt 3.7 18 11.5 8.8 8.7 Noble 2.8 16.8 3.2 9.4 8.9 Pam 3.5 16.1 7.9 6.3 7.9 Pineapple 3.4 15.7 8.7 6.8 6 Polyanna 3.4 18.2 9.3 7.1 7.4 Southern Home 4.1 19 6.5 5.1 6.7 Summit 3.2 18.7 8.5 7.4 8.1 Supreme 3.6 15.2 4.8 6.3 11.9 Sweet Jenny 3.3 16.7 12.6 6.4 7.2 Tara 3.3 15.6 9.3 5.9 7.3 Welder 3.8 17 6.5 8.9 6.5 Adapted from Andersen, P.C., Crocker, T.E., Breman, J., 2003 & Basha, S.M., Vasanthaiah, H.K.N., Kambiranda, D.M., Easwaran, K., Queeleym G., 2012.

117

CHAPTER 3 CAN ASPERGILLUS NIGER COLONIZE MUSCADINE GRAPES

Within the European Union, published reports have detailed the growth and isolation of the fungus Aspergillus niger from Vitis vinifera grapes within vineyards suited for wine production (Sage et al., 2002) (Logrieco et al., 2003). This species can infect before a wounding event, but primarily acts as an opportunistic or secondary bunch rot invader associated with sour rot (Rooney-Latham et al., 2008). These fungal isolates serve as an indicator that A. niger is capable of colonizing this grape variety and subsequently producing its carcinogenic secondary metabolites, fumonisin B2, B4, and B6, under the proper conditions. According to Hocking, et al. (2007), A. niger is by far the most common species of Aspergillus found on grapes even in the absence of disease. During the survey of six vineyards in Australia and Argentina, it was noted that the source of fungal inoculum included fallen dried berries, dead canes, vine bark, dried bunch stems, weeds and dead cover crop trash (Chuzle et al., 2006). Increased incidence of black Aspergillus spp. in vineyards was reported during hot, dry weather in southern-most regions, although higher temperatures combined with increased humidity and rainfall led to higher occurrences in similar areas also. This may partially explain higher incidences of FB2 contamination in wines from various southern regions in wine producing countries, however this climactic phenomenon was not observed in southern

Australian grape-growing regions (Varga and Kozakiewicz, 2006). The colonization of wine grapes via black Aspergillus spp. is known to increase well into berry growth from the berry setting stage until harvest, due to the inhospitable conditions caused by green immature berries and increased UV exposure from lighter leaf canopy cover. Berry skins soften and sugar content increases at “veraison” or berry ripening, supplying the

118

needed environment capable of supporting fungal colonization and toxin production, particularly if the berry is damaged. These same risks are also increased if berries are allowed to over-ripen on the vine due to late or delayed harvesting (Battilani et al.,

2006). Strains of this fungus have been shown to produce FB2 concentrations from 0.2

µg/g-293 µg/g in cultures after being isolated from grapes within vineyards. Further in vitro studies performed by Mogensen et al. (2010) demonstrated the capacity of the fungus to produce FB2 on inoculated V. vinifera grapes at concentrations of 171 µg/kg-

7841 µg/kg when placed under proper conditions. The growth of these fungal isolates and spore suspensions demonstrates that A. niger has the capability to produce FB2 within grape cultivation areas and within a laboratory environment when V. vinifera grapes act as the substrate. Fungal growth and mycotoxin contamination are not solely limited to the vineyard, the risk for contamination is also a concern within associated wineries. In southern Italy, twelve must samples were taken from the production of

“Primitivo” red wine in Apulia to assess possible toxin contamination. Of the twelve samples, two were found to be contaminated with FB2 at levels of 0.01 and 0.4 µg/mL

(Logrieco et al., 2009).

Although V. vinifera vines have historically not been very productive within the southeastern U.S. due primarily to Pierce’s and other diseases, Vitis rotundifolia or muscadine vines produce regular harvests. Consisting of a growing environment with comparable attributes to those that have experienced mycotoxin contamination in vineyards worldwide, it has become necessary to elucidate if similar contamination occurrences are taking place in the aforementioned region of the U.S. The objective of this study is to determine whether the muscadine grape, V. rotundifolia, can support the

119

colonization of the A. niger fungus and create a hospitable growing environment that leads to the production of the fumonisin B2 mycotoxin.

Methods and Materials

Safety

It must be noted that fumonisins are hepatotoxic, nephrotoxic and carcinogenic to rats and mice; the effects on humans are not fully known. Appropriate safety precautions were used, including personal protective equipment and the use of biological safety hood. Decontamination, with a 5% aqueous solutions of sodium hypochlorite for 5 mins, of the work area was always performed at the end of the work session. All hazardous waste was disposed of in labelled biohazard bags and containers.

Statistical Analysis

All statistical analysis during this study was performed using IBM Statistical

Package for the Social Sciences (SPSS) (Armonk, N.Y., USA).

Chemicals

All solvents used were high performance liquid chromatography (HPLC)-grade and purchased from Fisher Scientific (Waltham, MA). Water was purified by the Milli-Q system (Millipore, Bedford, MA, USA).

Fungal Hydration and Agar Production

According to Pitt and Hocking (1997), the most effective agar for the cultivation of

A. niger with some similarity to the investigated food matrix is Czapek’s Yeast Agar w/

20% sucrose. This agar was produced according to the protocol of Freshney (2010). To prepare the Czapek’s concentrate: 30g sodium nitrate, 5g potassium chloride, 5g

120

magnesium sulfate heptahydrate, 0.1g iron (II) sulfate heptahydrate, and 100mL water were combined and mixed thoroughly. A second mixture consisting of : 0.25g dipotassium phosphate, 1.25g yeast extract, 50g sucrose, 3.75g agar, and 500mL water was then produced. This mixture was then heated with stirring until translucent and slightly boiling. Next, the solution was autoclaved at 121°C for 20 min. After being allowed to cool, 2.5 mL Czapek’s concentrate was added and 20 mL medium was poured into 9 cm petri dish. Due to the iron component of the concentrate mixture, sterilization was not necessary.

The fungal species used in this experiment was Aspergillus niger van Tieghem, anamorph (WB 326) [CBS 554.65, IMI 50566, NRRL 326; 2766], and was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Prior to usage, the fungus was rehydrated from its freeze dried state by adding 1 mL deionized water to the glass ampoule, and shaken vigorously for 2 min. The solution was then transferred to a Falcon tube and combined with 5 mL deionized water and left to hydrate for 20 hours at 2-8°C. When fully hydrated, a total of 600 µL hydrate was used to inoculate each agar plate in three points. The plates were then incubated at 25-30°C for 7 days.

Upon completing the incubation period, each petri dish was flooded with (2) 15mL aliquots of deionized water, and scraped with a glass rod, then was further diluted with additional water totaling 90 mL to make a spore suspension. The suspension was then enumerated by combining 100 µL spore suspension aliquot with 100 µL 0.4%Trypan blue solution and injecting the solution into the Reichert Improved Neubauer (0.1 mm) hemacytometer (Buffalo, NY). Viable spores absorbed the solution and appeared blue under the light microscope for easier counting. Spores found in the four corner squares

121

and the center square where counted to determine spore density. Viable spore concentration was quantified by the following formulas:

푣푖푎푏푙푒 푐푒푙푙푠 (3-1) % 푣푖푎푏푙푒 푐푒푙푙푠 = ( ) ∗ 100 푡표푡푎푙 푐푒푙푙푠

푣푖푎푏푙푒 푐푒푙푙푠 푎푣푔# 푐푒푙푙푠 푝푒푟 푠푞푢푎푟푒 = (3-2) 푡표푡푎푙 푠푞푢푎푟푒푠(5)

푓푖푛푎푙 푣표푙. 퐷푖푙푢푡푖표푛 푓푎푐푡표푟 = (3-3) 푐푒푙푙 푣표푙.

푐푒푙푙 푐표푛푐푒푛푡푟푎푡푖표푛 = 푎푣푔# 푐푒푙푙푠 푝푒푟 푠푞푢푎푟푒 × 퐷퐹 × 104 (3-4)

The final viable spore suspension concentration was 1.89 x 108.

Grape Preparation and Extraction

Carlos and Noble grapes were sourced from Sirvent’s Vineyard (Florahome, FL,

USA) and were removed from bunches after being inspected for abrasions, mold, and good overall health. All selected grapes were disinfected by submersion in a 3% bleach solution (v:v) for five minutes, followed by thorough rinsing with deionized water. Each cultivar was then divided into three trials of three treatment groups: control, surface inoculated, and wounded, consisting of 25 grapes each. To wound the grapes, a 3-4mm incision was made at the grape calyx with a sterilized scalpel. All of the selected grapes, other than the controls, were inoculated with 20 µL of A. niger suspension either on the grape’s surface or within the wounded area. Each treatment was then moved to a moist chamber at 95% relative humidity, which was placed in an incubator for ten days at 25-

30°C without light. After the ten day incubation period expired, each trial was combined with 40mL 80% methanol (v:v) and homogenized for 90-120 seconds to produce a slurry using a two-speed Waring Commercial blender, model 17011S (Torrington, CT,

USA). The slurry was then centrifuged (Allegra X-15R, Beckman Coulter (Indianapolis,

122

IN)) for 15 min at 2500g at 4°C. The supernatant was then poured into a buchner funnel and filtered through a No. 1 Whatman filter (Marlborough, MA, USA) under a 25 in. Hg vacuum. A 10 mL extract aliquot was passed through a 30mg/3mL Phenomenex

StrataX-C33u Polymeric Strong Cation solid phase extraction column (Torrance, CA,

USA). The column was previously conditioned with 1 mL methanol and 1mL water. To further acidify the column, 1 mL water with 2% formic acid was added. Afterward, the column was washed with 1 mL methanol and the fumonisins were eluted with 1 mL methanol/water (1:4) with 2% NH4 OH (v:v). Extracts were then analyzed using HPLC-

MS/MS.

To evaluate the fumonisin production of the cultured fungus, agar plugs were extracted. Five agar plugs were removed from each of the six agar plates with a 6mm diameter cork borer. The plugs were subsequently placed in glass tube with 1.5 mL

75% methanol in water (75:25,v:v) The tubes were then sonicated for 120 min. When finished the samples were placed in syringes and filtered through 0.45µm PTFE filters.

The extract was then analyzed by HPLC-MS/MS.

LC-MS/MS Conditions

During this experiment, all samples were analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/MS/MS) via the

Agilent 1100 HPLC (Santa Clara, CA, USA) connected to a Thermo Scientific LCQ

Deca Ion Trap mass spectrometer (Waltham, MA, USA). Separations were conducted using an Agilent Zorbax SB C18 reversed phase column (2.1x 30mm; particle size

3.5µm) at a flow rate of 0.2 mL/min. A linear gradient program was used for the extract separation consisting of two mobile phases. Mobile phase A consisted of 0.1% formic acid in water (v:v), and mobile phase B was a solution of acetonitrile/methanol (80:20,

123

v:v) and 0.1% formic acid (v:v). The gradient program ran for a total of 20 mins as follows: 30-100%B (8 mins), 100%B (3 mins), 100-30% B (9 mins). Mass spectrometer acquisition was operated in positive ionization and multi reaction monitoring mode with nitrogen (N2) as the nebulizer gas with a source flow of 9 L/min at 350°C. The parameters for the MS/MS were: Capillary Temp-250.0 °C, Sheath Gas Flow-60, source

Voltage- 4.00 kV, Capillary voltage 29.00V,Full MS Target- 5e +007, SIM Target 2e

+007, MSN Target- 5e +007, Zoom Target 1e+007, Isolation width- 5.0, Collison energy- 37.5%, Activation time 30.00, Q-0.250, and 105-800 m/z scanning range.

Sample retention time was approximately 0-1 mins for agar plate samples, but ~6-7min. for contaminated grape samples due to pressure fluctuations. Detection was based on

LC-MS screening analysis with narrow ion traces around the protonated mass for FB2 with a mass to charge ratio (m/z) of 706.40, and created daughter ions at m/z 336.13 and 512.20. A 20µL aliquot was injected for each sample. Sample recoveries for the agar samples were determined by spiking three individual agar plugs with FB2 at the following concentrations: 0.96, 1.28, and 2.57 µg/mL. These samples were subjected to the same protocol as the agar plugs, following LC-MS/MS analysis toxin recovery from agar was 93%. The calibration curve showed good linearity in the range of 0- 5050

µg/mL, with R2 of 0.977. All chromatographic points on the calibration curve were run in triplicate. The relative standard deviation was less than 5%. A certified external standard of FB2 (51.4 µg/mL) (Romer Labs, Union, MO, USA) was used to construct the calibration curve. An approximate detection limit of 4.8 µg/mL fumonisin B2 and quantification limit of 14.7 µg/mL was calculated on the basis of blank sample response.

124

Result and Discussion

Fungal species produce hundreds of secondary metabolites characterized as mycotoxins, with the majority considered non-detrimental to human health due to being produced at trace levels, not coming in contact with food or feed, and only being produced under controlled laboratory conditions. With regard to understanding why these species produce such a multitude of secondary metabolites, researches have yet to pinpoint an exact causation for their production, however it is clear that these compounds have no function in overall cell growth or maintaining basic cellular function.

The mycotoxin fumonisin B2, a secondary metabolite of Aspergillus niger, was initially thought only to be associated with Fusarium spp. and was relegated to having a minimal role in the contamination of foodstuff. As research surrounding this mycotoxin has evolved, it is now noted that although Fusarium spp. produce fumonisin it does not have the capacity to produce fumonisins B2, B4, and B6 due to a genomic cluster impediment. Due to the association of FB2 and its carcinogenic nature, with A. niger the presence of this fungus in spoiled or adulterated foods is regarded as more than a physical filth.

All common grape types, wine, table and raisin are a few of the many commodities that this fungus is known to readily colonize. It has been reported to prefer warm, Mediterranean-like climates with sustained humidity due to its preference for environments with high water activity ranging from 0.95-0.99 which also includes its optimum aW for toxin production (Leong et al., 2006). The majority of the reports detailing the colonization of grapes and toxin production by this fungus have focused on

V. vinifera grapes. However, the climate within the southeastern U.S. provides all the attributes needed for A. niger to grow and proliferate, hence the need to investigate any

125

association between this fungus and the V. rotundifolia that are cultivated heavily in this region. It is possible that this fungus has been contaminating grapes and grape byproducts within his region unbeknownst to growers and the consumers for years.

Currently, no published reports detail the fungal growth or toxin production of A. niger with muscadine grapes as its substrate.

To ascertain whether this fungus has the propensity to colonize muscadine grapes Aspergillus niger, strain WB 326, was sourced and rehydrated from a lyophilized state. Strain WB 326 was chosen in particular because it is has been well characterized via amplified fragment length polymorphism analysis and DNA sequencing via Perrone et al. (2006), and is known to produce the fumonisin mycotoxin. According to Frisvad et al. (2007), after trying to cultivate strain WB 326 on the following agars: Czapek yeast autolysate (CYAS), yeast extract sucrose (YES), yeast extract (YEA), Czapek yeast autolysate with 5% NaCl (CYAS-5), CYAS with 20% sucrose, dichloran 18% glycerol(DG), glucose minimal medium (GMM), rice corn steep (RC), potato dextrose

(PD), potato carrot (PC), malt extract (MEA) and oatmeal (OAT), it was found to produce FB2 at the highest concentrations on CYAS-20, RC and CYAS. Of those agars, the strain was inoculated in three points on CYAS-20 plates to give the fungus a carbohydrate source to sustain viability and mimic the soluble solids content of common grape varietals. The fungus was incubated for 7 days and exhibited signs of a white mycelial mat by day seven as an indicator of healthy growth (Figure 3-1). After the careful removal of this mat, black conidia were discovered as expected. Following the removal of the fungal colonies via flooding with deionized water, and it was deemed necessary to determine if the cultured A.niger strain produced FB2 under the

126

aforementioned conditions. The method of removing agar plugs and extracting the mycotoxin to determine toxin production concentrations was also performed by

Palumbo et al. (2010) and Logreico et al. (2009), with FB2 concentrations ranging from

1.2-27µg/mL and 0.7-293 µg/mL, respectively. It must be noted that in these studies several different strains were being assessed. In this study only a single strain was cultured and toxin concentrations from the agar were found to be in agreement with the aforementioned studies with a range from 8.5-20.8 µg/mL (Figure 3-2). During the enumeration process, all of the spores were found to be viable, leading to a suspension at the 106 magnitude. With complete cell viability, it was unexpected that two of the six agar plates would have a substantially lower toxin production than the other plates.

Although the suspension was mixed thoroughly, it is possible that there was an uneven spore distribution on these plates. Several non-inoculated control plates were incubated under similar conditions at the same time to exclude the possibility of any interfering organisms.

Noble (Figure 3-9) and Carlos (Figure 3-10) muscadine grape cultivars were chosen as substrate to determine if A. niger would produce quantifiable concentrations of the fumonisin B2 toxin. These two cultivars are highly sought after for their consistency in wine production. The Carlos grape is measured at approximately ½ in. in diameter, and of a bronze color. It is a self -fertilizing cultivar and typical has yields high enough to support juice and wine operations. The Noble cultivar is also a self-fertilizing, medium-sized grape with black skins. Noble grapes are known to be produced in high yields, an advantage rendered by its high disease resistance. Its flavor is said to be less musky than other muscadines, and is noted for its color stability during processing. At

127

full maturity, the soluble solids content for the Noble and Carlos grape is 16.8° and

17.4° brix, respectively (Anderson et al., 1985). Brix levels such as these are one of the attributes that A. niger prefers from its host for survival and growth. This fungus is well- characterized as an opportunistic pathogen, taking advantage of damage done to its host by other organisms or physical abrasions. Being mindful of this characteristic, the muscadine grapes used for this study were subjected to two treatments, inoculation on the grapes surface and inoculation in a pre-made wound near the grape calyx. It was decided to make the wound near the calyx because in nature this position on the berry would be attached to the stem and inaccessible to colonization. In nature, fungi tend to focus all invasion activities on one weakened entry point. If the wound was made elsewhere on the berry this would provide multiple entry points for the pathogen, making this incubation scenario further from what is observed in the vineyard. It was hypothesized that the fungus would colonized the wounded grapes with relative ease via the pre-made entry point, but may not have the appresorial strength to overcome the skin turgor and pierce the skins of the surface inoculated samples. Before any samples were inoculated all grapes (controls, surface inoculated, and wounded) were surface sterilized with a 3% bleach solution to negate any interfering organisms. After incubation at 95% RH, it was unexpectedly observed that both control grape samples,

Noble and Carlos, exhibited signs of fungal sporulation (Figures 3-3, 3-6). This observation was unexpected due to the sterilization step, furthermore the containers used as moisture chambers were sterilized also. The source of moisture in the aforementioned containers was a moistened brown paper towel which was not sterilized. However, it is doubtful that the paper towel was a reservoir for the same

128

fungus used in the study and that it would disseminate itself evenly among all the control samples. With regard to the controls samples being contaminated via a source in the lab work space, all samples were prepared inside a Labgard Class II, Type A/B3 laminar flow biosafety cabinet (Plymouth, MN, USA), which prevents sample adulteration. Noonim et al. (2009), experienced a similar result when investigating the association of FB2 contamination by A. niger on Arabica and Robusta Thai coffee beans. The group observed what was described as a “high percentage of infection” by the fungus after surface sterilization of the coffee beans. It was concluded that this fungal species grows actively insideof certain agricultural commodities. Furthermore,

Aspergillus carbonarius and A. fumigatus have been noted to penetrate intact grapes and produce measurable ochratoxin A concentrations within the pulp without discernable damage to the grape skin (Battilani and Pietri, 2002). As for the grape samples, sporulation was noted visibly, but LC-MS/MS analysis did not yield quantifiable concentrations of the toxin from control grape samples. It is possible that if the control samples were given an extended incubation period that it would have produced FB2 in measureable quantities. This belief is held because visibly the infected control grapes lacked the typical white mycelial growth of a more mature fungal colony.

It is assumed that the observed sporulation was that of A.niger, however this was not confirmed via analytical identification,and could have been competeing organisms

After combining the incubated grape samples with an 80% methanol solution to extract the toxin, the mixture was homogenized into a slurry. The slurry was then centrifuged to separate the solid/liquid matrices leaving the toxin analyte in the liquid phase. To remove any large interfering components, the liquid phase was subjected to

129

vacuum-aided filtration. At this point, the focus became the removal of compounds that may have a negative effect on the chromatographic quantitation of the toxin. To address this issue, 10mL sample aliquots were subjected to further purification via strong cation- exchange solid phase extraction columns. This column was chosen based on the research of Mogensen et al. (2010), detailing the trials of more than five different solid phase extraction columns. The group reported that this column demonstrated better efficiency and reproducibility when compared to the other columns. Toxin recovery rates from the Mogensen study were 83% which was in agreement with the recoveries of

86% in the current study. Recovery rates were determined by spiking 10mL grape juice samples at the following concentrations: 0, 0.64, 0.96, 1.92 and 2.57 µg/mL, and subjecting the samples to SPE purification and LC-MS/MS analysis. The toxin load capacity of this column was also evaluated over a range from 0.1-1.500 µg/mL FB2 by the Mogenson group, and demonstrated a linear relationship with regard to the concentrations with no signs of reaching a maximal capacity. Furthermore, the amine located at C-2 of FB2 provides a functional group that can attach and take advantage of the cationic-nature of this column, yielding a purification that avoids anionic compounds.

Sample toxin identification and quantification was performed using high performance liquid chromatography with electrospray ionization ion trap tandem mass spectrometry. This quantitative method provided complete spectrometric data based on retention time, molecular weight, and corresponding fragment losses by the compound skeleton to represent a reliable chemical identification of injected analytes. The FB2 toxin was detected at a retention time of approximately 6-7 mins for grape samples, and

0-1min for agar plate analysis. The retention time variation was due to pressure

130

fluctuations in the HPLC system. The mass to charge ratio (m/z) for this compound is

706.40, however under MS/MS conditions the daughter ions of 336.13 and 512.20 were created and used for toxin chromatographic identification. It was observed that surface inoculated Carlos grape samples produced toxin levels that were very close to those of the wounded samples averaging 4.61µg/g and 3.98 µg/g FB2, respectively (Table 3-1).

This trend was also noted in the measurement of the noble samples, with surface inoculated samples averaging 3.28 µg/g and wounded noble samples averaging 3.36

µg/g FB2 (Table 3-1). The overall relative standard deviation was 17%. Initially, it was hypothesized that the wounded grapes would have the advantage with regard to higher levels of toxin production due to the perceived ease of fungal colonization. From these results, it is noted that with or without a provided opening in the cuticle of the muscadine grapes A.niger is capable of colonizing this grape cultivar and producing the FB2 toxin.

One of the most notable observations from this study was the variation of the fungal growth pattern in the wounded Noble and Carlos grapes. Mycelium completely covered the wounded Carlos samples (Figure 3-5), but was confined only to the wound area of the Noble grapes (Figure 3-8). This is attributed to the higher resveratrol (3, 5, 4- trihydroxy-trans-stilbene) content of the Noble grape leading to greater fungal resistance in the grape skins. A similar result was noted by Roldán et al. (2003), where it was observed that extensive growth of Botrytis cinerea on Palomino fino grapes caused resveratrol contents in the juice to decrease while increasing concentrations in the grape skins. This reseveratrol increase is due to the physiological stress experienced by the grapes. Lee and Lee (2015) detailed the anti-fungal mode of action of resveratrol against Candida albicans.They found that the grape polyphenol acted as an apoptosis-

131

inducer, exerting its effects from early to late stage apoptosis, subsequently leading to the loss of mitochondrial membrane potential. Cell mitochondrial dysfunction of this nature activates metacaspases, an apoptosis precursor, and promotes the release of cytochrome c, a required mitochondrial heme-protein.

A two-way ANOVA was conducted to examine the effects of grape cultivar and grape treatment on observed toxin levels. Outliers were assessed via the inspection of a boxplot and there were found to be none. Normality of the data was evaluated by

Shapiro-Wilk’s normality test and found to be normally distributed, p > 0.05. The assumption of homogeneity of variances was not violated as demonstrated by p=.250, according to Levene’s test for Equality of Error Variances. Overall, there was no statistically significant interaction between grape cultivar and treatment (surface inoculated/ wounded) for measured FB2 production in muscadine grapes. The fungus was found to produce slightly higher levels of toxin on Carlos grape samples due to their larger size, yielding more resources for colonization and growth. This result was expected also due the thinner skin of the Carlos muscadine versus that of the thicker skin of the noble grape. Furthermore, the Noble grape is known for its overall disease resistance, which led to the expectation that toxin levels for this grape may be lower than in the other studied cultivar. The mass of the grape samples were measured prior and post inoculation to determine if there was any correlation between the amount of fungi grown on the grape samples versus the concentration of toxin produced. The correlation for these two attributes was found to be very weak, establishing no means of predicting toxin concentration from fungal mass measurements.

132

Conclusion

From this study, it has become clear that the Noble and Carlos muscadine grape cultivars can serve as a substrate for the growth of and mycotoxin production by A. niger. Prior to this evaluation, there were no published reports addressing V. rotundifolia in association with possible FB2 contamination. Although only two muscadine cultivars were assessed, it is highly probable that these results can be transferred to other muscadine grape cultivars as well. With the knowledge that there is a positive association with this toxin and grape cultivar, it opens the discussion with regards to the fate and contamination concentrations of other muscadine products and by products.

133

Figure 3-1. Aspergillus niger on CYAS-20 Photo courtesy of author

Figure 3-2.Mean fumonisin B2 concnetrations extracted from CYAS-20 agar plug samples after A. niger cultivation

134

Figure 3-3. Control Carlos Muscadine after incubation at 25-30°C for 7 days Photo courtesy of author

Figure 3-4. Surface Inoculated Carlos Muscadine after incubation at 25-30°C for 7 days Photo courtesy of author

135

Figure 3-5. Wounded Carlos Muscadine after incubation at 25-30°C for 7 days Photo courtesy of author

Figure 3-6. Control Noble Muscadine after incubation at 25-30°C for 7 days Photo courtesy of author

136

Figure 3-7. Surface Inoculated Noble Muscadine after incubation at 25-30°C for 7 days Photo courtesy of author

Figure 3-8.Wounded Noble Muscadine after incubation at 25-30°C for 7 days Photo courtesy of author

137

Figure 3-9. Healthy Noble grape on vine Photo courtesy of author

Figure 3-10. Healthy Carlos grape on vine Photo courtesy of author

138

Table 3-1. Fumonisin B2 production by Aspergillus niger on Carlos and Noble muscadine grape cultivars after artificial inoculation with pre and post inoculation masses Cultivar Treatment Pre-inoculation (g) Post inoculation (g) change (+/-) FB2 (µg/g) Carlos control 194.7± 1.7 201.0± 14.4 6.3± 0.5 nd Carlos surface inoc. 214.3± 11.9 217.5± 10.0 3.2± 2.0 4.61± 3.2 Carlos wounded 188.9± 8.1 192.8± 8.0 3.9± 0.6 3.98± 7.1 Noble control 128.9± 3.2 133.8± 2.7 4.8± 0.4 nd Noble surface inoc. 131.9± 0.4 134.7± 1.0 2.7± 0.8 3.28± 2.5 Noble wounded 134.2± 2.1 137.3± 2.4 3.1± 1.2 3.26± 3.3 nd= not detected

139

RT: 0.00 - 14.97 0.60 NL: 100 1.12E8 95 TIC MS Plate4 90 85 80 75 70 65 60 55 50 45 40

RelativeAbundance 35 30 25 20 15 10 5 0.79 1.08 1.66 2.57 3.33 3.66 4.73 5.39 6.07 6.88 7.36 8.10 8.73 9.09 9.64 10.33 11.01 11.95 12.20 12.91 13.74 14.08 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Time (min) Figure 3-11.Plate 4 chromatogram

Plate4 #26 RT: 0.60 AV: 1 NL: 2.60E7 T: + c ESI Full ms2 [email protected] [190.00-1000.00] 688.31 100 95 90 85 80 75 70 65

60 530.29 55 50 512.28 45 40

RelativeAbundance 35 30 670.25 25 20 336.23 494.21 15

10 548.26 5 318.26 220.19 261.03 301.35 374.29 396.09 476.62 563.91 620.05 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z Figure 3-12. Plate 4 total ion chromatogram, FB2 daughter ions 336.23 and 512.28

140

CHAPTER 4 STORAGE IMPACTS ON FUMONISIN CONCENTRATIONS IN MUSCADINE GRAPE JUICE

Within the Unites States, consumers have a wide array of beverages from which to choose. The industry itself is trending toward beverages that are perceived as healthy or emphasize positive health benefits. The removal of demonized ingredients such as aspartame have also become common place, as has the introduction of smaller product bottles and cans to reduce consumer sugar intake (Taylor, 2016). One trend that has held firm over the past 12 years, according to the State of the Plate Report 2015,

(Produce for Better Health Foundation), is that the highest consumption of fruit juice is by children under the age of six at 158 annual eatings per capita (AEPC), followed by children ages 6-12 with 119 AEPC in 2014. Annual eatings per capita is defined as the number of times the “average” person consumes a product annually (across users and non-users). With one of the most vulnerable subsets of the population having the highest consumption rate of this product, its safety must be guaranteed. To address this consumption concern, the U.S. FDA enforces a policy stating that single strength apple juice, reconstituted single strength apple juice (if the food is an apple juice concentrate), or the single strength apple juice component of the food must not contain concentrations of the mycotoxin patulin (4-hydroxy-4H-furo[3,2c]pyran, 2[6H]-one) at or above 50 ppb. This mycotoxin is produced by Penicillium, Aspergillus, and

Byssochlamys spp. as a secondary metabolite, and is commonly associated with the contamination of pears, apples and apple products in the form of blue rot (Rice et al.,

1977). Lindroth (1980) reported patulin to be carcinogenic, teratogenic, and mutagenic in multiple species. Like most other mycotoxins, patulin is heat stable and can withstand the pasteurization regimes to which most fruit juices are subjected. However, its stability

141

in apple and grape juice have been reported to be diminished over periods from 10 days to five weeks with reductions ranging from 50% to complete non-detection in juice conditions that included sulfhydryl and protein amino groups in the product matrix

(Pohland and Allen, 1970) (Ough and Corison, 1980) (Harwig et al., 1977).

The Aspergillus species is not only responsible for producing the patulin mycotoxin; Aspergillus niger has been reported to produce the toxic secondary metabolites fumonisin B2, B4, and B6 .Of these three toxins, B2 is observed most often during the occurrence of commodity contamination. Although much is known about this mycotoxin, the distinct purpose of its production by this fungus has yet to be elucidated

(Frisvad et al., 2007). This fungus and its toxins have been reported to contaminate commodities including: peanuts, onions, corn and corn-based foods, animal feed, beer, coffee, grapes, and wine (Noomin et al.,2009) (Logreico et al., 2013)(Scott et al., 1995)

(Mogensen et al. 2010). Furthermore, chronic exposure to this toxin has caused spontaneous disease in pigs and horses. Equine leukoencephalomalacia and porcine pulmonary edema are diseases caused by FB2 contaminated feed consumption, targeting the brain matter of horses and the lungs of pigs, with cardiotoxicity serving as a precursor (Smith et al., 1999). The International Agency for Research on Cancer classified the B analog of fumonisin within class 2B as “possibly carcinogenic to humans”. In 2001, the Joint FAO/WHO Expert Committee of Food Additives recommended that the provisional maximal tolerable daily intake of total fumonisin,

B1+B2+B3, alone or in combination be set at 2µg/kg body weight (Bolger et al., 2001).

The presence of A. niger within vineyards has been documented for some time, however it was overlooked as researchers focused their efforts towards other

142

Aspergillus species and toxins such as ochratoxin A. The A. niger “aggregate” with regard to food processing was in the past given GRAS (generally regarded as safe) status for its production of citric acid (Visconti et al. 2008). It has been well-established that A. niger is one of the most frequently isolated fungi from grapes within wine- producing vineyards in the E.U. These strains have been confirmed as producers of fumonisin and efforts have been made to reduce contamination levels within wines

(Perrone et al., 2007).

Within the southeastern region of the United States the muscadine grape, Vitis rotundifolia, have gained considerable popularity. This popularity is in part due to the consistent yields of cultivars used in wine and juice-making, such as Noble and Carlos.

Fresh fruit cultivars such as Doreen, Ison, and Fry have also enjoyed a strong market presence among consumers. Many of these cultivars are known for their high resistance to plant diseases that would stunt the yields and quality of vinifera grapes. Muscadine grapes are known to have thicker skins, large seeds, and a pulp that is characterized as soft, with a musky flavor. Berries vary in size ranging from ¼- 1 ½ inches in diameter and 4-15 grams in weight, containing oblong seeds. Grape cultivars vary over a range of colors from white, also known as bronze, to pink, red, blue, nearly black, and purple

(Olein, 2001). The overall phenolic and resveratrol concentrations of muscadine grapes was found to be higher than bunch and Florida hybrid bunch grapes. Resveratrol has been associated with anticancer properties against blood, colon, and prostate cancers

(Basha et al., 2004) (Mertens-Talcott and Percival, 2005). These grapes are grown in a region that is bordered by the Gulf of Mexico to the south and the Atlantic Ocean to the east, providing humidity that when paired with warm temperatures creates an

143

environment very similar to those observed in the European Union that favored the cultivation of A. niger in wine grape producing vineyards. It has been established that muscadine grapes have ability to support Aspergillus fungal growth and toxin production. Now, the question posed is will the fumonisin B2 toxin react as patulin was observed to in apple juice, degrading over time and in the presence of sulfhydryl groups. Currently, there is no FDA policy governing the mycotoxin contamination concentrations within grape juice. The objective of this study is to determine whether the fumonisin B2 toxin is affected by varying storage conditions over a six month period.

Methods and Materials

Safety

Chemicals

All solvents used were high performance liquid chromatography (HPLC)-grade and purchased from Fisher Scientific (Waltham, MA). Water was purified by the Milli-Q system (Millipore, Bedford, MA, USA).

Juice Pasteurization and Sample Preparation

Single strength Carlos grape juice with 50 ppm potassium sulfite was used during this study. The juice samples were batch pasteurized according to the U.S. FDA

144

Guidance for Industry: Juice HACCP Hazards and Controls Guidance (2004) inside a

Labgard Class II, Type A/B3 laminar flow biosafety cabinet (Plymouth, MN, USA)

(Figure 4-1). 1000 mL media bottles were used as pasteurization vessels, and were filled then placed in water baths. Heat was applied to the water baths once all vessels were filled, and the temperature of the baths and the juice were both monitored with thermometers. As the process commenced the water was covered, taking care not to let the temperature of the water bath exceed 79-112°C. When the temperature of the juice reached 71.1°C for 6 sec, the juice samples were removed from the heat, covered, and allowed to cool.

Pasteurized and non-pasteurized juice was divided and inoculated at three concentration levels of FB2: 0, 350 or 530 µg/L. All three treatment levels were stored under three conditions: -20°C, 3°C, and 20°C over a six month period in the dark.

During that period, 10 mL sample aliquots were removed each month and underwent

SPE extraction. The aliquot was passed through a 30mg/3mL Phenomenex StrataX-

C33u Polymeric Strong Cation solid phase extraction column (Torrance, CA, USA). The column was previously conditioned with 1 mL methanol and 1mL water. To further acidify the column, 1 mL water with 2% formic acid was added. Afterward, the column was washed with 1 mL methanol and the fumonisins were eluted in 1 mL methanol/water (1:4) with 2% NH4 OH (v:v). Toxin recovery rates observed by

145

capacity of this column was also evaluated over a range from 0.1-1.500 µg/mL FB2 by the Mogenson group, and demonstrated a linear relationship with regard to the concentrations with no signs of reaching a maximal capacity. Extracts were finally analyzed using HPLC-MS/MS to determine if concentrations of FB2 remained at initial level.

LC-MS/MS Conditions

All samples were analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/MS/MS) via the Agilent 1100 HPLC (Santa

Clara, CA, USA) connected to a Thermo Scientific LCQ Deca Ion Trap mass spectrometer (Waltham, MA, USA). Separations were conducted using an Agilent

Zorbax SB C18 reversed phase column (2.1x 30mm; particle size 3.5µm) at a flow rate of 0.2 mL/min. A linear gradient program was used for the extract separation consisting of two mobile phases. Mobile phase A consisted of 0.1% formic acid in water (v:v), and mobile phase B was a solution of acetonitrile/methanol (80:20, v:v) and 0.1% formic acid (v:v). The gradient program ran for a total of 20 mins as follows: 30-100%B (8 mins), 100%B (3 mins), 100-30% B (9 mins). Mass spectrometer acquisition was operated in positive ionization and multi reaction monitoring mode with nitrogen (N2) as the nebulizer gas with a source flow of 9 L/min at 350°C. The parameters for the

MS/MS were: Capillary Temp-250.0 °C, Sheath Gas Flow-60, source Voltage- 4.00 kV,

Capillary voltage 29.00V,Full MS Target- 5e +007, SIM Target 2e +007, MSN Target-

5e +007, Zoom Target 1e+007, Isolation width- 5.0, Collison energy- 37.5%, Activation time 30.00, Q-0.250, and 105-800 m/z scanning range. Sample retention time was approximately 5-6 min. for the contaminated grape juice samples. Detection was based on LC-MS screening analysis with narrow ion traces around the protonated mass for

146

FB2 with a mass to charge ratio (m/z) of 706.40, and created daughter ions at m/z

336.13 and 512.20. A 20µL aliquot was injected for each sample. The calibration curve showed good linearity in the range of 0- 5050 µg/mL, with R2 of 0.977. All chromatographic points on the calibration curve were run in triplicate, leading to a relative standard deviation less than 5%. A certified external standard of FB2 (51.4

µg/mL) (Romer Labs, Union, MO, USA) was used to construct the calibration curve. An approximate detection limit of 4.8 µg/mL fumonisin B2 and quantification limit of 14.7

µg/mL was calculated on the basis of blank sample response.

Results and Discussion

The fungal toxin patulin is widely known to contaminate fresh apples and apple- based products through colonization by Penicillium, Aspergillus, and Byssochlamys spp.

Once the products, beverages in particular, are infused with this fungal secondary metabolite, its stability is finite. This was illustrated by Pohland and Allen (1970), who reported that after adding 8 mg/L patulin to commercial apple juice, the toxin’s stability only lasted for the first ten days of the evaluation. Scott and Somers (1968) found that the addition of 4 mg/L patulin was moderately stable in both grape and apple juices with a 15-75% reduction after heating at 80°C for 10 min and 50-80% reduction after five weeks. This same research group also noted that patulin was found to be even less stable in orange juice. In instances where patulin contaminated apple juice was subjected to extreme conditions such as during the study by Kryger (2001), the toxin concentration was reduced by 33% after heating at 100°C for 3 hours. The product quality attributes were not described after the prolonged treatment, but it is likely that these attributes would be wholly unfavorable to consumers after such a treatment. The additions of ascorbic acid at 500 ppm and sulfur dioxide at 100 ppm were also found to

147

decrease toxin concentrations by 25%, according to Aytaç and Acar (1992). Although the mechanism by which patulin degrades is not completely clear, it is hypothesized that the metal-catalyzed oxidation of ascorbate or ascorbic acid occurs. Singlet oxygen or free radicals generated by these reactions may attack the conjugated double bonds of patulin, changing its structure and accounting for its concentration decrease (Brackett and Marth, 1979). Samples of patulin contaminated apple cider with the addition of 5 mg/mL activated charcoal reduced patulin levels from 30 ppm initially, to non-detectable levels (Sands et al., 1976). Hartwig et al. (1977), proposed that patulin’s reaction with sulfhydryl groups and the amine groups of proteins and other molecules could be a link to the breakdown mechanism. In conjunction with these additions, the alcoholic fermentation of apple cider reduced the levels of patulin by 99% (Stinson et al., 1978).

During fermentation, patulin has been observed to be converted to ascladiol by top- fermenting yeast such as Saccharomyces cervisea (Moss and Long, 2002). This conversion product is noted to have one-quarter the acute toxicity of patulin (Suzuki et al., 1971). Similar results were obtained by Inoue et al. (2013) when the research group found that patulin demonstrated a substantial decline in concentration to nearly undetectable levels over 18 days of beer fermentation. The decline in concentration was attributed to enzymatic degradation which left the toxin being reduced to less than 10% of its initial concentration within 48 hours and complete decomposition within 4 days.

According to Ekşi and Koca (2004), during storage studies of patulin contaminated apple juice concentrates at 64, 105, and 146 ppb by the fourth month of the study all samples had undetectable concentrations of the toxin. Each concentration level was stored at 22 and 30°C, and after the first month of storage, toxin reductions were

148

between 45-64% and 66-86% respectively. During the second month of storage, patulin reductions were approximately 75-86% at 22°C and 78-100% at 30°C. Although the exact mechanism for the decrease in patulin stability is unknown. Researchers such as

Linroth (1980), hypothesize that the metal-catalyzed oxidation of ascorbate or ascorbic acid may occur. The free radicals generated from the aforementioned reactions could possibly attack the conjugated double bonds of patulin, leading to decomposition and accounting for the structural changes with concentration reductions. The reduction of this toxin in apple products seems to be a positive outcome, however, when posed with the decision of whether to discard contaminated products or let the toxin “self-degrade” within contaminated product meant for the market, this situation could give way to unscrupulous manufacturers. The detoxification or “disappearance” of patulin within the beverage matrix during storage periods raises the question of whether other toxins may be affected by a similar time –dependent mechanism of degradation.

The fungal secondary metabolite fumonisin B2, has slowly become associated with the contamination of products produced with grapes, and dried vine fruit. This relationship between the toxin and vineyards has become highly established due its production by multiple strains of Aspergillus niger isolated from vineyards. Vineyards within the southeastern U.S. that produce muscadine grapes experience similar climatic conditions to those witnessed by vineyards in the E.U. where these fumonisin-producing fungi are found. Sharing this common attribute, it is expected that vineyards within this region may also have detectable levels of mycotoxin contamination within products derived from these growing areas. The stability of this mycotoxin and its propensity for patulin-like degradation within the juice matrix has yet to be explored. To elucidate this

149

situation, samples of the FB2 contaminated pasteurized and non-pasteurized Carlos muscadine juice were analyzed over a six month period to determine its stability within the juice matrix at -20°, 3°, and 20° C.

Both pasteurized and non-pasteurized grape juice samples contained a sulfite addition of 50 ppm. This addition was to stabilize juice color, and reduce any browning that may occur within the juice samples, thereby removing the possible interference.

The sulfite treatment was added per industry practice before learning of the effects of sulfhydryl groups in the degradation of patulin. All samples were subjected to solid phase extraction, and analyzed via LC-MS/MS, using an external FB2 standard for quantification purposes. FB2 was identified via a retention time between 6-7 min, and the mass of its daughter ions. Sample aliquots for analysis were taken from the larger batch of juice in the case of room temperature (20°C) and refrigerated juice (3°C), frozen samples (-18°C) were prepared and stored individually in 10 mL aliquots to remove any freeze/thaw interactions that may occur. Storage of grape juice at 20°C is not ideal or a standard practice of the juice-industry, but was included for a more well- rounded and robust study. Both pasteurized and non-pasteurized juice were spiked at the 350 and 530 ppb levels. Although grape juice samples were subjected to an FDA pasteurization protocol, it was hypothesized that this step would have little to no effect on FB2 concentrations. The purpose of pasteurization with regard to fruit juices is to achieve a five-log reduction for oocysts of Cryptosporidium parvum, which are more heat resistant than E.coli O157:H7 and controls bacterial pathogens (FDA, 2004). This hypothesis was proven to be correct, after the first month of storage, the only decrease in toxin retention was observed in the pasteurized 3°C sample at approximately 3%

150

(Figure 4-3), however it is doubtful that this reduction can be attributed to pasteurization since it was not observed consistently over all samples. The stability of the fumonisin B2 toxin and lack of degradation in Carlos juice is illustrated by the mean concentration levels over the six month storage period (Tables 4-1 – 4-4). Pasteurized juice samples were observed to have higher toxin retention percentages (85-99%), when compared to non-pasteurized (85-64%) samples over the six month storage period (Figures 4-3 and

4-2). The highest levels of toxin retention were demonstrated in the pasteurized, frozen

530ppb samples at 98.6% (Figure 4-4). This finding was quite counter-intuitive since pasteurization is a method employed to degrade pathogenic organisms. It is possible that the heat-treatment removed any competing compounds or organisms within the samples, subsequently creating an environment for the mycotoxin to survive solely within the juice matrix. Pasteurization temperatures are typically far too low to have an adverse effect on mycotoxin concentrations as was demonstrated by the pasteurization of FB2 spiked milk. After pasteurization, FB2 recoveries of the spiked milk samples ranged from 82.4-85.9% (Maragos and Richard, 1994). According to Jackson et al.

(1996), FB2 decomposition begins at or above 150°C, with 90% FB2 loss at temperatures >175°C. At both concentrations within this study, FB2 levels were noted to be reduced over the six month period. Other than the reduced toxin concetrations, indicators of FB2 hydolysis products from degradation were noted in sample chromatograms. As a result of fragmentation, the product ions were identified at m/z

372 and 301. Although the mechanism for the fragmentation is unknown, Bartók et al.

(2006) identified the following scheme for the fragmentation: TCA (tricarballylic acid) is gradually eliminated, water is relased with the formation of its corresponding anhydride,

151

TCA-anhydride, followed by its elimination; the splitting-off of TCA in the form of its ketene and water, yielding fumaric and acetic acids (Figure 4-19). However, toxin retention percentages demonstrated the overall stability of the fumonisin B2 toxin in muscadine grape juice. The mean retention percentage for pasteurized samples at the

350 and 530ppb level was between 96.3-93% and 96-100%, respectively. For non- pasteurized samples, retention percentages for the 350 and 530ppb concentrations was

89.3-93.0% and 77.6-93.4%, respectively. These mean values serve as a prime indication of the stability of FB2 in white muscadine grape juice. The greatest toxin reductions were observed in 20°C non-pasteurized samples at the 350 and 530 ppb spike-levels at 17 (Figure 4-5) and 36% (Figure 4-6), respectively. As mentioned previously, all juice samples were dosed with a sulfite addition of 50ppm. However, this addition did not have the same degradative effect that was demonstrated in juice samples contaminated with patulin. Similar retention of FB2 in the liquid matrix was reported by Visconti et al. (1994), where they evaluated FB1 and FB2 stability in a 1:1 acetonitrile :water solution with 2% toxin loss in pure solution. Overall, toxin degradation in grape juice samples was consistent and gradual during the six month evaluation period. Maragos and Richard (1996) reported similar observations when evaluating the storage stability of fumonisin in spiked whole milk at 50 ng/mL for an 11 day period.

The slope produced by the toxin retention data provided a strong context for the rates of sample degradation observed, yielding an inverse relationship between the slope and observed retention rates. Non pasteurized 530 ppb juice stored at 20°C experienced the sharpest decline in mycotoxin degradation (Figure 4-5) which was

152

attributed to its warmer storage temperature, and increased fermentation effects.

Pasteurized 350 ppb (Figure 4-3) and non-pasteurized 530 ppb juice (Figure 4-6) both displayed the highest toxin retention rates for the frozen (-20°C) juice samples and the lowest rates of retention in samples stored at room temperature (20°C). Non pasteurized 350 ppb juice samples stored at refrigeration and freezer temperatures exhibited similar rates of toxin retention; however neither sample’s retention percentage dropped below 85% over the six month study period (Figure 4-5). Pasteurized 3°C samples spiked at the 530ppb level were observed to have the lowest toxin retention rate among the treatments at this concentration level (Figure 4-4). This observation was unexpected due to the refrigerated temperature this sample was held at. The lower temperatures were hypothesized to aid in the stabilization of the mycotoxin since heat is known to intensify the degradation of most compounds. Under these sustained storage conditions for the six month period, attributes such as flavor quality would be expected to decline, hence this condition in particular is thought of as being non-ideal for juice storage. Room temperature juice spiked at 350 ppb FB2 was noted to exhibit a possible increase in measured toxin concentration between months 5 and 6 (Figure 4-5). When the standard deviation (±8.2 ppb) of the mean is taken into consideration the lower concentration reduces the apparent toxin increase.

Conclusion

From this study, it has been noted that the pasteurization protocol to which fruit juices in the U.S. are subjected has minimal effect on fungal toxin stability or degradation as previously believed. The six month storage of mycotoxin contaminated

Carlos grape juice at three different temperatures, has demonstrated that none of the storage conditions could replicate the toxin degradation demonstrated by patulin-

153

contaminated apple juice. The addition of sulfites to the Carlos juice did not assist or intensify FB2 degradation either. Previous studies have used other liquid matrices such as milk and acetonitrile/water solutions to examine fumonisin stability. When introduced to muscadine grape juice, fumonisin B2 was observed to be a stable contaminant showing no real signs of degrading to safe levels. It could be argued that if stored for longer periods the toxin may degrade to safe levels, however storage times longer than six months are far from practical with regard to industry operations. Additionally, in the context of validation of preventive controls for human food (FDA, 2015) this would be a tenuous means of enhancing food safety.

154

Figure 4-1. Pasteurization apparatus 1 for Carlos grape juice processing Photo courtesy of author

Figure 4-2. Pasteurization apparatus 2 for Carlos grape juice processing Photo courtesy of author

155

Table 4-1. Mean fumonisin B2 content of pasteurized Carlos grape juice over six month period storage in three different storage environments after 350 ppb artificial inoculation Storage Period (Month) Storage Temp °C 1* 2* 3* 4* 5* 6* -20 359.8 ±4.68† 336.9 ±7.06 334.7 ±7.41 331 ±6.2 331 ±2.68 330.9 ±4.68 3 340.9 ±3.79 340.3 ± 4.63 333.7 ±4.25 314.3 ±2.92 308.3 ±1.75 299 ±3.55 20 366.4 ± 3.14 341.7 ± 6.47 319.3 ±5.19 311.3 ±3.11 308.9 ±4.6 301.2 ±3.12 *=parts per billion †= standard deviation

Table 4-2. Mean fumonisin B2 content of pasteurized Carlos grape juice over six month period in three different storage environments after 530 ppb artificial inoculation Storage Period (Month) Storage Temp°C 1* 2* 3* 4* 5* 6* -20 536.1 ±9.93† 536.8 ±38.4 536.7 ±13.65 536.5 ±8.27 529.6 ±5.36 524.4 ± 5.27 3 538.0 ±9.53 520.8 ±1.13 503.1 ±7.66 490.3 ±1.20 488.2 ±7.66 484.9 ±1.20 20 532.0 ±8.14 514.8 ±8.86 514.3 ±7.11 498.9 ±11.8 497.1 ± 1.82 494.6 ±5.67 *=parts per billion †= standard deviation

156

Table 4-3. Mean fumonisin B2 content of non-pasteurized Carlos grape juice over six month period in three different storage environments aftert 350 ppb artificial inoculation Storage Period (Month) Storage Temp°C 1* 2* 3* 4* 5* 6* -20 360.30 ±3.63† 341.3 ±6.60 327.7 ±4.13 320.0 ±20.1 301.6 ±0.32 300.1 ±1.34 3 346.4 ±1.59 326.5 ±4.78 310.8 ±6.59 298.4 ±3.34 294.30 ±2.80 298.7 ±6.54 20 354.3 ±2.79 342.9 ±9.99 334.1 ±13.04 320.9 ±10.89 291.2 ±0.48 297.8 ±8.2 *=parts per billion †=standard deviation

Table 4-4. Mean fumonisin B2 content of non-pasteurized Carlos grape juice over six month period in three different storage environemnts after 530 ppb artificial inoculation Storage Period (Month)

Storage Temp°C 1* 2* 3* 4* 5* 6*

-20 535.0 ±2.42† 531.6 ±3.12 510.2 ±6.42 495.2 ±1.51 494.1 ±3.54 491.8 ±14.15

3 522.5 ±31.8 508.5 ±10.13 501.6 ±5.96 497.5 ±16.31 471.7 ±63.0 468.6 ±12.9

20 511.3 ±3.5 454.8 ±16.05 408.4 ±9.23 397.0 ±6.25 357.7 ±9.49 340.8 ±8.82 *=parts per billion †=standard deviation

157

110

100 RETENTION % RETENTION

2 2 90 FB

80 0 1 2 3 4 5 6 7 MONTH

20C 3C -20C

Figure 4-3. Mean fumonisin B2 retention percentages in pasteurized Carlos grape juice over six months after storage in three different temperaturesfollowing 350 ppb artificial inoculation

102

100

94 FB2 RETENTION %FB2

90 0 1 2 3 4 5 6 7 MONTH

20 3 -20

Figure 4-4. Mean fumonisin B2 retention percentages in pasteurized Carlos grape juice over six months after storage in three different temperatures following 530 ppb artificial inoculation

158

110

100

90 FB2 RETENTION % FB2

80 0 1 2 3 4 5 6 7 MONTH

20 3 -20

Figure 4-5. Mean fumonisin B2 retention percentages in non-pasteurized Carlos grape juice over six months after storage in three different temperatures following 350 ppb spike level artificial inoculation

110

100

80 RETENTION % RETENTION

2 70 FB

50 0 1 2 3 4 5 6 7 MONTH

20 3 -20

Figure 4-6. Mean fumonisin B2 retention percentages in non-pasteurized Carlos grape juice over six months after storage in three different temperatures following 530 ppb artificial inoculation

159

RT: 0.00 - 19.99 6.70 NL: 100 9.68E5 95 TIC MS NPastRT10 90 0A_6_6 85 80 75 70 65 60 55 50 45 40

RelativeAbundance 35 30 25 20 13.42 15 12.33 13.33 13.56 13.75 10 11.58 11.29 13.89 5 0.72 10.06 14.27 0.83 1.99 2.91 3.76 4.23 5.04 5.82 8.19 8.55 9.83 15.34 16.00 17.47 18.27 19.19 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (min) Figure 4-7. Non-pasteurized (20°C) 530 ppb month 6 chromatogram

NPastRT100A_6_6 #283 RT: 6.68 AV: 1 NL: 1.76E5 T: + c ESI Full ms2 [email protected] [190.00-1000.00] 688.29 100 95 90 85 80 75 70 65 512.30 60 55 50 530.26 45 40

RelativeAbundance 35 30 25 670.32 20 336.34 354.29 15 494.20 10 548.34 318.56 5 372.34 219.36 246.97 301.22 476.30 622.58 651.62 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z Figure 4-8. Non-pasteurized (20°C) 530 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28

160

RT: 0.00 - 19.98 6.70 NL: 100 8.89E5 95 TIC MS NPastFree 90 50A_6_6 85 80 75 70 65 60 55 50 45 40

RelativeAbundance 35 30 25 20 12.28 12.92 13.34 13.41 15 11.57 13.67 10 0.72 11.21 13.84 5 0.79 9.87 11.02 14.05 1.07 3.34 3.48 4.19 4.95 5.87 6.53 7.55 8.64 9.13 15.90 16.42 17.83 19.13 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (min) Figure 4-9. Non-pasteurized (-20°C) 530 ppb month 6 chromatogram

NPastFree50A_6_6 #284 RT: 6.70 AV: 1 NL: 2.39E5 T: + c ESI Full ms2 [email protected] [190.00-1000.00] 688.28 100 95 90 85 80 75 70 65 60 512.33 55 50 45 40 530.24

RelativeAbundance 35 30 25 670.34 20 336.33 15 354.32 494.27 10 548.26 5 318.27 372.21 428.10 600.05 652.25 704.61 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z Figure 4-10. Non-pasteurized (-20°C) 530 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28

161

RT: 0.00 - 19.98 6.65 NL: 100 6.50E5 95 TIC MS NPastFri10 90 0A_6_6 85 80 75 70 65 60 55 50 45 40

RelativeAbundance 35 30

25 12.25 12.32 13.58 20 11.63 15 13.79 11.47 10 11.04 13.93 0.60 10.52 0.71 9.34 14.19 5 2.11 3.03 4.03 5.54 6.36 7.03 8.59 14.85 16.77 17.48 18.16 18.28 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (min) Figure 4-11. Non-pasteurized (3°C) 530 ppb month 6 chromatogram

NPastFri100A_6_6 #282 RT: 6.65 AV: 1 NL: 1.75E5 T: + c ESI Full ms2 [email protected] [190.00-1000.00] 688.22 100 95 90 85 80 75 70 65 60 55 512.27 50 45 530.22 40

RelativeAbundance 35 30 670.28 25 20 15 336.35 10 494.24 548.25 5 318.36 219.07 273.44 372.12 477.28 592.78 615.14 742.30 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z Figure 4-12. Non pasteurized (3°C) 530 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28

162

RT: 0.00 - 19.99 6.67 NL: 100 9.76E5 95 TIC MS PastFree50 90 A_6_6 85 80 75 70 65 60 55 50 45 40

RelativeAbundance 35 30 25 20 12.24 15 0.57 11.61 13.57 13.69 10 0.74 11.49 11.01 13.92 5 9.46 9.86 1.00 1.61 2.63 3.77 4.55 5.49 6.46 8.21 8.80 15.31 16.52 17.27 17.91 19.12 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (min)

Figure 4-13.Pasteurized (-20°C) 350 ppb month 6 chromatogram

PastFree50A_6_6 #282 RT: 6.65 AV: 1 NL: 2.00E5 T: + c ESI Full ms2 [email protected] [190.00-1000.00] 688.24 100 95 90 85 80 75 70 65 512.26 60 55 50 45 530.26 40

RelativeAbundance 35 30 670.31 25 20 336.34 15 354.29 494.23 10 548.20 5 318.32 372.28 205.15 291.32 439.41 619.29 660.60 705.17 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z Figure 4-14. Pasteurized (-20°C) 350 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28

163

RT: 0.00 - 19.98 6.66 NL: 100 6.55E5 95 TIC MS PastFri50A 90 _6_6 85 80 75 70 65 60 55 50 45 40

RelativeAbundance 35 30

25 11.58 12.98 13.43 13.57 20 13.66 15 0.57 11.46 13.81 10 0.74 11.04 0.79 10.40 13.95 8.74 9.07 5 1.38 2.18 3.50 4.33 5.93 6.50 7.09 14.28 15.34 16.67 17.69 19.03 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (min) Figure 4-15. Pasteurized (3C) 350 ppb month 6 chromatogram

PastFri50A_6_6 #283 RT: 6.66 AV: 1 NL: 1.68E5 T: + c ESI Full ms2 [email protected] [190.00-1000.00] 688.25 100 95 90 85 80 75 70 65 60 512.25 55 50 530.21 45 40

RelativeAbundance 35 30 670.23

25 336.25 20

15 354.25 494.30 10 548.29 5 318.34 372.35 247.37 450.64 476.35 554.70 630.40 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z Figure 4-16. Pasteurized (3°C) 350 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28

164

RT: 0.00 - 20.00 6.64 NL: 100 7.62E5 95 TIC MS PastRT50B 90 _6_6 85 80 75 70 65 60 55 50 45 40

RelativeAbundance 35 30 25 12.24 12.88 20 11.60 13.35 15 0.58 11.51 13.83 10 0.72 13.95 6.80 10.94 5 0.77 8.60 10.40 14.42 1.24 2.35 3.56 3.92 4.91 6.16 6.35 6.94 7.68 14.98 16.19 17.54 18.37 19.95 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (min) Figure 4-17. Pasteurized (20°C) 350 ppb month 6 chromatogram

PastRT50B_6_6 #281 RT: 6.64 AV: 1 NL: 1.94E5 T: + c ESI Full ms2 [email protected] [190.00-1000.00] 688.26 100 95 90 85 80 75 70 65 512.27 60 55 50 530.25 45 40

RelativeAbundance 35 30 670.31 25 20 336.33 15 354.31 494.29 10 548.33 5 318.47 301.24 372.30 437.21 652.35 706.50 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z Figure 4-18. Pasteurized (20°C) 350 ppb month 6 total ion chromatogram, FB2 daughter ions 336.23 and 512.28

165

Tricarballylic acid (TCA) (176 Da) Acetic acid (60Da)

Ketene form of tricarballylic acid (158 Da)

Tricarballylic anhydride (158 Da) Fumaric acid (116 Da)

Figure 4-19. Structures of compounds formed during fumonisin B2 degradation

166

CHAPTER 5 ARE COMMERCIALLY AVAILABLE MUSCADINE WINES A THREAT TO HUMANS?

The Aspergillus genus has demonstrated both beneficial and detrimental characteristics within human society. The beneficial nature of this fungus is demonstrated during its production or over-production of citric acid (Moyer, 1953), fermentation of Puer tea (Mo et al., 2005), and extracellular enzyme production (Wösten et al., 2007). Aspergillus is currently considered one of the most economically important genera of micro fungi due to its collection of value-added traits. However, Aspergillus spp. are known to form mycotoxins of public health significance, including ochratoxins and fumonisins. The substrate for FB2 contamination is wide-ranging, including corn and corn-based products (Marasas, 2001), beer (Shephard et al., 2005) and black tea leaves

(Martins et al., 2001). According to previous work from Logrieco (2010), and Mogensen

(2010), it has been established that FB2 produced by A. niger is a wine contaminant of

Vitis vinifera wines in Europe. Based on this observation, we found it justifiable to assess possible contamination levels and its prevalence in Vitis rotundifolia wines.

Currently within the United States, there have been no published reports with regard to mycotoxin contamination within beverages, particularly wines. Many of the studies detailing this type of contamination are generated outside of the U.S., in wine- making regions with a climate and overall growing environment very similar to that of the southeastern United States.

Within the southeastern region of the United States, Vitis rotundifolia Michx., or the muscadine grape as it is commonly known, has been cultivated as a native grape for centuries (Andersen et al., 2003). This grape species is well-suited to the heat and humidity of a region that typically favors plant disease development. Furthermore, it

167

withstands Pierce’s disease, a grape disease that has devastated the table wine grape

Vitis vinifera (Gray, 2008), which is also susceptible to anthracnose and downy mildew when grown in the southeastern United States (Andersen et al., 2003).

During cultivation, muscadines are susceptible to colonization by black aspergilli such as Aspergillus carbonarius, A. tubingensis, and A. niger, common fungal pathogens responsible for the postharvest decay of many types of fruit (Pitt and

Hocking, 1997). These Aspergillus species are opportunistic pathogens ubiquitous in vineyards and are known to cause grape berry and bunch rot (Varga et al., 2004).

According to Zimmerli and Dick (1996), grapes and associated products contaminated by these fungal species also exhibited concentrations of Ochratoxin A (OTA) between

11-296 pg/mL. OTA has been shown to be nephrotoxic, immunotoxic and teratogenic in animals; causing liver and kidney tumors in mice. Further exploration of A. niger contaminated commodities i.e. coffee beans and raisins, led to the discovery of

fumonisin B2 and fumonisin B4 contamination of these products (Mogensen et al., 2009).

FB2 is considered to be a Group 2B carcinogen (possibly carcinogenic in humans) by the International Agency for Research on Cancer (IARC, 2002) and has been associated with an increased risk of esophageal cancer in the former Transkei region of

South Africa and China, and possible neural tube defects. (Lerda et al., 2005) (Stack,

1998). Once harvested, muscadines are used to produce many value-added products including wine, juice, vinegar, jellies, and jams. Wine and juice are products of concern due to the risk that there is a chance of high fumonisin contamination.

Although previous studies have cited the discovery of fumonisin contaminated wines in the E.U. The U.S. Food and Drug Administration has no legal limit set for total

168

fumonisin contamination (FB1+FB2+FB3) for beverages. However, contamination limits have been set for corn products for human consumption such as corn meal (2ppm), popcorn (3 ppm), and cleaned corn for masa (4ppm) (FDA, 2001). The threat of FB2 contamination in grape products is not one that has been thoroughly investigated within the Southeastern United States, although major risks factors leading to fungal growth such as moisture, temperature, and high soluble solids content are prevalent in this geographical area. The objective of this experiment was to evaluate the fumonisin B2 contamination levels in commercially-available muscadine wine and juice products and to determine if these concentrations potentially pose a risk to human health and /or require further research.

Materials and Methods

Safety

Wine Samples

Sixty-two bottled wines (22red, 32 white, 7 blush, 1 sparkling) and six bottled juice samples were randomly purchased from grocery and liquor retailers in Florida,

Georgia, Alabama, North Carolina, Texas, and Louisiana. The muscadine varietals

169

included in these samples were Carlos, Magnolia, Scuppernong, Noble, Hunt, and Ison.

Blanc du bois wine samples were also included in the analyzed samples due to their hardiness against Pierce’s disease and cultivation within southeastern states.

Statistical Analysis

All statistical analysis for this project was performed using SAS 9.4 (Cary, NC,

USA).

Chemicals

All solvents used were high performance liquid chromatography (HPLC)-grade and purchased from Fisher Scientific (Waltham, MA, USA). Water was purified on a

Milli-Q system (Millipore, Bedford, MA, USA). Fumonisin B2 standards were purchased from Romer Labs (Union, MO, USA) and Neogen Corp (Lansing, MI, USA) at the following concentrations: 0, 50, 100, 300, 600 ppb. These standards were a part of the

Veratox competitive direct ELISA Immunoassay kit for total fumonisin, which also included: enzyme-labeled fumonisin conjugate, substrate, stop solution, mixing wells, and antibody wells. 2M tris buffer was used for sample pH adjustment. Clarification of wine samples was obtained via use of a proprietary clarification compound provided by

Neogen Corp.

Sample Preparation

A 5 mL aliquot of each wine sample was removed and diluted with 15 mL of methanol. Diluted samples were then mixed by inversion. Each sample was pH adjusted to 6.5-7.0 drop wise with 2M tris buffer, and gently shaken to ensure proper mixing. 20 mL of clarification compound was added to the solution, and vortexed for one min. The samples were then centrifuged at 1000g for 2 min using an Allegra X-15R,

Beckman Coulter (Indianapolis, IN, USA) to pellet the clarification compound. Finally,

170

the aliquots were decanted and filtered through a Neogen Corp. filtering syringe, into amber vials and used for final fumonisin B2 analysis.

Fumonisin Analysis

All ELISA reagents and FB2 controls were used at room temperature. After preparation, samples and controls were subjected to the addition of 100µL of conjugate to each 100µL diluted aliquot in ELISA mixing wells. Samples were then mixed and

100µL was transferred to antibody wells and allowed to incubate at room temp for 10 min. After incubation completion, sample wells were rinsed five times with deionized water, wells were tapped on absorbent paper to ensure dryness. 100µL of substrate was then added to the antibody wells via 12 channel pipettor and allowed to incubate for

10 min. To bring the incubation to an end, 100µL of stop solution was added to the test wells. All wells were analyzed for optical densities via the Neogen Stat Fax 321 Plus

Microwell Strip Reader (Lansing, MI, USA) with a 650 nm filter. The optical densities of the controls formed the standard curve, and the sample optical densities were plotted against the curve to calculate exact contamination concentrations. Wine sample analysis was replicated three times from three independent aliquots. Sample run results were rejected if the correlation coefficient of the standard curve was <0.98. The limit of detection of the ELISA assay was 50 ppb with a range of quantification at 50-600 ppb.

To extend the range of quantification of the assay, a stock solution of FB2 standard was diluted to a concentration of 960 ppb to capture highly contaminated samples within the standard curve for accurate quantification. The principle governing this assay pertains to the free fumonisin from the samples and controls being allowed to compete with enzyme-labeled fumonisin for the antibody binding sites. The addition of substrate, and its reaction with bound conjugate determines color change/development or optical

171

density levels. Samples within this study were validated for FB2 content via the previously described LC-MS/MS methodology. Spiked agar samples used for toxin recovery were also used to compare the LC-MS/MS and ELISA methods. The validated toxin concentrations were 0.96, 1.28, 2.57 µg/mL. Two of these samples are above the

LOQ for the ELISA kit, so sample dilution was performed to address this. The rates of toxin recovery for both methods when compared were found to be 82-86%.

Results and Discussion

From our trials, it was found that all sampled Blanc du Bois wine and muscadine wine/ juice samples (n= 68) contained measurable levels of the fumonisin B2 mycotoxin

(Table 5.2). According to a statistical analysis, significant differences (p<0.05) were found when comparing wine contamination levels from wines produced in each state.

North Carolina and Louisiana had the highest concentrations of contamination at 0.62

µg/mL and 0.52 µg/mL over multiple samples, respectively (Table 5.2). Georgia and

Florida were found to have the lowest contamination concentrations of the states surveyed with sample means of 0.25 µg/mL and 0.35 µg/mL over multiple samples, respectively (Table 5.2). The highest levels of contamination were noted in red wine samples across all sampled states (Table 5.1). Typical fungal colonization of plant substrate begins on the outermost plant regions, i.e. grape skins, so it stands to reason that red wines had higher contamination levels due to the common red wine vinification procedure of allowing grape juice to remain in contact with the pomace(skins, pulp, and seeds) for several days. This allowed contact time contributes directly to an environment favorable for A. niger growth (higher soluble solids content and higher water activity) and any pre-formed toxin. Although there is a pH difference between that of grape skins and juice, A. niger has been shown to grow over a pH range of 1.4-9.8

172

(Schuster et al., 2002). Furthermore, this technique is not administered in the making of white wines where there is minimal contact between grape skins and juice. Samples of

Blanc du Bois taken from North Carolina and Louisiana were found to have the highest contamination levels amongst all states, respectively (Table 5.1). With regard to juice contamination, white muscadine juice samples were found to have higher FB2 concentrations than red juice samples. The vintage of the various wines sampled was unavailable, hence making yearly production comparisons and correlations more difficult. This information could prove useful in elucidating the growing conditions of the tested wines and the contributions of climatic factors on fungal growth in concert with wine contamination. Across all states, no one grape cultivar stood out as the most highly contaminated, but higher concentrations were noted in wines that used red cultivars i.e. blush and red blends, with the only outlier being the white grape blended wines of Alabama. Comparing the grape cultivars amongst each other, contamination levels were found to be statistically different. However, due to the great variation of contamination levels within the samples, correlating these factors proved difficult.

The controls in the ELISA kit used for sample analysis in this study were combined fumonisins (FB1+FB2+FB3). Aspergillus niger has been cited in the production of fumonisins B4 and B6 (Mogensen et al., 2010) also, however we did not quantify these two toxins because they are produced in lesser quantities by A. niger than FB2. It is believed that the source of toxin concentrations found in the sampled wines were from Aspergillus spp. for several reasons. The Fusarium genus is not typically associated with grape or vineyard infection. Previous research from Falk et al., (1996), demonstrated that the direct inoculation of grape vines with Fusarium proliferatum as a

173

biocontrol against downy mildew did not present detectable concentrations of fumonisin contamination of grapes or produced juice. Furthermore, A. niger lack the FUM 12 gene which is directly related to the production of FB1 and FB3 via the encoding of monooxygenase P450, that catalyzes hydroxylation at the fumonisin backbone at

carbon 10, without this gene the fungus is left to produce FB2, FB4 and , FB6 (Proctor et al., 2006).

From this research study, we found that all of the collected wine and juice samples had some level of quantifiable fumonisin B2 contamination. These contamination levels varied by state and grape cultivar. With regard to human health for a 70 kg person, if we assume wine consumption of 300 ml (1-2 glasses) as recommended by the World Health Organization for a positive health response and take into account the average FB2 contamination level from this study of 0.4 mg/mL, FB2 exposure from wine consumption would be 0.12 mg/ 300mL of wine. This contamination level is below the guideline associated with the risk characterization by the WHO-FAO for average wine consumption. The joint expert committee for food and additives

(JECFA) has established a provisory maximum tolerable daily intake (PMTDI) of 2µg/kg of body weight based on the NOEL (no observed adverse effect level) of 0.2 mg/kg body weight per day and a safety factor of 100. When using the FDA guidelines as the standard for fumonisin contamination limits, all of the sampled beverages were well under these established limits for food for human consumption by at least one magnitude at 2-4ppm. Since maize and corn are the most common substrates for this type of mycotoxin contamination, pairing contaminated beverages such as these in a diet with high levels of contaminated maize, corn, or other fumonisin contaminated

174

foodstuff may be detrimental to human health. Although the U.S. is one of the largest producers of corn, 13.016 billion bushels in 2014 (Capehart,2013), the majority of this crop is used for ethanol and feed production with only 10-11% for human consumption, of which the majority is mainly processed foods that significantly reduces FB2 contamination levels in the diet (Siantar et al., 2008). Furthermore, the locales where the majority of these grape-based beverages are consumed the average diet consists of minimal levels of corn or maize negating or depressing the risk associated with the mycotoxin contamination.

Conclusion

From these tests, it can be suggested that the fermentation/ vinification process does not completely destroy the mycotoxin since it was found readily in such a high percentage of tested samples. A large percentage of the tested wines excluded a vintage which hindered any valid comparisons between the wines, including production year, climate, rainfall, and humidity levels within the vineyards. Information such as this may assist in drawing conclusions as to the higher contamination concentrations in some wines as opposed to others. It can also be presumed that some of the wines sampled may be blends from differing harvests or years as well, which would compound sample variation. One factor not explored, was the environment/facilities in which the wines were produced. It was noted by Zimmerli and Dick (1996), that poor winery sanitation may lead to mold growth in barrels and other equipment, thus yielding a contaminated final product. Further testing on the fate of fumonisin throughout the wine-making process would aid in the understanding of the behavior of this mycotoxin and efforts to lessen human exposure.

175

Although this study focused on V. rotundifolia-based wines and juices, contamination levels of V. vinifera grapes should also be investigated to ensure FB2 contamination levels are within thresholds deemed safe for the consuming public. This genus of grapes is also grown in areas where the elements for potential contamination are present.

176

Table 5-1. Meanfumonisin B2 concentrations in contaminated wine by grape cultivar (n=68) State Cultivar Product µg/mL Florida Carlos wine 0.39± 0.02 Florida Carlos wine 0.37± 0.10 Florida Rose wine 0.49± 0.08 Florida Noble wine 0.34± 0.02 Florida red wine 0.52± 0.13 Florida Noble wine 0.50± 0.05 Florida Magnolia wine 0.26± 0.02 Florida white juice 0.12± 0.03 Florida white wine 0.42± 0.01 Florida red wine 0.37± 0.01 Florida red wine 0.47± 0.02 Florida white wine 0.35± 0.31 Florida BDB wine 0.26± 0.08 Florida BDB wine 0.39± 0.07 Florida BDB wine 0.40± 0.03 Georgia white wine 0.15± 0.03 Georgia white wine 0.18± 0.07 Georgia white wine 0.33± 0.02 Georgia red wine 0.11± 0.02 Georgia red wine 0.42± 0.02 Georgia white juice 0.19± 0.03 Georgia Noble wine 0.38± 0.05 Georgia Carlos wine 0.35± 0.08 Georgia Magnolia wine 0.16± 0.02 Georgia red juice 0.14± 0.02 Georgia Noble wine 0.22± 0.03 Georgia white wine 0.37± 0.02 Georgia red juice 0.14± 0.05 Georgia white juice 0.20± 0.03 Georgia red wine 0.40± 0.01 Alabama Carlos wine 0.26± 0.02 Alabama Noble wine 0.26± 0.00 Alabama Magnolia wine 0.46± 0.06 Alabama Blush wine 0.31± 0.00 Alabama red wine 0.54± 0.06 Alabama white wine 0.75± 0.15 Alabama Blush wine 0.44± 0.00 Alabama Blush wine 0.41± 0.03 Alabama Blush wine 0.41± 0.06 Alabama white wine 0.45± 0.03

177

Table 5-1. continued State Cultivar Product µg/mL Alabama white wine 0.40± 0.02 N. Carolina red wine 0.50± 0.02 N. Carolina red juice 0.19± 0.03 N. Carolina Hunt wine 0.57± 0.02 N. Carolina Hunt wine 0.56± 0.00 N. Carolina Magnolia wine 0.61± 0.02 N. Carolina Carlos wine 0.61± 0.26 N. Carolina BDB wine 0.55± 0.05 N. Carolina Scuppernong wine 0.56± 0.03 N. Carolina red wine 0.58± 0.08 N. Carolina red wine 0.41± 0.00 N. Carolina Scuppernong wine 0.42± 0.06 N. Carolina Scuppernong wine 0.36± 0.04 Louisiana Magnolia wine 0.51± 0.04 Louisiana red wine 0.58± 0.03 Louisiana Carlos wine 0.34± 0.07 Louisiana Blush wine 0.44± 0.02 Louisiana Blush wine 0.41± 0.01 Louisiana Carlos wine 0.40± 0.04 Louisiana red wine 0.65± 0.02 Louisiana Noble wine 0.62± 0.07 Louisiana BDB wine 0.37± 0.04 Louisiana Ison wine 0.63± 0.09 Louisiana white wine 0.46± 0.11 Texas BDB wine 0.38± 0.01 Texas Noble wine 0.85± 0.07 Texas Magnolia wine 0.30± 0.03 Texas BDB wine 0.19± 0.01 BDB=Blanc du bois * safety threshold- WHO/FAO 140 µg/kg/day E.U./U.S.-2-4mg/kg (commodity dependent)

178

Table 5-2. Mean fumonisin B2 concentrations in contaminated wine by sampled state Florida (n)* Georgia (n)* Louisiana (n)* Alabama (n)* N. Carolina (n)* Texas (n)* Red wine 0.44±0.36 (5) 0.31±0.13 (5) 0.62± 0.03(4) 0.40±0.20 (2) 0.52± 0.07(5) 0.85(1) White wine 0.36± 0.28(5) 0.25± 0.10(6) 0.43± 0.07(4) 0.39± 0.09(4) 0.51± 0.11(5) 0.31 (1) Blush/Rose 0.49(1) n/a 0.42± 0.03(2) 0.40± 0.20(4) n/a n/a Blanc du bois 0.35± 0.07(3) n/a 0.37 (1) n/a 0.55 (1) 0.28±0.13(2) Red juice n/a 0.28± 0.03(2) n/a n/a 0.19 (1) n/a White juice 0.12(1) 0.39± 0.01(2) n/a n/a n/a n/a Sparkling wine n/a n/a n/a 0.75 (1) n/a n/a *=parts per million (n)=number of bottles

179

1200

1000

800

600 ppb

400

200

0 Noble Carlos Red blend White blend Hunt Magnolia Ison Scuppernong Blanc du bois Blush/Rose Florida Georgia Lousiana Alabama N. Carolina Texas

Figure 5-1.Mean fumonisin B2 contamination concentrations in wine by grape cultivar for each sampled state (n=68)

=FAO/WHO Provisional Maximum Tolerable Daily Intake for FB2 (70kg person)

=U.S. FDA Maximum limit for total Fumonisin in foods for human consumption

180

CHAPTER 6 DOES VINIFICATION EFFECT MYCOTOXIN CONCENTRATIONS IN MUSCADINE WINES?

A large number of commodities have been documented as being contaminated by the toxic secondary metabolites of fungi, although they are not associated with gross or highly visible fungal colonization. This can be problematic, as there are few to no overt symptoms or signs of their contamination. The toxic effects of these xenobiotics are typically non-acute and are demonstrated over an extended time period after being ingested on a continual basis (Sanchez and Carrillo, 2010). It is not unusual for those affected by this sort of malaise to be uncertain from whence it originated. Furthermore, many of the most vulnerable populations, such as those in developing countries, do not have access to resources that would enable them to assess possibly contaminated foodstuffs. The importance of good manufacturing and agricultural practices cannot be understated.

One such toxin that acts in silence is fumonisin B2 (FB2), which is commonly associated with the contamination of maize, one of the most important crops worldwide.

Generally, this toxin is produced by Aspergillus niger and Fusarium spp. strains. FB2 can accumulate to harmful levels in maize kernels when climatic conditions favor kernel rot, yielding spoiled, unusable crops meant as sustenance for humans and livestock.

The difficulty in controlling these fungal strains is that they can colonize commodities during pre-harvest, harvest or post-harvest under storage conditions (Susca et al.,

2014). Their preference for substrates with Aw 0.97-0.985, high NaCl and high sucrose contents offers a wide range of commodities in which to colonize (Mogensen et al.

2009). According to Prelusky et al. (1996) studies indicated that although fumonisin absorption from feed was relatively low in animal models, the toxin remains in the

181

tissues over an extended period. Absorbed fumonisin tends to accumulate in the liver and kidneys with enterohepatic recirculation contributing to the toxin’s biological half-life of approximately 96 min. in orally dosed pigs. The structure of fumonisin also contributes to its toxic mode of action. Its structural similarities to the sphingoid bases sphinganine and sphingosine are essential to its ability to disrupt cellular metabolism.

These bases act to protect the surface of the cell via the formation of a mechanistically stable and chemically resistant outer portion of the plasma membrane lipid bilayer.

Cellular recognition and signaling depend on the intact nature of this bilayer. Fumonisin acts by directly replacing portions of the bilayer and disrupting cellular signaling involving apoptosis, necrosis, proliferation, stress responses, senescence, and differentiation (Spiegel and Milstein, 2002). Evidence of these disruptions has been illustrated in effected horses (equine leukoencephalomalacia) and pigs (porcine pulmonary edema) (Harrison et al., 1990). In humans, the consumption of foodstuff contaminated with FB2 has been epidemiologically linked to esophageal cancer and neural tube defects (Marasas et al., 1988), leading to an International Agency for

Research on Cancer classification of Group 2B, possibly carcinogenic to humans

(IARC, 2002). Based on this evidence, subchronic toxicity, and long-term carcinogenicity studies in rat models equivalent to 0.2 and 0.25 mg/kg b.w./day, respectively, a Joint FAO/WHO Expert Committee on Food Additives concluded that a

PMTDI of 2 µg/kg b.w./day of FB1-3 alone or in combination on the basis of the NOEL of

0.2 mg/kg b.w. with a safety factor of 100 was sufficient for human health.

Further study has revealed that although maize is one of the most affected crops by fumonisin contamination, other commodities are deeply affected also. Organic

182

matter and field debris serve as a reservoir for soil-derived inoculum surviving throughout growing areas. The FB2 toxin has been isolated from rice (Park et al., 2005), black tea leaves (Martins et al., 2001), pine nuts (Main et al., 20007) and asparagus

(Logrieco et al., 1998). Thai Arabica and Robusta coffee beans were found to be contaminated with fumonisin-producing A. niger strains and yielded FB2 concentrations from 1.3-9.7ng/g. Raisins, dates, figs and currants sourced from various countries were evaluated for FB2 contamination. After fungal identification and isolation, toxin levels in these fruit averaged 7.22 mg/kg, with a range from 4.55-35.49 mg/kg (Vargas et al.

2010). Grapes used in the production of wine have also been targeted as substrate for

A. niger colonization and subsequent toxin production. Upon evaluating the FB2 contamination levels of Portugese wines, Abrunhosa et al. (2011) found that strains of

A. niger isolated from vineyards in the wine regions of Douoro, Ribatejo, Alentejo, Vinho

Verde, and Madiera produced 0.15-1.39 mg/kg FB2. Wine must collected from Italian wine-producers yielded FB2 contents of 0.01-0.4 mg/L (Logrieco et al., 2009), and red wine samples contained levels from 0.4-2.4 µg/L (Logreico et al., 2010). According to

Mogensen et al. (2010), wines sourced from various producing regions across the E.U. ranged in FB2 contamination concentrations of 1 to 25 µg/L.

From these studies it can be surmised that the risk of FB2 contamination within wines is highly probable. However, none of these studies addresses the fate of the fumonisin toxin during the vinification process. It is evident that FB2 is quantifiable in the final product, but this does not offer any indication of whether the final observed quantity has remained consistent over the entire process or if there was any decrease due to processing or chemical reaction. The objective of this study is to investigate the fate of

183

the FB2 toxin throughout the wine-making process to understand if there is any reduction or possible increase in toxin quantity. To initiate this objective, muscadine grapes (V. rotundifolia) are the chosen model or substrate. These grape, indigenous to the south eastern United States are cultivated in vineyards under similar climactic conditions which are suitable for the growth, colonization and toxin production of A. niger.

Materials and Methods

Safety

Statistical Analysis

All statistical analysis during this study was performed using IBM Statistical

Package for the Social Sciences (SPSS) (Armonk, N.Y., USA).

Chemicals

All solvents used were high performance liquid chromatography (HPLC)-grade and purchased from Fisher Scientific (Waltham, MA, USA). Water was purified on a

184

Veratox competitive direct ELISA Immunoassay kit for total fumonisin, which also included: enzyme-labeled fumonisin conjugate, substrate, stop solution, mixing wells, and antibody wells. Samples that were observed to be of higher concentrations were diluted and reanalyzed.

Sample Preparation

Muscadine wine was produced from Carlos and Noble grapes sourced from

Sirvent’s Vineyard (Florahome, FL, USA). Each wine variety was spiked with fumonisin

B2 at the 200 and 1500 ppb levels, with unspiked controls for both grape varieties.

Following each major vinification step 3-10 mL sample aliquots were removed for toxin analysis. Each aliquot was passed through a 30mg/3mL Phenomenex StrataX-C33u

Polymeric Strong Cation solid phase extraction column (Torrance, CA, USA) for toxin extraction. The column was previously conditioned with 1 mL methanol and 1mL water.

To further acidify the column, 1 mL water with 2% formic acid was added. Afterward, the column was washed with 1 mL methanol and the fumonisin was eluted with 1 mL methanol/water (1:4) with 2% NH4 OH (v:v). Toxin recovery rates observed by

Mogensen et al. (2010), were 83% which was in agreement with the recoveries of 86% in the current study. Recovery rates were determined by spiking 10mL grape juice samples at the following concentrations: 0, 0.64, 0.96, 1.92 and 2.57 µg/mL, and subjecting the samples to SPE purification and LC-MS/MS analysis. The toxin load capacity of this column was also evaluated over a range from 0.1-1.500 µg/mL FB2 by the Mogenson et al., (2010) and demonstrated a linear relationship with regard to the concentrations with no signs of reaching a maximal capacity.

185

Wine lees extraction

After grape crushing, Noble must (juice with seeds and skins) was allowed to stand in vessels for flavor and color maturation. Following this period, the grape must was pressed, separating the juice from the seeds and skins. A portion of this fraction was collected for toxin analysis. To extract FB2, 50g lees (yeast sediment) samples were homogenized via two-speed Waring Commercial blender, model 17011S

(Torrington, CT, USA) with 120 mL 70% methanol for 2 min on low setting. The slurry was then centrifuged for 15 min at 2500g and 4°C using an Allegra X-15R, Beckman

Coulter (Indianapolis, IN). The supernatant was then decanted and filtered through a

Whatman No. 1 filter (Marlborough, MA, USA). Samples were stored at -18°C until further analysis via ELISA.

Yeast sediment extraction

After wine samples were racked the yeast sediment left behind was collected and analyzed for possible FB2 concentrations. 30mL of 70%methanol was added to the 20 mL sediment samples. Samples were then sonicated for 20 min and centrifuged for

15min at 2500g and 4°C. The supernatant was decanted and filtered through 5µm

PTFE filter. Samples were held at -18°C until further analysis was performed via ELISA.

Titratable acidity, pH, and °Brix

The titratable acidity, pH, and brix were measured in the raw juice before being made into wine and after as a final wine product (TA and pH). A final brix reading was not taken for wine samples as ethanol affects the refractive index, yielding measurements that tend to be erroneously high. For pH measurements, the Accumet

Basic-AB15plus (Waltham, MA) was used. The pH meter was standardized with buffers

2, 7, and 10. 150mL aliquots were collected for pH analysis and measured while stirring.

186

To measure titratable acidity, 5mL juice or wine was diluted with 150mL distilled water. 0.1N NaOH was added dropwise to the sample until reaching an endpoint of pH

8.2 which was measured in the above described method. Acidity was calculated as

(푚퐿 푁푎푂퐻)(0.1)(0.075)(1000) (w/v) tartaric acid using the following calculation: . 5

As the concentration of soluble solids increases in solution, the refractive index increase is simultaneous. The more molecules found in solution, the more the light passing through it is bent or refracted. °Brix was measured using the Sinotech

Handheld Refractometer (Fujian, China) by placing several small drops of sample and reading the point where the white and blue colors met. All analyzed samples were measured when they reached 20°C.

Alcohol content

To determine the alcohol content of the various wines produced during this study, an ebuillometer (Alpha tech, Auckland New Zealand) was employed. The measurements were based on %volume of alcohol produced in the wines. The measurement range of the instrument was from 0-17% by volume, with an uncertainty of ±0.15% by volume, and an accuracy of 0.1% by volume. This analysis is based on

Raoult’s law relationship of boiling point depression and was performed according to

Zoecklein et al. (1999) Aliquoted samples for each wine treatment were measured in triplicate.

Noble wine production

Noble grapes were harvested by hand over a two day period in Florahome,

Florida, between harvests grapes were stored under refrigerated conditions at 3°C. No more than two days after harvesting, the grapes were rinsed of any visible debris and

187

crushed. Destemming was not necessary as this grape cultivar is easily shaken from the stem at harvesting. After crushing, the grapes were divided into sample replicates and allowed to stay in contact with pomaces at 3°C for 4 days. Juice samples were removed to run several analysis on the juice including brix, titrateable acidity, and pH.

Immediately after crushing, each batch was spiked with FB2 at 200 µg/kg and 1500

µg/kg concentration levels. Each wine batch was dosed with 18% potassium metabisulfite at 0.25mL/kg, and pitched with Saccharomyces bayanus (Red Star

Premier Cuvee-Prise de Mousse, Fermentis Cedex France) and held at 21°C for four days. To prepare the yeast, 250mL D.I. water was heated to 40°C, 5g of yeast was added to the water and swirled to form a suspension hydrating over 20 min. Three 10 mL samples were removed from each batch for FB2 analysis. After this period, each batch of noble must was pressed, separating the skins and grape seeds. Sucrose was then added to the must for chaptalization, to raise the soluble solids content of each replicate batch to 20%. After this addition, noble must was then stored in gallon bottles with airlocks at 13°C. All samples were racked after six weeks, reserving yeast sediments (lees), sulfites were added at 0.25 mL/kg, and the headspace of each bottle was filled with nitrogen. To determine if fermentation was completed, the airlock of each bottle was inspected for CO2 escape and each sample was subjected to the

Clintest System (Elkhart, IN, USA) to assess reducing sugar levels. Reducing sugar concentrations at 0.2-0.3% were deemed dry enough for fermentation completion. The mechanism of the reaction is similar to that in copper reductions, except that the heat required is provided internally by the neutralization reaction of NaOH and citric acid

(Zoecklein et al., 1999). The airlocks were replace with screw caps and 10mL aliquots

188

were removed, samples were stored at 4°C. Samples were racked for a second time after 11 weeks, reserving sediment, collecting 10mL samples, adding sulfites, and filling the headspace with nitrogen. After eight months of aging, 10ml aliquots were removed for analysis.

Carlos wine production

Carlos grapes were harvested by hand over a two day period in Florahome,

Florida; between harvest days grapes were stored under refrigerated conditions at 3°C.

Grapes were then cleared of debris, removing wounded, sub-par grapes and rinsed.

Destemming was not needed, as the grapes were hand harvested and shaken from the stems. Carlos grapes were crushed then pressed, separating juice from skins. A

250mL aliquot was removed to analyze pH, °Brix, and titratable acidity. The juice was sulfited with 18% potassium metabisulfite at 0.50mLSO2/L juice, then divided and spiked with FB2 at the 200 and 1500ppb levels, reserving juice for controls also.

Sucrose was added as a chaptalization step to raise juice soluble solids content to 20%, according to Zoecklein et al. (1999). Each batch was pitched with Saccharomyces bayanus (Red Star Premier Cuvee-Prise de Mousse, Fermentis Cedex France) at

12.5mL/L and held at 20°C for seven days. To prepare the yeast, 250mL D.I. water was heated to 40°C, 5g of yeast was added to the water and swirled to form a suspension hydrating for 20 mins. After the seven days of room temperature fermentation, the juice was transferred to glass vessels, capped with airlocks, and stored at 13°C. During fermentation, the samples were racked, reserving yeast sediment, collecting samples, adding sulfites at 25ppm, and filling the headspace with nitrogen. To determine whether the fermentation was complete, the airlock of each bottle was inspected for CO2 escape and each sample was subjected to the Clintest System (Elkhart, IN, USA) to assess

189

reducing sugar levels. Reducing sugar concentrations at 0.1-0.2% or below were deemed dry enough for fermentation completion. This determination was made in reference to a color code in the system, with a sensitivity level of 0.05% (The Ames

Company, 1978). However, measured levels will never reach 0% due to the pentoses in the wines, even though they are not fermentable. The airlocks were replace with screw caps and 10mL aliquots were removed, samples were stored at 4°C. Samples were racked for a second time after 11 weeks, reserving sediment, collecting 10mL samples, adding sulfites, and filling the headspace with nitrogen. After eight months of aging,

10ml aliquots were removed for analysis.

Fumonisin Analysis

10 min. To bring the incubation to an end, 100µL of stop solution was added to the test wells. All wells were analyzed for optical densities via the Neogen Stat Fax 321 Plus

190

detection of the ELISA assay was 50 ppb with a range of quantification at 50-600 ppb.

To extend the range of quantification of the assay, a stock solution of FB2 standard was diluted to a concentration of 960 ppb to capture highly contaminated samples within the standard curve for accurate quantification. Samples quantified outside of this range were diluted and reanalyzed. The principle governing this assay pertains to the free fumonisin from the samples and controls being allowed to compete with enzyme-labeled fumonisin for the antibody binding sites. The addition of substrate, and its reaction with bound conjugate determines color change/development or optical density levels.

Results and Discussion

The Noble and Carlos muscadine grapes used to evaluate the fate of the fumonisin B2 toxin throughout the wine-making process were harvested in mid- to late

September. During this period in north central Florida, climate temperature highs remained within the range of optimal A. niger growth (30-37°C) five weeks before harvesting. Approximately two weeks before harvesting, the highest climate temperature remained ±4°C within the temperature optimum (25-30°C) for fumonisin B2 production by the A. niger fungus. Conditions such as these and the ubiquitous nature of A. niger create all the needed attributes for grape contamination within the vineyard.

The Carlos grape harvest was 72.6 kg and yielded 34.8L grape juice, whereas the

Noble harvest was 62.3kg and yielded approximately 59.4L juice. The variation in juice yields was possibly due to the white Carlos grapes being slightly chilled before crushing and pressing as higher juice yields have been associated with processing at warmer temperatures as reported by Threlfall (2002). As is typical in red wine production, the

Noble grapes were rested after crushing on the grape pomace (skins and seeds), to drawing out more juice and for increased color maturation. The initial brix

191

measurements for both Noble and Carlos harvests were 15.1 and 11.8, respectively, both of which were lower than in previously reported harvests. However, pH measurements for both grape cultivars reflected previously reported data. Aliquot samples were removed at key points of the wine making process and held at -18°C until subjected to SPE extraction and ELISA analysis. Figures 6-7 and 6-8 detail the wine production process and identifies where samples were drawn.

From the wine sample analysis during production, natural contamination was observed in Carlos and Noble samples with the white grapes measuring at slightly higher levels of toxin concentration. During the harvest and subsequent grape rinsing and sorting there were no signs of fungal colonization, however there was collecting debris i.e. dead leaves and over-ripened grapes on the vineyard floor and between rows, well established source of fungal inoculum. Due to natural contamination at approximately 494.9 ppb in control samples, FB2 levels at the early pressed juice stage of spiked Carlos samples were higher than anticipated. This same natural toxin contamination was also noted in Noble grape samples, with a mean of 274.9 ppb. It was observed that the mean contamination for Carlos control grapes was higher than

Noble grapes, this is attributed to the interference of the seed and skin fraction of the must the samples were taken from. Without this interference, it is highly possible that the control sample mycotoxin levels would have been closer. After fermentation, toxin concentrations decreased in all Carlos samples except one control sample (Table 6-2).

Furthermore, the trend in decreasing toxin levels after the fermentation of white wine was also noted by Anli et al. (2011), who observed a 26% decrease in OTA concentrations during wine production in Turkey. The mean FB2 loss of all Carlos

192

control samples after fermentation was 12.4% (Figure 6-3). The inverse was noted in the Noble samples after fermentation, with an increase in measured toxin concentration across all Noble treatments. Possible mycotoxin reduction effects based on fermentation where possibly lessened due to a low addition of sucrose in the Noble wine samples during chaptalization. The lacking of additional sucrose is evident from the low final alcohol content percentages of the Noble wine. The low alcohol contents may be due to the underestimation of red juice volume of the Noble must. Typical red wine alcohol percentages ranges are measured minimally at 12-13%. In this study the average alcohol percentage of all produced red wine was 7.1%. Even at such a low alcohol content U.S. regulations would still classify this beverage possibly as table wine.

However, the according to the International Organization of Vine and Wine (O.I.V), a minimum of 8.5% (vol /vol) alcohol content must be reached to be considered wine

(Zoecklein et al.,1999). The higher measured toxin levels is in direct response to the aforementioned interference from the pomace fraction of the red wine must. In Table 6-

3, the pomace fraction for all Noble samples demonstrated higher toxin concentrations than the previous must fraction. Similar observations were noted by Ratola et al. (2005), when assessing ochratoxin A content of port wine microvinification. The higher toxin concentrations in the pomace was attributed to toxin adsorption to the pomace fraction of the red wine. In both Noble and Carlos samples the FB2 concentrations in spiked samples decreased over a range of 11.6-72% and 14.8-81.6% respectively, after two

Noble rackings and a single Carlos racking. However, the toxin concentrations rose

19.6% for the Noble controls after the first racking, this observation did not follow the earlier trend of toxin adsorption to the dead yeast matter. Despite the initial increase

193

after the first racking, FB2 levels were lessened by 12.1% during the subsequent racking. The highest decrease in toxin concentration from the second racking was observed in the 1500ppb spiked Noble sample at 72.1%. Toxin reductions for the

1500ppb spike level Carlos samples were measured at 76.5% after a single racking

(Figure 6-3). All wine samples were allowed to age in the bottle over an eight month period. After this aging, toxin concentration losses were exhibited across all wine treatments and cultivars. A 17% mycotoxin reduction was reported by Grazoli et al,

2006, when evaluating the fate of OTA in Negroamaro wines in Italy after aging. During

Noble wine production, the by-products or lees generated by alcoholic fermentation and natural settling generally exhibited FB2 concentrations that were higher than any other fraction measured indicating that the physical removal of the toxins had a greater effect on decontamination than chemical degradation. A similar phenomena was reported by

Inoue et al, 2013, who noted higher levels of fumonisin detoxification via the adsorption to spent grain during an 18 day fermentation of clear lager after spiking malt with 14 different mycotoxins. During the 3-day fermentation of maize for the production of ethanol by Bothast et al, (1992) extremely minor FB2 degradation was observed as most of the initial fumonisin was recovered the distiller’s grain, thin stillage, and distillers’ soluble fractions. Fernandes et al., 2007 also cited the solid fraction removal and filtration of wines as a method of removing measurable quantities of fungal toxin contamination. This research group also noted that malolactic fermentation aids in toxin reduction via the action of lactic acid bacteria that also function in the deacidification of wines. A 3% mycotoxin recovery was observed from collected wine lees after malolactic fermentation. According to Niderkorn et al. (2009), lactic acid bacteria bind FB2 via the

194

bacteria’s peptidoglycan binding site, attracting one of fumonisins multiple tricarballylic moieties to initiate the binding. Although malolactic fermentation was not performed during these fermentations, per industry standards, it is evident that final mycotoxin level reduction would have benefited from this processing step based on these findings.

The produced Noble wine had measured pH levels between 2.9-3.0, a typical pH target for this style of wine is 3.3-3.5, malolactic fermentation would have facilitated a pH increase and possibly removed some of the measured mycotoxin. A one-way ANOVA was performed to compare the mycotoxin levels at the various Noble and Carlos wine production steps. A statistically significant difference (p< 0.05) was found amongst all the wine making steps for all Carlos and Noble wine treatments. To further identify where the differences in toxin level were a Tukey’s mean separation was performed.

Across all Carlos samples the toxin levels found after fermentation and after raking were consistently of the same grouping, demonstrating no statistical difference (Table 6-3).

The affects of fermentation and physical removal were shown to act in a similar fashion with regard to mycotoxin concentration depreciation. The findings for the Noble wine samples also followed this similar pattern with the after fermentation and racking toxin concentrations demonstrating the same outcomes (Table 6-5). However, among the

Noble and Carlos samples contaminated at higher concnetrations, there was a distinct separation between toxin levels found after fermentation and within the sediment. At these toxin levels, the sediment was found to consistenly have higher quantities of mycotoxin.

The use of a sensitive indirect competitive ELISA for fumonisin contamination quantification of fermented beverages was also performed by Scott et al, (1997) in their

195

survey of beers available for commercial consumption in Spain, Canada, and Kenya.

The principle governing this assay is dependent upon the free fumonisin from the samples and controls being allowed to compete with enzyme-labeled fumonisin for antibody binding sites. The addition of substrate, and its reaction with bound conjugate determines color change/development or optical density levels at 650nm. The relationship between the optical densities and contamination concentrations is inverse, as the density increases, contamination levels are decreased.

As it pertains to human health, the PMTDI for fumonisins 1-3 alone or in combination has been set at 2 µg/kg b.w./day by JEFCA. If a person of 70 kg is considered, this allows ingest of up to 140 µg of fumonisin/day. In this study, the two contamination levels for both grape cultivars was 200 and 1500 µg/L. After being subjected to the vinification process including bottle aging, neither grape cultivar at either spiked treatment level was measured at a mycotoxin concentration below the accepted levels for safety. However, during the wine-making process, malolactic fermentation and a final filtration were not included in the process. It is possible that the combination of these chemical and physical treatments may have lowered the naturally contaminated Noble and Carlos controls to levels below the safety threshold. Although the mycotoxin spike levels in this study were relatively low, they were consistent with

FB2 concentrations produced by isolated A. niger and in vineyard selected grapes in previous studies (Abrunhosa et al., 2011) (Hocking et al., 2007) (Logrieco et al., 2009,

2010) (Mogensen et al., 2010).

Conclusion

From this study is evident that fermentation alone does not serve as an adequate means to detoxify or decontaminate wines that have come in contact with mycotoxins.

196

However, the combination of wine racking, alcoholic and malolactic fermentation, and fining together can remove a fraction of the toxin concentrations. It is also important to consider the growing conditions surrounding the harvest because warmer weather is conducive to increased fungal growth and colonization, with subsequent toxin production. Furthermore, diligence in the vineyards to reduce debris such as dead vines and dropped berries that may act as substrate for fungal inoculum is important as well.

The mycotoxin contamination of wines should be considered by producers even if only to verify that toxin levels are below the PMTDI set forth by JEFCA.

197

Table 6-1. Noble and Carlos muscadine wine physical attributes before and after production Sample pH °Brix Alcohol % Titratable acidity (g/100mL) Carlos initial final initial v/v Initial* Final* Control 3.36± 0.07 3.08± 0.02 11.9± 0.1 12.1± 0.2 0.47± 0.04 0.66± 0.2 200 3.4± 0.1 3.1± 0.03 11.9± 0.1 11.5± 0.5 0.44± 0.07 0.34± 0.1 1500 3.5± 0 3.1± 0.01 11.8± 0.06 12.0± 0.61 0.41± 0.03 0.31± 0.02 Control 3.5± 0.08 3.0± 0.03 14.6± 0 6.5± 0.2 0.34± 0 0.30± 0.1 200 3.6± 0.09 3.0± 0.06 15.1± 0.2 7.0± 0.3 0.35± 0.2 0.33± 0.05 1500 3.6± 0.1 3.0± 0.06 15.1± 0.8 7.2± 0.6 0.34± 0.03 0.4± 0.04

198

Table 6-2. Mean fumonisin B2 concentrations in wine and wine fractions during Carlos wine production after artificial inoculation with 200 and 1500ppb FB2 After Carlos Sample Juice* fermentation* Sediment* Rack* Sediment* After Aging* control juice 1 484.6 ±96.3 615 ±24.5 170.6 ±12.0 409.5 ±65.4 278.5 ±34.2 183.4 ±42.5 control juice 2 546.5 ±35.2 352.5 ±39.3 198.1 ±61.2 406 ±56.6 524.3 ±23.7 211 ±62.3 control juice 3 453.6 ±66.4 330.7 ±82.4 278.5 ±31.1 447.9 ±71.2 nd 187.3 ±69.0

200-1 1071.2 ±39.1 659.88 ±84.0 468.6 ±54.1 541.36 ±81.2 916.2 ±47.1 165.41 ±58.1 200-2 1105.2 ±85.7 564.62 ±59.1 396.6 ±58.0 367.9 ±25.5 743.6 ±62.0 194.2 ±89.6 200-3 1209.9 ±11.6 698.21 ±18.6 400.6 ±35.0 496.97 ±41.5 1032 ±59.4 236.1 ±22.0

1500-1 1966.69 ±64.8 1396.97 ±13.7 555.4 ±18.4 475.64 ±75.8 1417.6 ±77.1 391.15 ±74.2 1500-2 2284.2 ±46.6 1341.77 ±15.4 627 ±13.2 492.36 ±68.8 1321.6 ±65.4 316.41 ±36.9 1500-3 1863.3 ±79.6 1313.23 ±27.0 584.4 ±15.6 468.6 ± 50.3 1131.8 ±47.7 416.75 ±152.1 *=parts per billion

Table 6-3. One-way analysis of variance applied to fumonisin B2 content (ppb) of samples collected in different Carlos winemaking steps Control 200 1500 Juice 494.9 b 1128.7 c 2038.1 c After Fermentation 432.7 b 640.9 b 445.3 a Overall Rack 421.3 b 468.7 ab 478.6 a Overall Sediment (2) 290 a 659 b 659 b After aging 193.9 a 198.5 a 374.7 a (n)=number of times performed during wine making Means followed by the same letter are not significantly different (p<0.05)

199

2500

2000

1500 ppb 1000

500

0 JUICE AFTER FERMENTATION AFTER RACKING AFTER AGING

control 200 1500

Figure 6-1. Mean fumonisin B2 toxin concentrations throughout Carlos wine production after artificial inoculation at 200 and 1500ppb FB2

3000

2500

2000

1500 ppb

1000

500

0 control 200 1500

Juice After fermentation lees After racking lees

Figure 6-2. Mean fumonisin B2 concentrations in wine yeast sediment during Carlos wine production after artificial inoculation at 200 and 1500ppb FB2

200

2600 2400 2200 2000 1800 1600 1400

1200 ppb 1000 800 43.2 12.4 14.8 58.4 600 55.7 76.5 82.4 81.6 400 60.7 200 0 -200 Juice After Fermentation After Racking After Aging

CONTROL 200 1500 % FB2 LOSS

Figure 6-3.Evolution of fumonisin B2 in two artificially inoculated samples during the Carlos wine-making process

201

Table 6-4. Fumonisin B2 concentrations in wine and wine fractions during Noble wine production after artificial inoculation with 200 and 1500 ppb FB2 After Noble Sample Must* Pomace* Fermentation* 1st rack* Sediment* 2nd rack* Sediment* After Aging*

control must 1 269 ±47.3 642.2 ±39.1 575.37 ±48.1 776.56 ±28.7 210.9 ±18.7 473.64 ±42.1 131.4 ±14.7 316.7 ±36.0 control must 2 253.8 ±19.2 597 ±6.7 669.78 ±39.5 740.55 ±40.1 104.4 ±44.6 517.93 ±39.4 nd 297 ±55.2 control must 3 301.8 ±41.4 591 ±18.7 576.96 ±40 662.25 ±48.4 nd 610.73 ±26.1 450.3 ±11.8 150.6 ±29.5

200-1 433.34 ±32.4 869.2 ±37.0 651.72 ±18.9 610.82 ±47 908.6 ±21.3 640.01±38.3 115.26 ±33.5 344.87 ±11.0 200-2 465.79 ±36.7 522.2 ±38.4 857.23 ±185.9 800.74 ±56.3 645.2± 28.6 601.34 ±86.9 157.16 ±28.2 360.92 ±14.0 200-3 463.04 ±22.3 370.6 ±21.2 817.93 ±162.4 645.73 ±36.42 nd 697.08 ±71.8 nd 327.63 ±18.8

1500-1 373 ±35.5 685.2 ±13.6 2427.6 ±58.8 1092.24 ±60.7 769 ±55.2 570.08 ±84.2 194.7 ±49.0 473.1 ±30.9 1500-2 452.36 ±63.6 1233.4±51.2 2280.6 ±144.7 1609.95 ±23.7 731.8 ±29.7 754.44 ±39.0 185.2 ±34.4 510 ±76.1 1500-3 464.4± 65.4 1113.4 ±39.4 2547.36 ±235.2 932.62 ±70 405.4 ±50.1 672.51 ±40.4 172.9 ±29.1 658.2 ±58.5 *=parts per billion

Table 6-5. One-way analysis of variance applied to fumonisin B2 content (ppb) of samples collected in different Noble winemaking steps Control 200 1500 Pomace 610.0 b 987.3 b 1010.6 b After Fermentation 607.4 b 775.6 b 2418.5 c Overall Rack (2) 630.2 b 665.9 b 938.64 ab Overall Sediment (2) 120.7 a 224.2 a 409.34 a After aging 254.7 a 254.7 a 547.1 a (n)=number of times performed during wine making Means followed by the same letter are not significantly different (p<0.05).

202

3000

2500

2000

1500 ppb

1000

500

0 MUST AFTER FERM. 1 ST R AC K 2 ND R AC K AFTER AGING

control 200 1500

Figure 6-4. Mean fumonisin B2 toxin concentrations throughout Noble wine production after artificial inoculation at 200 and 1500ppb FB2

1600

1400

1200

1000

800 ppb

600

400

200

0 Control 200 1500

Must Pomace 1st rack lees 2nd rack lees

Figure 6-5. Mean fumonisin B2 concentrations in wine yeast sediment during Noble wine production after artificial inoculation with 200 and 1500ppb FB2

203

2380 2180 1980 1780 1580 1380 11.6

1180 ppb 49.3 980 12.1 -19.6 72.1 780 16.7 55.6 77.1 580 58.1 380 180 -20 Must After ferm. 1st rack 2nd rack After Aging

CONTROL 200 1500 % FB2 LOSS

Figure 6-6. Evolution of fumonisin B2 in two artificially inoculated samples during the Noble wine-making process

204

Noble Grapes

Must with pomace Sampling pomacdppomace

Maceration (alcoholic fermentation 24-26°C)

End of maceration Sampling pomace only

Pressing

Wine (end of alcoholic Sampling fermentation)

Sampling First racking

Second racking Sampling

After bottling aging Sampling

Figure 6-7. Noble wine production process and sampling protocol

205 Carlos Grapes

Before Maceration (juice) Sampling

Fermentation Sampling

after

Racking Sampling

Bottle aging Sampling

Figure 6-8. Carlos wine production process and sampling protocol

206

CHAPTER 7 OVERALL CONCLUSIONS AND FUTURE WORK

The importance of agriculture in the United States and around the world as a source of food and materials needed or required in our daily lives can never be understated. And from this significance, is derived the expressed vigilance needed in following good agricultural and manufacturing practices. The sheer volume of pathogenic organisms and compounds that can contaminate/adulterate agricultural products is staggering. However, through research and innovation the scientific community has ventured to keep these threats at bay. One such threat, fumonisin B2 was initially only associated with the Fusarium fungal species and known as a prolific contaminant of corn and corn products. Now it is recognized that this mycotoxin is also produced by Aspergillus niger and is a contaminant of dried fruit, grapes and grape products, i.e. wine and juice. Reports of FB2 contamination within vineyards have been published since 2010, however, all of these reports have focused on popular wine regions of the European Union. Several grape growing regions in the U.S. have climates that mirror the wine-producing regions mentioned in the reports of FB2 contamination. A. niger is ubiquitous in nature and has the ability to colonize vegetation over a wide range of temperatures, although it will only produce its toxin within a much narrower temperature span. This temperature window (25-30°C) is commonly experienced during the growing season in vineyards of the southeastern United States.

Since all of the factors needed for FB2 production by A. niger were consistently present, the evaluation of the toxin in this environment was the logical subsequent step in characterizing the hazard presented by this mycotoxin in this food system.

207

Initially, it was important to identify whether muscadine grapes (Vitis rotundifolia) could serve as a viable substrate for A. niger colonization and support toxin production.

From the experiments performed, it was found that the Noble and Carlos muscadine grape cultivars can serve as a substrate for the growth and mycotoxin production.

Although these were the only two cultivars considered in the study, it is highly probable that these results can be translated onto other V. rotundifolia cultivars due to their similar horticultural production systems and biochemical/physiological characteristics.

Fungal toxin contamination of fruit juice is well demonstrated by the adulteration of apple juice by patulin. Contamination concentrations for this beverage have been limited to 50 ppb by several governments and food safety organizations worldwide due to its consumption by highly vulnerable populations (young children). Through the study of patulin interactions with apple juice it was reported that this mycotoxin degrades at storage temperatures over time. Furthermore, the addition of potassium sulfite also enhances the patulin degradation demonstrated in apple juice. Both of these factors were evaluated, in our studies, in Carlos muscadine juice with an additional pasteurization step. The grape juice treatments were exposed to the following storage conditions: freezer (-18°C), refrigerator (4°C), and room temperature (20°C) for a variety of storage times. It was found that none of these conditions in tandem with, or without the U.S. FDA juice pasteurization protocol had a significant effect in the reduction of FB2 contamination to levels deemed safe for human consumption. The sulfite additions to the contaminated grape juice did not have the same degradative effects demonstrated in patulin-contaminated apple juice. The fumonisin B2 toxin can be classified as being a stable contaminant in the juice matrix.

208

With this knowledge of the toxin stability within juices, an assessment of other grape beverages, such as wine was needed. Muscadine wines available for commercial purchase and consumption were collected from six states within the southeastern United States. In all of the tested wine samples there was some measurable level of FB2 contamination. However, no one grape cultivar could be classified as the most highly contaminated, but wines that used red grapes as opposed to white did have higher concentrations overall. With regard to human health, considering a person of 70kg and the PMTDI of 2µg/kg of body weight set forth by

JEFCA, all of the sampled wines were below a harmful concentration when considered independent of other food intake. If this person were consuming a diet of other foods contaminated by FB2, then additive effects could exceed this threshold.

When evaluating the fate of the FB2 mycotoxin during the wine-making process, it was observed that fermentation alone does not serve as an adequate means to detoxify wines contaminated with mycotoxins. The combination of racking, alcoholic and malolactic fermentation, coupled with fining were found to remove a substantial fractions of toxin concentrations. Although physical means removed measurable toxin quantities, samples deliberately contaminated and subjected to various processing steps did not reach a level that would be completely safe for human consumption based on the guidelines per FAO/WHO. During periods where vineyard climate is not conducive to fungal toxin production traditional vinification methods are appropriate, but in years contrary to this wine makers must take extra precaution to reduce contamination to favorable levels.

209

With regard to future research endeavors to expand the knowledge base of fungal toxin contamination within muscadine wines, observing the mechanism and quantifying concentrations of FB2 removed from these wines via malolactic fermentation would be beneficial. Malolactic fermentation was not performed during the FB2 fate study described in Chapter 6, as this type of fermentation is typically not employed in the production of muscadine wine. The removal of ochratoxin A from wines was facilitated by this type of fermentation and it is very likely similar results may be obtained for FB2 in muscadine wines. Traditional fining methods with materials such as bentonite, isinglass, gelatin, or casein were also not performed during muscadine wine production, but have been found to remove portions of ochratoxin A contamination from wines.

Although Ochratoxin A and FB2 have different chemical structures, it is reasonable to believe that this method of physical removal would also have success in FB2 contaminated wines as the two mycotoxins both exhibited similar adsorption properties during physical removal in earlier wine-making steps.

A survey of commercial wines produced in wine-producing regions of the western U.S. for fumonisin contamination would also be beneficial. The reports of fungal wine contamination in the European Union (E.U.) were in association solely with V. vinifera grapes; this genus of grape is also produced in California and western U.S. wine regions. Climactic conditions in some wine-producing regions of this area are also similar to those affected in the E.U. If it is found that there is a substantial risk for fumonisin contamination in this region, assessing the efficacy of current fungicide protocols and their usefulness against A. niger would also be beneficial to the knowledge base. Although the climate conditions that support mycotoxin production are

210

known, no study has undertaken actually investigating and comparing the grapes and wines of similar vineyards under normal growing condition versus more extreme, toxin bolstering conditions. From a study such as this, wine-producers can be more assured of specific conditions where higher toxin concentrations in their final product may be a larger issue. It is understood that the wine-producing industry in various parts of the country is large, and producing this product has spanned many years and is art as much as it is science. However, considerations should be taken to limit mycotoxin contamination in wine, ensuring a safe and acceptable product for consumers to enjoy without fear or hesitation.

211

LIST OF REFERENCES

Abbax, H., Gelderblom, W., Cawood, M., Shier, W., 1993. Biological activities of fumonisins, mycotoxins from Fusarium moniliforme, in jimsonweed (Datura stramonium L.) and mammalian cell cultures. Toxicon 31, 345-353.

Abdulrazzaq, Y.M., Osman, N., Yousif, Z.M., Al- Falahi, S., 2003. Aflatoxin M1 in breast- milk of UAE women. Annals of Tropical Paediatrics International Child Health 23, 173.

Abel, S., Gelderblom, W.C.A., 1998. Oxidative damage and fumonisin B1 induced toxicity in primary rat hepatocytes and rat liver in vivo. Toxicology 131, 121-131.

Abrunhosa, L., Calado, T., Venâncio, A., 2011. Incidence of Fumonisin B2 production by Aspergillus niger in Portuguese wine regions. Journal of Agricultural and Food Chemistry 59, 7514.

Abrunhosa, L., Paterson, R., Kozakiewicz, Z., Lima, N., Venâncio, A., 2001. Mycotoxin production from fungi isolated from grapes. Letters in Applied Microbiology 32, 240-242.

Ahmed, K., Matsuya, Y., Nemoto, H., Zaidi, S.F., Sugiyama, T., Yoshihisa, Y., Shimizu, T., Kondo, T., 2009. Mechanism of apoptosis induced by a newly synthesized derivative of macrosphelides with a thiazole side chain. Chemico Biological Interactions 177, 218-226.

Ahmedullah, M., Himelrick, D.G., 1989. Grape management. , In G.J. Galletta, D.G. Himelrick (Eds.), Small Fruit Crop Management. Prentice Hall, Englewood Cliffs, N. J, pp. 383.

Ainsworth, G.C., 1976. Introduction to the History of Mycology. Cambridge University Press, Cambridge.

Akkerman, R., Farahani, P., Grunow, M., 2010. Quality, safety and sustainability in food distribution: a review of quantitative operations management approaches and challenges. OR Spectrum 32, 863-904.

Alberts, J.F., Gelderblom, W.C.A., Thiel, P.G., Marasas, W.F.O., Van Schalkwyk, D.J., Behrend, Y., 1990. Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme. Applied Environmental Microbiology 56, 1729.

Alberts, A.W., 1998. Discovery, biochemistry and biology of lovastatin. American Journal of Cardiology 62, 10J.

Alexander, N., Proctor, R., McCormick, S., 2009. Genes, gene clusters, and biosynthesis of trichothecenes and fumonisins in Fusarium. Toxin Reviews 28, 198.

212

Ames Company, 1978. Dextrocheck Test for reducing sugar: Information Summary. Elkhardt, IN.

Anand, R., Kulothungan, S., Keerthana, V., 2010. In vitro and in vivo assay of selected fungicides against Aspergillus niger. IUP Journal of Life Sciences 4, 7-18.

Andersen, P.C., Bryan, M.W., Baker, L.H., 1985. Effect of two wire vertical and Geneva double curtain training systems on berry quality and yield of muscadine grapes. Proceedings of the Florida State Horticultural Society 98, 175-178.

Andersen, P.C., Crocker, T.E., Breman, J. 2003. The muscadine grape. Retrieved 1/29/2013, from http://edis.ifas.ufl.edu/hs100.

Arora, R.G., Frolen, H., Fellner-Feldegg, H., 1983. Inhibition of ochratoxin A teratogenesis by zearalenone and diethylstilbestrol. Food and Chemical Toxicology 21, 779.

Aytaç, S.A., Moss, M.O., 1992. Production and storage of apple juice during kükürtd - oxide and 1- patulin effect on the stability of the ascorbic acid. Journal of Küke 15, 11-17.

Bacon, C.W., Porter, J.K., Norred, W.P., 1995. Toxic interaction of FB1 and fusaric acid measured by injection into fertile chicken egg. Mycopathologia 129, 29.

Bailey, G.S., 1994. Role of aflatoxin-DNA adducts in the cancer process. In D.L. Eaton, J.D. Groopman (Eds.), The Toxicology of Aflatoxins: Human Health, Veterinary, and Agricultural Significance. Academic Press, San Diego, CA, pp. 137-148.

Barrier-Guillot, B., 2006. Prevention of Fusarium toxins in cereals— situation of maize. European Commission, Brussels.

Bartók, T., Árpád, S., Szekeres, A., Mesterházy, Á., Bartók, M., 2006. Detection of new fumonisin mycotoxins and fumonisin-like compounds by reversed-phase high- performance liquid chromatography/electrospray ionization ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 20, 2447-2462.

Basha, S.M., Musingo, M., Colova, V.S., 2004. Compositional differences in the phenolic compounds of muscadine and bunch grape wines. African Journal of Biotechnology 3, 523-528.

Basha, S.M., Vasanthaiah, H.K.N., Kambiranda, D.M., Easwaran, K., Queeley, M. G., 2012. Genetic variation in sugar composition among muscadine, Florida hybrid bunch and bunch grape genotypes. International Journal of Wine Research 4, 15-23.

Bates, R.P., Morris, J.R., Crandall, P.G., 2001. Principles and Practices of Small and Medium-Scale Fruit Juice Processing. Food and Agriculture Organization, Rome, Italy.

213

Battilani, P., Magan, N., Logrieco, A.F., 2006. European research on Ochratoxin A in grapes and wine. International Journal of Microbiology 111, S2-S4.

Battilani, P., Pietri, A., Bertuzzi, T., Languasco, L., Giorni, P., Kozakiewicz, Z., 2003. Occurrence of ochratoxin A–producing fungi in grapes grown in Italy. Journal of Food Protection 66, 633-636.

Battilani, P., Pietri, A. 2002. Ochratoxin A in grapes and wine. European Journal of Plant Pathology 108, 639-643.

Beckerman, J.L., 2011. Fungicide mobility. Nursery management 3, 25-26.

Belli, M., 2006. Ochratoxin A and ochratoxigenic molds in grapes, must and wine. Ecophysiological Studies University of Lleida.

Bennett, J.W., Klich, M., 2003. Mycotoxins. Clinical Microbiology Reviews 16, 497-516.

Bennett, J.W., Lee, L.S., Vinnett, C., 1971. The correlation of aflatoxin and norsolorinic acid production. Journal of American Oil Chemist 48, 368-370.

Bennett, J., 1987. Mycotoxins, mycotoxicoses, mycotoxicology and mycopathologia. Mycopathologia 100, 3.

Bennett, J., Klich, M., 2003. Mycotoxins. Clinical microbiology reviews 16, 497.

Betina, V., 1984. Mycotoxins as secondary metabolites. In V. Betina (Ed.), Mycotoxins- Production, Isolation, Separation and Purification. Elsevier Science Publishers, Amsterdam, pp. 13.

Betina, V., Binovska, Z., 1979. Diphasic production ofcitrinin by Penicillium janthinellum and its regulation. Biologia (Bratislava) 34, 461.

Bezuidenhout, S.C., Gelderblom, W.C.A., Gorsl-Allman, C.P., Horak, R.M., Marasas, W.F.O., Spiteller, G., VIeggaar, R., 1988. Structure elucidation of the fumonisins,mycotoxins from Fusarium moniliforme.. Journal of Chemical Society, Chemical Communications 198B, 743.

Bhat, R.V., Beedu, S.R., Ramakrishna, Y., Munski, K.L., 1989. Outbreak of tricothecene mycotoxicosis associated with consumption of mold-damaged wheat production in Kashmir Valley, India. Lancet 1, 35.

Bhat, R.V., Sherry, P.H., Amruth, R.P., Sudershan, R.V., 1997. A foodborne disease outbreak due to the consumption of mouldy sorghum and maize containing fumonisin mycotoxins. Journal of Clinical Toxicology 35, 249.

Bittencourt, A.B.F., Oliveira, C.A.F., Dilkin, P., Corre, B., 2005. Mycotoxin occurrence in corn meal and flour traded in Sao Paulo, Brazil. Food Control 16, 117.

214

Bladt, T.T., Frisvad, J.C., Knudsen, P.B., Larsen, T.O., 2013. Anticancer and antifungal compounds from Aspergillus, Penicillium and other filamentous fungi. Molecules 18, 11338-11376.

Bolger, P.M., Carrington, C.D., Henry, S.H., 1996. Risk assessment for risk management and regulatory decision-making at the U.S. Food and Drug Administration. , In A. Fan, L. Chang (Eds.), Toxicology and Risk Assessment: Principles, Methods, and Application. Marcel Dekker, Inc., New York,

Bolger, M., Coker, R.D., Dinovi, M., Gaylor, D., Gelderblom, W.C., Olsen, M., Paster, N.R., R.T., Shephard, G., Spiers, G.J.A., 2001. Fumonisins. Safety evaluation of certain mycotoxin in food.47, 103-279.

Bordin, K., Rosim, R.E., Neeff, D.V., Rottinghaus, G.E., Oliveira, C.A.F., 2014. Assessment of dietary intake of fumonisin B1 in São Paulo, Brazil. Food Chemistry 155, 174.

Borrow, A., Jefferys, E., Kessel, R., Llods, E., Lloyd, P., Nixon, I., 1 961. The metabolism of Gibberlla fujkuroi in stirred culture. Canadian Journal of Microbiology 7, 227.

Bothast, R.J., Bennett, G.A., Vancauwenberge, J.E., Richard, J.L., 1992. Fate of fumonisin B1 in naturally contaminated corn during ethanol fermentation. Applied and Environmental Microbiology 58, 233-236.

Bothast, R.J., Bennett, G.A., Vancauwenberge, J.E., Richard, J.L., 1992. Fate of fumonisin B1 in naturally contaminated corn during ethanol fermentation. . Applied and Environmental Microbiology 58, 233-236.

Brackett, R.E., Marth, E.H., 1979. Ascorbic acid and ascorbate cause disappearance of patulin from buffer solutions and apple juice. Journal of Food Protection 11, 864- 866.

Brown, D., Butchko, R., Busman, M., Proctor, R., 2077. The Fusarium verticillioides FUM gene cluster encodes a Zn (II) 2Cys6 protein that affects FUM gene expression and fumonisin production. Eukaryotic Cell 6, 1210.

Bryden, W.L., 1982. Aflatoxins and animal production: an Australian perspective. Food Technology in Australia 34, 216.

Bryden, W.L., 2007. Mycotoxins in the food chain: human health implications. Asia Pacific Journal of Clinical Nutrition 16, 95.

Bu’Lock, J.D., 1961. Intermediary metabolism and antibiotic synthesis. Advances in Applied Microbiology 3, 293-342.

215

Buhler, W. (2015). Mechanisms Of fungicide resistance. Retrieved 11/28/2015, from http://pesticidestewardship.org/resistance/FungicideResistance/Pages/Mechanis ms-of-Fungicide-Resistance.

Bu'Lock, J.D., 1967. Essays in Biosynthesis and Microbial Development. John Wiley, New York.

Bu'Lock, J.D., 1975. Industrial Mycology. In D.R. Berry, J.E. Smith (Eds.), The Filamentous Fungi. Vol. 1. Edward Arnold Ltd, London, pp. 33.

Bu'Lock, J.D., 1961. Intermediary metabolism and antibiotic synthesis. Advances in Applied Microbiology 3, 293.

Butchko, R.A., Plattner, R.D., Proctor, R.H., 2006. Deletion analysis of FUM genes involved in tricarballylic ester formation during fumonisin biosynthesis. Journal of Agricultural and Food Chemistry 54, 9398.

Cabras, P., Conte, E., 2001. Pesticide residues in grapes and wine in Italy, Food Additives & Contaminants. Food Additives & Contaminants 18, 880-885.

Caldas, E.D., Silva, A.C.S., 2007. Mycotoxins in corn-based food products consumed in Brazil: An exposure assessment for fumonisins. Journal of Agricultural and Food Chemistry 55, 74-79.

Calvo, A.M., Wilson, R.A., Bok, J.W., Keller, N.P., 2002. Relationship between secondary metabolism and development. Microbiology and Molecular Biology Reviews 66, 447-459.

Campbell, L.R., Dayton, D.H., Sohal, G.S., 1986. Neural tube defects: A review of human and animal studies on the etiology of neural tube defects. Teratology 34, 171-187.

Capehart, T. (2015). Corn background. Retrieved 1/1/2013, from http://www.ers.usda.gov/topics/crops/corn/background.aspx.

Castella, G., Bragulat, M.R., Cabanes, F.J., 1999. Surveillance of fumonisins in maize- based feeds and cereals from Spain. . Journal of Agriculture and Food Chemistry 47, 4707.

Castelo, M.M., Katta, S.K., Sumner, S.S., Hanna, M.A., Bullerman, L.B., 1998. Extrusion cooking reduces recoverability of fumonisin B1 from extruded corn grits. Journal of Food Science 63, 696.

Cawdell-Smith, A.J., Edwards, M.J., Bryden, W.L., 2007. Mycotoxins and abnormal embryonic development. In K.E. Panter, T.L. Wierenga, J.A. Pfister (Eds.), Poisonous Plants: Global Research and Solutions. CABI Publishing, Wallingford, UK. , pp. 525.

216

Chalfant, C.E., Spiegel, S., 2005. Sphingosine 1-phosphate and ceramide 1-phosphate: expanding roles in cell signaling. Journal of Cell Science 118, 4605-4612.

Chang, W.J., Rothberg, K.G., Kamen, B.A., Anderson, R.G., 1992. Lowering the cholesterol content of MA 104 cells inhibits receptor-mediated transport of folate. . Journal of Cellular Biology 118, 63-69.

Cheeke, P.R., 1998. Mycotoxins in cereal grains and supplements. In P.R. Cheeke (Ed.), Natural Toxicants in Feeds, Forages, and Poisonous Plants. Interstate Publishers, Inc, Danville, IL, pp. 87-136.

Choudhury, S.R., Nair, K.K., Kumar, R., Gogoi, R., Srivastava, C., 2010. Nanosulfur: A potent fungicide against food pathogen, Aspergillus niger.1276, 154.

Christensen, C.M., 1974. Storage of Cereal Grains and Their Products. American Association of Cereal Chemists, St. Paul, Minnesota.

Chulze, S.N., Magnoli, C.E., Dalcero, A.M., 2006. Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America. International Journal of Food Microbiology 111, 5-9.

Cirillo, T., Ritieni, A., Galvano, F., Amodio Cocchieri, R., 2003. Natural co-occurrence of deoxynivalenol and fumonisins B1 and B2 in Italian marketed foodstuffs. Food Additives & Contaminants 20, 566-571.

Cole, M., 2004. Food safety objectives: concept and current status.95, 13-20.

Cole, R.J., 1986. Occurrence and clinical manifestation of rubratoxins A& B and cyclopiazonic acid. , In J.L. Richard, J.R. Thurston (Eds.), Diagnosis of Mycotoxicoses. Martinus Nijhoff, Boston, 54-67.

Cole, R.J., 1986. Etiology of turkey “X” disease in retrospect: a case for the involvement of cyclopiazonic acid. Mycotoxin Research 2, 3-7.

Council for Agricultural Science and Technology, 2003. Mycotoxins: Risks in Plant, Animal and Human Systems. Ames, IA. 139.

Cox, R.J., 2007. Polyketides, proteins and genes in fungi: programmed nano-machines begin to reveal their secrets. Organic and Biomolecular Chemistry 5, 2010-2015.

Dalbo, M.A., Weeden, N.F., Reisch, B.I., 2000. QTL analysis of disease resistance in interspeciﬁc hybrid grapes. Acta Horticulturae 528, 215-219. de Swarte, C., Donker, R.A.,2005. Towards an FSO/ALOP based food safety policy. Food Control 6, 825-830.

Dermen, H.,1 964. Cytogenetics in hybridization of bunch and muscadine-types grapes. Society for Economic Botanist 18, 137-148.

217

Desjardins, A.E., 2006. Fusarium Mycotoxins: Chemistry, Genetics and Biology, American Phythopathological Society Press, St. Paul, MN.

Dijksterhuis, J., Samson, R.A., 2007. Food mycology: a multifaceted approach to fungi and food, CRC Press, Boca Raton, FL.

Dodds, D.R., Gross, R.A., 2007. Chemicals from biomass. Science 318, 1250.

Doko, M.B., Rapior, S., Visconti, A., Schjøth, J.E., 1995. Incidence and levels of fumonisin contamination in maize genotypes grown in Europe and Africa. Journal of Agriculture and Food Chemistry 43, 429.

Dorrenhaus, A., Flieger, A., Golka, K., Schulze, H., Albrecht, M., Degen, G.H., Follman, W., 2000. Induction of unscheduled DNA synthesis in primary human urothelial cells by the mycotoxin ochratoxin A. Toxicological Sciences 53, 271-277.

Dragan, Y.P., Bidalack, W.R., Cohen, S.M., Goldsworthy, T.L., Hard, G.C., Howard, P.C., Riley, R.T., Voss, K.A., 2001. Implications of apoptosis for toxicity, carcinogenicity, and risk assessment: fumosisin B1 as an example Journal of toxicology science 61, 6-17.

Dutcher, J., 2001. Insect Pests. In F.M. Basiouny, D.G. Himelrick (Eds.), Muscadine Grapes. ASHS Press, Alexandria, VA.

Eaton, D.L., Groopman, J.D., 1994. The Toxicology of Aflatoxins, Academic Press, New York.

Elis, J., DiPaolo, J.A., 1967. Aflatoxin B1induction of malformation. Archives of Pathology 83, 53. el-Nezami, H.S., Nicoletti, G., Neal, G.E., Donohue, D.C., Ahokas, J.T.,1995. Aflatoxin M1 in human breast milk samples from Victoria, Australia and Thailand. Food Chemistry and Toxicology 33, 173.

Elortza, F., Nuhse, T., Foster, L.J., Stensballe, A., Peck, S.C., Jensen, O.N., 2003. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins. . Molecular & Cellular Proteomics 2, 1261-1270.

Emmett, R.W., Parbery, D.G., 1975. Appressoria. Annual Review of Phytopathology 13, 147-165.

Enongene, E.N., Sharma, R.P., Bhandari, N., Miller, J.D., Meredith, F.I., Voss, K.A., Riley, R.T., 2002. Persistence and reversibility of the elevationin free sphingoidbases induced by fumonisin inhibition of ceramide synthase. Toxicological Sciences 67, 173-181.

218

Falk, S.P., Pearson, R.C., Gadoury, D.M., Seem, R.C., Sztejnberg, A., 1996. Fusarium proliferatum as a biocontrol agent against grape downy mildew. Phytopathology 86, 1010-1017.

FAO (Food and Agriculture Organization of the United Nations). 2006. Food Safety risk analysis. A guide for national food safety authorities. Retrieved 5/15/15, from http://www.who.int/foodsafety/publications/micro/riskanalysis06.pdf.

Fernandez-Surumay, G., Osweiler, G.D., Yaeger, M.J., Rottinghaus, G.E., Hendrich, S., Buckley, L.K., 2005. Fumonisin B–glucose reaction products are less toxic when fed to swine. Journal of Agricultural and Food Chemistry 53, 4264.

Fernandes, A., Ratola, N., Cerdeira, A., Alves, A., Venâncio, A., 2007. Changes in Ochratoxin A concentration during winemaking. American Journal of Enology and Viticulture 58, 92-96.

Fishel, F.M., Dewdney, M.M., 2015. Fungicide Resistance Action Committee's (FRAC) Classification Scheme of Fungicides According to Mode of Action. #PI94. Retrieved 5/29/2013, from http://edis.ifas.ufl.edu/pi131.

Freshney, R.I., 2010. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications John Wiley and Sons, Hoboken, NJ, USA.

Frisvad, J.C., Larsen, T.O., Thrane, U., Meijer, M., Varga, J., Samson, R.A., Nielsen, K.F., 2011. Fumonisin and ochratoxin production in industrial Aspergillus niger strains. PLoS One 6, e23496.

Frisvad, J.C., Smedsgaard, J., Samson, R.A., Larsen, T.O., Thrane, U., 2007. Fumonisin B2 production by Aspergillus niger. Journal of Agricultural and Food Chemistry 55, 9727-9732.

Frost, G.M., Moss, D.A., 1987. Production of enzymes by fermentation. , In H.J. Rehm, G. Reed (Eds.), Biotechnology. VCH, Weinheim, pp. 65.

Fujii, I., Watanabe, A., Sankawa, U., Ebizuka, Y., 2001. Identification of Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans. Chemistry & Biology 8, 189.

Fung, F., Clark, R.F., 2004. Health effects of mycotoxins: a toxicological overview. Journal of Toxicology Clinical Toxicology 42, 217-234.

Galvano, F., Galofaro, V., Galvano, G., 1996. Occurrence and stability of aflatoxin M1 in milk and milk products. A worldwide review. Journal of Food Protection 59, 1079.

García-Cela, E., Ramos, A.J., Sanchis, V., Marin, S., 2012. Emerging risk management metrics in food safety: FSO, PO. How do they apply to the mycotoxin hazard? Food Control, 797-808.

219

Gareis, M., Martlbauer, E., Bauer, J., Gedek, B., 1988. Determination of ochratoxin A in human milk. Z. Lebensm. Unters. Forsch. 186, 114.

Gelderblom, W.C.A., Riedel, S., Burger, H.M., Abel, S., Marasas, W.F.O., 2008. Carcinogensis by the Fumonisins: Mechanism, Risk analysis, and Implications. In D.P. Siantar, M.W. Trucksess, P.M. Scott (Eds.), Food Contaminants-mycotoxins and food allergens. . American Chemical Society, Michigan, pp. 80-95.

Gelderblom, W.:Cawood,M.: Snyman,S.:Vleggaar,R:Marasas,W.,1993. Structure- activity relationships of fumonisins in short-term carcinogenesis and cytotoxicity assays. Food Chemistry and Toxicology 31, 407.

Gelderblom, W.C., Jaskiewicz, K., Marasas, W.F., Thiel, P.G., Horak, R.M., Vleggaar, R., Kriek, N.P., 1988. Fumonisins--novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme. Applied and Environmental Microbiology 54, 1806-1811.

Gerber, R., Lou, L., Du, L., 2009. A PLP-dependent polyketide chain releasing mechanism in the biosynthesis of mycotoxin fumonisins in Fusarium verticillioides. Journal of the American Chemical Society 131, 3148.

Girolamo, A., Solfrizzo, M., Holst, C., Visconti, A., 2001. Comparison of different extraction and clean-up procedures for the determination of fumonisins in maize and maize-based food products. . Food Additives and Contaminants 18, 59.

Gong, Y.Y., Cardwell, K., Hounsa, A., Egal, S., Turner, P.C., Hall, A.J., Wild, C.P., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. British Medical Journal 325, 20.

Grando, M.S., Bellin, D., Madini, A., Stefanini, M., Pozzi, C., Velasco, R., 2000. Construction of an AFLP and SSR genetic map of Vitis from an interspeciﬁc hybrid population.

Grassin, C., Fauguenbergue, P., 1999. Enzymes, fruit juice processing. In M.C. Flickinger, S.W. Drew (Eds.), Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation. Wiley & Sons, Inc, New York,

Gray, D.J.(2008. New plants for florida: Grapes. Retrieved 1/1/13, from http://edis.ifas.ufl.edu/ag208.

Gutleb, A.C., Morrison, E., Murk, A.J., 2002. Cytotoxicity assays for mycotoxins produced by Fusarium strains: a review. Environmental Toxicology and Pharmacology 11, 309-320.

Guzman-de Peña, D., Aguirre, J., Ruiz-Herrera, J., 1998. Correlation between the regulation of sterigmatocystin biosynthesis and asexual and sexual sporulation in Emericella nidulans. Antonie Leeuwenhoek 73, 199-205.

220

Hakomori, S., Igarashi, Y., 1995. Functional role of glycosphingolipids in cell recognition and signaling. Journal of Biochemistry (Tokyo) 118, 1091-1103.

Hanson, J.R., 2008. The chemistry of fungi, Royal Society of Chemistry, Cambridge, UK.

Harrison, L.R., Colvin, B.M., Green, J.T., Newman, L.E., Cole, J.R., 190. Pulmonary edema and hydrothorax in swine produced by fumonisin B1. A toxic metabolite of fusarium monililorme. Journal of Veterinary Diagnostic Investigation 2, 217.

Harwig, J., Blanchfield, B.J., Jarvis, G., 1977. Effect of aw on disappearance of patulin and citrinin from grains. Journal of Food Science 42, 1233.

Haschek, W.M., Motelin, G., Ness, D.K., Harlin, K.S., Hall, W.F., Vesonder, R.F., Peterson, R.E., Beasley, V.R., 1992. Characterization of fumonisin toxicity in orally and intravenously dosed swine. Mycopathologia 117, 83-96.

Haschek, W.M., Gumprecht, L.A., Smith, G., Tumbleson, M.E., Constable, P.D., 2001. Fumonisin toxicosis in swine: an overview of porcine pulmonary edema and current perspectives. Environmental Health Perspectives 109 Suppl 2, 251-257.

Hawksworth, D.L., 1991. The fungal dimension of biodiversity: Magnitude, significance, and conservation. Mycological Research 95, 641-655.

Hayden, N., Maude, R., 1992. The role of seed‐borne Aspergillus niger in transmission of black mould of onion. Plant Pathology 41, 573-581.

Hayes, A.W., Hood, R.D., Lee, H.L., 1974. Teratogenic effects of ochratoxin A in mice. Teratology 9, 93.

He, L., Nan, M.H., Oh, H.C., Kim, Y.H., Jang, J.H., Erikson, R.L., Ahn, J.S., Kim, B.Y., 2011. Asperlin induces G2/M arrest through ROS generation and ATM pathway in human cervical carcinoma cells. Biochemical and Biophysical Research Communications 409, 489-493.

Heathcote, J.G., Hibbert, J.R., 1978. Aflatoxins: Chemical and Biological Aspects, Elsevier, Amsterdam.

Hlywka, J.J., Bullerman, L.B., 1999. Occurrence of fumonisin B1 and B2 in beer. Food Additives & Contaminants 16, 319-324.

Hocking, A.D., Leong, S.L., Kazi, B.A., Emmett, R.W., Scott, E.S., 2007. Fungi and mycotoxins in vineyards and grape products. International Journal of Food Microbiology 119, 84-88.

221

Hopwood, D.A., 1997. Genetic contributions to understanding polyketide synthases. Chemical Reviews 97, 2465.

Huang, C., Dickman, M., Henderson, G., Jones, C., 1995. Repression of protein kinase C and stimulation of cyclic AMP response elements by fumonisin, a fungal encoded toxin which is a carcinogen. Cancer Research 55, 1655-1659.

Humpf, H.U., Schmelz, E.M., Meredith, F.I., Vesper, H., Vales, T.R., Menaldino, D.S., Liotta, D.C., Merrill, A.H., Merrill, A.H.,1998. Acylation of naturally occurring and synthetic 1-deoxysphinganines by ceramide synthase: formation of N-palmitoyl- aminopentol (PAP1) produces a toxic metabolite of hydrolyzed fumonisin (AP1), and a new category of ceramide synthase inhibitor. Journal of Biological Chemistry 273, 19060-19064.

Humpf, H.U., Voss, K.A., 2004. Effects of thermal food processing on the chemical structure and toxicity of fumonisin mycotoxins. Molecular Nutrition and Food Research 48, 255.

Hur, J.M., Yun, H.J., Yang, S.H., Lee, W.Y., Joe, M.H., Kim D., 2008. Gliotoxin enhances radiotherapy via inhibition of radiation-induced GADD45a, p38 and NFkappaB activation. Journal of Cellular Biochemistry, 2174-2184.

Inoue, T., Nagatomi, Y., Uyama, A., Mochizuki, N., 2013. Fate of mycotoxins during beer brewing and fermentation. Bioscience, Biotechnology, and Biochemistry 77, 1410-1415.

Instituto Brasileiro de Geografia e Estatística. (2013). Pesquisa de orçamentos familiares. Retrieved 05/17, 2013, from www.ibge.gov.br/home/estatistica/populacao/condicaodevida/pof/2008_2009_aq uisicao/tabelas_pdf/ tab111.pdf.

International Agency for Research on Cancer, 2002. IARC monographs on the evaluation of carcinogenic risks to humans.82, 301-366.

Jackson, L.S., Hlywka, J.J., Senthil, K.R., Bullerman, L.B., 1996. Effects of thermal processing on the stability of fumonisin B2 in an aqueous system. Journal of Agriculture and Food Chemistry 44, 1984-1987.

Jackson, L.S., Katta, S.K., Fingerhut, D.D., DeVries, J.W., Bullerman, L.B., 1997. Effects of baking and frying on the fumonisin B1 content of corn-based foods. Journal of Agriculture and Food Chemistry 45, 4800.

Jager, A.V., Tedesco, M.P., Souto, P.C., Oliveira, C.A., 2013. Assessment of aflatoxin intake in São Paulo, Brazil. Food Control 33, 87.

Jaskiewicî, K., van Rensburg, S.J., Marasas, W.F.O., Gelderblom, W.C.A., 1987. Carcinogenicity of Fusarium moniliforme culture material in rats. Journal of the National Cancer Institute 78, 321.

222

Joint Expert Committee on Food Additives (JEFCA). (2005). Fumonisins. Retrieved 9/25, 2015, from http://www.inchem.org/documents/jecfa/jecmono/v47je03.htm#6.0.

Jones, R.K., Duncan, H.E., Payne, G.A., Leonard, K.J., 1980. Factors influencing infection by Aspergillus flavus in silk-inoculated corn. Plant Disease Journal 64, 859.

Kabak, B., 2009. The fate of mycotoxins during thermal food processing. Journal of the Science of Food and Agriculture 89, 549.

Kawamura, C., Tsujimoto, T., Tsuge, T., 1999. Targeted disruption of a melanin biosynthesis gene affects conidial development and UV tolerance in the Japanese pear pathotype of Alternaria alternata. Molecular Plant-Microbe Interactions Journal 12, 59-63.

Kendricks, K., 1999. Fumonisins and neural tube defects in South Texas. Epidemiology 10, 198-200.

Khoury, M.J., Beaty, T.H., Liang, K.Y., 1988. Can familial aggregation of disease be explained by familial aggregation of environmental risk factors. American Journal of Epidemiology 127, 674-683.

Kim, E.K., Scott, P.M., Lau, B.P., 2003. Hidden fumonisin in corn-flakes. Food Additives and Contaminants 20, 161.

Kintz, P., 2004. Value of hair analysis in post mortem toxicology. Forensic Science International 142, 127-134.

Knudsen, P.B., Mogensen, J., M., Larsen, T.O., Nielsen, K.F., 2011. Occurrence of fumonisins B2 and B4 in retail raisins. Journal of Agriculture and Food Chemistry 59, 772.

Koca, N., Ekşi, A., 2005. Reduction of patulin in apple juice concentrates during storage. Journal of Food Safety 25, 1-8.

Kolb, E., 1984. Recent knowledge on the mechanism of action and metabolism of mycotoxins. Zeitschrift fur die Gesamte Innere Medizin und Ihre Grenzgebiete 39, 353-358.

Krapels, I.P., Pooij, I.A., Wevers, R.A., Zielhuis, G.A., Spauwen, P.H., Brussel, W., Steegers-Theuissen, R.P., 2004. Myo-inositol, glucose, and zinc status as risk factors for non-syndromic cleft lip with or without cleft palate in offspring: A case- control study. BJOG: An International Journal of Obstetrics and Gynecology 111, 661-668.

223

Kriek, N.P., Kellerman, S., Marasas, W.F.O., 1981. A comparative study of the toxicity of Fusarium verticiliioides to horses, primates, pigs, sheep and rats. Onderstepoort Journal of Veteranary Research 48

Kryger, R.A., 2001. Volatility of patulin in apple juice. Journal of Agriculture and Food Chemistry 49, 4143.

Lacey, J., Ramakrishna, N., Hamer, A., Magan, N., Marfleet, I.C., 1991. Grain fungi. In D.K. Aurora, G. Mukerji, E.H. Marth (Eds.), Handbook of Applied Mycology. Marcel Dekker, Inc., New York, pp. 121-177.

Leblanc, J.C., Tard, A., Volatier, J.L., Verger, P., 2005. Estimated dietary exposure to principal food mycotoxins from The First French Total Diet Study. Food Additives & Contaminants 22, 652-672.

LeBreton, E., Frayssinet, C., Lafarge, C., deRecondo, A.M., 1964. Aflatoxine- mecanisme de l’action. Food Cosmetics 2, 675-680.

Lee, D.S., Jeong, G.S., Li, B., Lee, S.U., Oh, H., Kim, Y.C., 2011. Asperlin from the marine-derived fungus Aspergillus sp. SF-5044 exerts anti-inflammatory effects through heme oxygenase-1 expression in murine macrophages. Journal of Pharmacological Sciences 116, 283-295.

Lee, J., Lee, D.G. 2015. Novel antifungal mechanism of resveratrol: Apoptosis inducer in Candida albicans. Current Microbiolgy 3,383-389.

Lehninger, A.L., Nelson, D.L., Cox, M.M., 2005. Lehninger principles of biochemistry, W.H. Freeman, New York.

Lemke, S.L., Ottinger, S.E., Ake, C.L., Mayura, K., Phillips, T.D., 2001. Deamination of fumonisin B1 and biological assessment of reaction product toxicity. Chemical Research in Toxicology 14, 11-15.

Leong, S., Hocking, A.D., Pitt, J.I., 2004. Occurrence of fruit rot fungi (Aspergillus section Nigri) on some drying varieties of irrigated grapes. Australian Journal of Grape and Wine Research 10, 83-88.

Leong, S.L., Hocking, A.D., Scott, E.S., 2006. Effect of temperature and water activity on growth and ochratoxin A production by Australian Aspergillus carbonarius and A. niger isolates on a simulated grape juice medium. International Journal of Food Microbiology 110, 209-216.

Lerda, D., Bistoni, B.M., Peralta, N., Ychari, S., Vazquez, M., Bosio, G., 2005. Fumonisins in foods from Cordoba (Argentina), presence and genotoxicity. Food Chemistry and Toxicology 43, 691-698.

224

Li, Z.T., Dhekney, S.A., Dutt, M., Van Aman, M., Tattersall, J., Kelley, K., Gray, D.J., 2007. Isolation and characterization of the 2S albumin gene and promoter from grapevine. Acta Horticulturae 738, 765.

Liao, L.L., Grollman, A.P., Horwitz, S.B., 1976. Mechanism of action of the 12, 13- epoxytrichothecene, anguidine, an inhibitor of protein synthesis. Biochimica et Biophysica Acta 454, 273-284.

Liao, W.Y., Shen, C.N., Lin, L.H., Yang, Y.L., Han, H.Y., Chen, J.W., Kuo, S.C., Wu, S.H., Liaw, C.C.,2012. Asperjinone, a nor-neolignan, and terrein, a suppressor of ABCG2-expressing breast cancer cells, from thermophilic Aspergillus terreus. Journal of Natural Products 75, 630-635.

Lindahl, B.D., Ihrmark, K., Boberg, J., Trumbore, S.E., Hogberg, P., Stenlid, J., Finlay, R.D., 2007. Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New Phytologist 173, 611-620.

Lindroth, S., 1980. Occurrence, formation and detoxification of patulin mycotoxin. Technical Centre of Finland, VTT, Espoo, Finland, 215.

Logrieco, A., Bottalico, A., Mule, G., Moretti, A., Perrone, G., 2003. Epidemiology of toxigenic fungi and their associated mycotoxins for some Mediterranean crops. European Journal of Plant Pathology 109, 645-667.

Logrieco, A., Doko, B., Moretti, A., Frisullo, S., Visconti, A., 1998. Occurrence of fumonisin B1 and B2 in Fusarium proliferatum infected asparagus plants. Journal of Agricultural and Food Chemistry 46, 5201-5204.

Logrieco, A., Ferracane, R., Visconti, A., Ritieni, A., 2010. Natural occurrence of fumonisin B2 in red wine from Italy. Food Additive Contamination Part A, 1136- 1141.

Logrieco, A., Ferracane, R., Haidukowsky, M., Cozzi, G., Visconti, A., Ritieni, A., 2009. Fumonisin B2 production by Aspergillus niger from grapes and natural occurrence in must. Food Additive Contamination Part A 26, 1495.

Logrieco, A.F., Haidukowski, M., Susca, A., Mulè, G., Munkvold, G.P., Moretti, A., 2013. Aspergillus section Nigri as contributor of fumonisin B2 contamination in maize. Food Additive Contamination Part A 31, 149.

Logrieco, A.F., Ferracane, R., Cozzi, G., Haidukowsky, M., Susca, A., Mulè, G., Ritieni, A., 2011. Fumonisin B2 by Aspergillus niger in the grape–wine chain: an additional potential mycotoxicological risk. Annals of microbiology 61, 1-3.

Lu, J., Schell, L., Lamikanra, S., 1993. Introgression of seedlessness from bunch grapes into muscadine grapes. Proceedings of the Florida State Horticultural Society 106, 124.

225

Maksimova, E., Yie, T.A., Rom, W.N., 2008. In vitro mechanisms of lovastatin on lung cancer cell lines as a potential chemopreventive agent. Lung 186, 45-54.

Malone, B.M., Richard, J.L., Romer, T., Johnasson, A.S., Whitaker, T., 1998. Fumonisin reduction in corn by cleaning during storage discharge. In L. O'Brien, A.B. Blakeney, A.S. Ross, C.W. Wrigley (Eds.), Cereals 98, Proceedings of the 48th Australian Cereal Chemistry Conference. Royal Australian Chemical Institute., North Melbourne, Australia, pp. 372-379.

Mansson, M., Klejnstrup, M.L., Phipps, R.K., Nielsen, K.F., Frisvad, J.C., Gotfredsen, C.H., Larsen, T.O., 2010. Isolation and NMR characterization of fumonisin B2 and a new fumonisin B6 from Aspergillus niger. Journal of Agricultural and Food Chemistry 58, 949-953.

Maragos, C.M., Richard, J.L., 1994. Quantitation and stability of fumonisins B1 and B2 in milk. Journal of AOAC International 77, 1162.

Marasas, W.F.O., 2001. Discovery and Occurence of the fumonisin: A historical perspective. Environmental Health Perspectives 109, 239-243.

Marasas, W., Kellerman, T.S., Gelderblom, W., Coetzer, J.A., Thiel, P., Van der Lugt, Jaco J, 1988. Leukoencephalomalacia in a horse induced by fumonisin B1 isolated from Fusarium moniliforme. Onderstepoort Journal of Veterinary Research 55:4,197-203.

Marasas, O., Nelson, P., Toussoun, T., 1984. Toxigenic Fusarium Species Identity and Mycotomcology. Pennsylvania State University Press, University Park, PA.

Marasas, W.F.O., Riley, R.T., Hendricks, K.A., Stevens, V.L., Sadler, T.W., Gelineau- van Waes, J., Missmer, S.A., Cabrera, J., Torres, O., Gelderblom, W.C.A., Bryden, W.L.,2004. Fumonisins disrupt sphingolipid metabolism, folate transport, and neural tube development in embryo culture and in vivo: a potential risk factor maize for human neural tube defects among populations consuming fumonisin- contaminated maize. Journal of Nutrition 134, 711.

Marin, S., Ramos, A.J., Vazquez, C., Sanchis, V., 2007. Contamination of pine nuts by fumonisin produced by strains of Fusarium proliferatum isolated from Pinus pinea. Letters in Applied Microbiology 44, 68-72.

Marocco, A., Gavazi, C., Pietri, A., Tabaglio, V., 2008. On fumonisin incidence in monoculture maize under no-till, conventional tillage and two nitrogen fertilization levels. Journal of the Science of Food and Agriculture 88, 1217.

Martins, M.L., Martins, H.M., Bernardo, F., 2001. Fumonisin B1 and B2 in black tea and medicinal plants. Journal of Food Protection 64, 1268-1270.

Martins, M.L., Martins, H.M., Bernardo, F., 2001. Fumonisins B1 and B2 in black tea and medicinal plants. Journal of Food Protection 64, 1268-1270.

226

Martirosyan, A., Clendening, J.W., Goard, C.A., Penn, L.Z., 2010. Lovastatin induces apoptosis of ovarian cancer cells and synergizes with doxorubicin: potential therapeutic relevance. BMC Cancer 10, 103.

Matossian, M.A.K., 1989. Poisons of the past: molds, epidemics, and history, Yale University Press, New Haven.

Mayura, K., Parker, S., Berndt, W.O., Phillips, T.D., 1984. Ochratoxin A induced teratogenisis in rats: partial protection by phenylalanine applied and environmental microbiology. Applied and Environmental Microbiology 48, 1186.

Mbugua, S.K., Gathumbi, J.K., 2004. The contamination of Kenyan lager beers with Fusarium mycotoxins. Journal of the Institute of Brewing 110, 227-229.

Merrill, A., Wang, E., Vales, T., Smith, E., Schroeder, J., Menaldino, D., Alexander, C., Crane, H., Xia, J., Liotta, D., 1996. Fumonisin: toxicity and sphingolipid biosynthesis. Advanced Experiments in Medical Biology 392, 297.

Merrill, A.H., Sweeley, C.C., 1996. Sphingolipids: metabolism and cell signaling. Lipoproteins and Membranes. In D.E. Vance, J.E. Vance (Eds.), Biochemistry of Lipids. Elvesier, New York, NY, pp. 43-73.

Merrill, A.H., Sullards, M.C., Wang, E., Voss, K.A., Riley, R.T., 2001. Sphingolipid metabolism: Roles in signal transduction and disruption by fumonisins. Environmental Health Perspectives 109, 283-289.

Mertens-Talcott, S.U., Percival, S.S., 2005. Ellagic acid and quercetin interact synergistically with resveratrol in the induction of apoptosis and cause transient cell cycle arrest in human leukemia cells. Cancer Letters 218, 141-151.

Micco, C., Ambruzzi, M.A., Miraglia, M., Brera, C., Onori, R., Benelli, L., 1991. Contamination of human milk with ochratoxin A. International Agency on Research on Cancer Scientific Publications 115, 105.

Mo, H., Xu, X., Yan, M., Zhu, Y., 2005. Microbiological analysis and antibacterial effects of the indigenous fermented Puer tea. Agro Food Industry Hi-Tech 16, 16-18.

Mogensen, J.M., Larsen, T.O., Nielsen, K.F., 2010. Widespread occurrence of the mycotoxin fumonisin B2 in wine. Journal of Agricultural and Food Chemistry 58, 4853-4857.

Mogensen, J.M., Frisvad, J.C., Thrane, U., Nielsen, K.F., 2010. Production of Fumonisin B2 and B4 by Aspergillus niger on grapes and raisins. Journal of Agriculture and Food Chemistry 58, 954.

Mogensen, J.M., Nielsen, K.F., Samson, R.A., Frisvad, J.C., Thrane, U., 2009. Effect of temperature and water activity on the production of fumonisins by Aspergillus niger and different Fusarium species. BMC Microbiology 9, 281.

227

Molinie´, A., Faucet, V., Castegnaro, M., Pfohl-Leskowicz, A., 2005. Analysis of some breakfast cereals on the French market for their contents of ochratoxin A, citrinin and fumonisin B1: Development of a method for simultaneous extraction of ochratoxin A and citrinin. Food Chemistry 92, 391.

Moreno, E.C., Garcia, G.T., Ono, M.A., Vizoni, E., Kawamura, O., Hirooka, E.Y., 2009. Co-occurrence of mycotoxins in corn samples from the Northern region of Paraná State, Brazil. Food Chemistry 116, 220.

Morgavi, D.P., Riley, R.T., 2007. An historical overview of field disease outbreaks known or suspected to be caused by consumption of feeds contaminated with Fusarium toxins. Animal Feed Science and Technology 137, 201-212.

Morris, J.R., Brady, P.L., 2004. Temperature effects on produce degradation. In O. Lamikanra (Ed.), Produce Degradation: Reaction Pathways and Their Prevention. CRC Press, Boca Raton, Fl.,

Moss, M.O., M.T., 2002. Fate of patulin in the presence of the yeast Saccharomyces cerevisiae. Food Additives & Contaminants 19, 387-399.

Moyer, A.J., 1953. Effect of alcohols on the mycological production of citric acid in surface and submerged culture. I. Nature of the alcohol effect. Applied Microbiology 1, 1-7.

Munkvold, G.P., Desjardins, A.E., 1997. Fumonisins in maize: Can we reduce their occurrence? Plant Disease Journal 81, 556-565.

Murphy, M.M., Pickett, T.A., Cowart, F.F. 1938. Muscadine grapes: Culture, varieties, and some properties of juices. Georgia Agricultural Experiment Station Bulletin 199

Musser, S.M., Gay, M.L., Mazzola, E.P., Plattner, R.D., 1996. Identification of a new series of fumonisins containing 3-hydroxypyridine. Journal of natural products 59, 970-972.

Navarro, S., Noyes, R., Jayas, D.S., 2002. Stored Grain Ecosystem, and Heat and Moisture Transfer in Grain Bulks, In S. Navarro, R. Noyes (Eds.), The Mechanics and Physics of Modern Grain Aeration Management. CRC Press, Boca Raton, pp. 56.

Niderkorn, V., Morgavi, D.P., Aboab, B., Lemaire, M., Boudra, H., 2009. Cell wall component and mycotoxin moieties involved in the binding of fumonisin B1 and B2 by lactic acid bacteria. Journal of Applied Microbiology 106,977-985.

Noonim, P., Mahakarnchanakul, W., Nielsen, K.F., Frisvad, J.C., Samson, R.A., 2009. Fumonisin B2 production by Aspergillus niger in Thai coffee beans. Food Additive Contamination Part A 26, 94-100.

228

Norred, W.P., Plattner, R.D., Chamberlain, W.J., 1993. Distribution and excretion of [14C] fumonisin B1 in male Sprague–Dawley rats. Natural Toxins, 341-346.

Olien, W.C., 2001. Introduction to the muscadine. , In F.M. Basiouny, D.G. Himelrick (Eds.), Muscadine Grapes. ASHS Press, Alexandria, VA., pp. 150-167.

Olien, W., Hegwood, C., 1990. Muscadine-A classic southeastern fruit. HortScience 25, 726.

Orsolic, N., 2012. Bee venom in cancer therapy. Cancer Metastasis Review, 173-194.

Oswald, I.P., Martin, D.E., Bouhet, S., Pinton, P., Taranu, I., Accensi, F., 2005. Immunotoxiological risk of mycotoxins for domestic animals. Food Additive Contamination Part A 22, 354.

Ough, C.S., Corison, C.A., 1980. Measurement of patulin in grapes and wine. Journal of Food Science 45, 476-478.

Palencia, E.R., Klich, M.A., Glenn, A.E., Bacon, C.W., 2009. Use of a rep-PCR system to predict species in the Aspergillus section Nigri. Journal of Microbiological Methods 79, 1-7.

Palencia, E.R., Hinton, D.M., Bacon, C.W., 2010. The black Aspergillus species of maize and peanuts and their potential for mycotoxin production. Toxins 2, 399.

Park, J.W., Choi, S.Y., Hwang, H.J., Kim, Y.B., 2005. Fungal mycoflora and mycotoxins in Korean polished rice destined for humans. International Journal of Microbiology 103, 305-314.

Park, J.W., Scott, P.M., Lau, B.P., Lewis, D.A., 2004. Analysis of heat-processed corn foods for fumonisins and bound fumonisins. Food Additives and Contaminants 21, 1168.

Paszkowski, U., 2006. Mutualism and parasitism: the yin and yang of plant symbioses. Current Opinion in Plant Biology 9, 364-370.

Payne, G.A., Hagler, W.M., Adkins, C.R., 1988. Aflatoxin accumulation in inoculated ears of field-grown maize. Plant Disease Journal 72, 422-424.

Pazoutova, S., Pokorny, V., Rehacek, Z., 1977. The relationship between conidiation and alkaloid production in saprophytic strains of Claviceps purpurea. Canadian Journal of Microbiology 23, 1182-1187.

Perkins- Veazie, P., 2002. Grapes (Muscadine). , In K.C. Gross, C.Y. Yang, M. Saltveit (Eds.), The Commercial Storage of Fruits, Vegetables, and Florist and Nursery Crops. Agricultural Research Service, Beltsville, MD.

229

Perrone, G., Mule, G., Susca, A., Battilani, P., Pietri, A., Logrieco, A., 2006. Ochratoxin A production and AFLP analysis of Aspergillus carbonarius, A. tubigensis and A. niger strains isolated from grape in Italy. Applied Environmental Microbiology 72, 680-685.

Perrone, G., Susca, A., Epifani, F., Mule, G., Logrieco, A.F., 2007. Biodiversity of Aspergillus Section Nigri from grapes in Europe. CBS, Utrecht, The Netherlands, 39-41.

Pestka, J.J., Zhou, R.R., Moon, Y., Chung, Y.J., 2004. Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other tricothecenes; unraveling a paradox. Toxicology Letters 153, 61.

Petrini, O., G.B., 1994. Host Wall Alterations by Parasitic Fungi, APS Press, St. Paul, MN.

Pitt, J.I., Hocking, A.D., 2009. Fungi and food spoilage, Springer, New York.

Pittet, A., Parisod, V., Schellenberg, M., 1992. Occurrence of fumonisin B1 and B2 in corn-based products from the Swiss market. Journal of Agriculture and Food Chemistry 40, 1352.

Pohland, A.E., Allen, R., 1970. Stability studies with patulin. Journal of the Association of Official Analytical Chemist 53, 688.

Prelusky, D.B., Miller, J.D., Trenholm, H.L., 1996. Disposition of 14C-derived residues in tissues of pigs fed radio-labelled fumonisin B1. Food Additive Contamination Part A 13, 155-162.

Prelusky, D.B., Savard, M.E., Trenholm, H.L., 1995. Pilot study on the plasma pharmacokinetics of fumonisin B1 in cows following a single dose by oral gavage or intravenous administration. Natural Toxins 3, 389-394.

Prelusky, D.B., Trenholm, H.L., Savard, M.E., 1994. Pharmacokinetic fate of 14C- labelled fumonisin B1 in swine. Natural Toxins, 73-80.

Proctor, R.H., Brown, D.W., Plattner, R.D., Desjardins, A.E., 2003. Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis. Fungal Genetics and Biology 38, 237-249.

Proctor, R.H., Plattner, R.D., Desjardins, A.E., Busman, M., Butchko, R.A., 2006. Fumonisin production in the maize pathogen Fusarium verticillioides: genetic basis of naturally occurring chemical variation. Journal of Agricultural and Food Chemistry 54, 2424-2430.

230

Produce for Better Health Foundation. 2015. State of the plate, 2015 study on America’s consumption of fruit and vegetables. Retrieved 2/2/16, from http://www.PBHFoundation.org

Puel, O., Galtier, P., Oswald, I.P., 2010. Biosynthesis and toxicological effects of patulin. Toxins 2, 613-631.

Ramljak, D., Calvert, R.J., Wiesenfeld, P.W., Diwan, B.A., Catipovic, B., Marasas, W.F., Victor, T.C., Anderson, L.M., Gelderblom, W.C.A., 2000. A potential mechanism for fumonisin B1- mediated hepatocarcinogenesis: cyclin D1 stabilization associated with activation of Akt and inhibition of GSK-3beta activity. Carcinogenesis, 1537-1546.

Raper, K.B., Fennell, D.I., 1965. The genus Aspergillus. Williams & Wilkins Co., Baltimore, MD.

Ratola, N., Abade, E.E., Simoes, T., Venancio, A., Alves, A., 2005. Evolution of ochratoxin A content from must to wine in port wine microvinification. Analytical and Bioanalytical Chemistry, Special Issue 382, 405-411.

Ren, Z., Lu, J., 2003. Muscadine vine dieback: Emerging problem and possible control measures. Proc. Florida State Horticultural Society. Proceedings of the Florida State Horticultural Society 116, 8-10.

Rheeder, J.P., Marasas, W.F., Vismer, H.F., 2002. Production of fumonisin analogs by Fusarium species. Applied and Environmental Microbiology 68, 2101-2105.

Riaz, S., Meredith, C.P., 2000. A microsatellite marker based linkage map of Vitis vinifera.

Rice, S.L., Beuchat, L.R., Worthington, R.E., 1977. Patulin production by Byssochlamys spp. in fruit juices. Applied Environmental Microbiology 34, 791-796.

Richard, J.L., Meerdink, G., Maragos, C.M., Tumbleson, M., Bordson, G., Rice, L.G., Ross, P.F., 1996. Absence of detectable fumonisins in the milk of cows fed Fusarium proliferatum (Matsushima) Nirenberg culture material. Mycopathologia 133, 123-126.

Riley, R.T., Hinton, D.M., Chamberlain, W.J., Bacon, C.W., Wang, E., Merrill, A.H., Voss, K.A., 1994. Dietary fumonisin B1 induces disruption of sphingolipid metabolism in Sprague-Dawley rats: a new mechanism of nephrotoxicity. Journal of Nutrition 124, 594-603.

Riley, R.T., Voss, K.A., 2006. Differential sensitivity of rat kidney and liver to fumonisin toxicity: organ-specific differences in toxin accumulation and sphingoid base metabolism. Toxicological Sciences, 335-345.

231

Riley, R., Enongene, E., Voss, k., Norred, W., Meredith, F., Sharma, R., Spitsbergen, J., Williams, D., Carlson, D., Merrill, A., 2001. Sphingolipid perturbations as mechanisms for fumonisin carcinogenesis. Environmental Health Perspectives 109, 301.

Rio, B., Lautraite, S., Parent-Massin, D., 1997. In vitro toxicity of trichothecenes on human erythroblastic progenitors. Human & Experimental Toxicology 16, 673- 679.

Rocha, L.O., Nakai, V.K., Brahini, R., Reis, T.A., Kobashigawa, E., Corrêa, B., 2009. Mycoflora and co-occurrence of fumonisins and aflatoxins in freshly harvested corn in different regions of Brazil. International Journal of Molecular Science 10, 5090.

Rooney-Latham, S., Janousek, C.N., Eskalen, A., Gubler, W.D., 2008. First report of Aspergillus carbonarius causing sour rot of table grapes (Vitis vinifera) in California. Plant Disease Journal 92, 651.

Ruediger, G.A., Pardon, K.H., Sas, A.N., Godden, P.W., Pollnitz, A.P., 2004. Removal of pesticides from red and white wine by the use of fining and filter agents. Australian Journal of Grape and Wine Research 10, 8-16.

Ruijter, G.J.G., Kubicek, C.P., Vissler, J., 2002. Production of organic acids by fungi. In H.D. Osiewacz (Ed.), The Mycota. Heidelberg: Springer-Verlag,pp. 213.

Sage, L., Krivobok, S., Delbos, E., Seigle-Murandi, F., Creppy, E.E., 2002. Fungal flora and ochratoxin A production in grapes and musts from France. Journal of Agricultural and Food Chemistry 50, 1306-1311.

Samarajeewa, U., Sen, A.C., Cohen, M.D., Wei, C.I., 1990. Detoxification of aflatoxins in foods and feeds by physical and chemical methods. Journal of Food Protection 53, 489.

Samson, R.A., 1992. Current taxonomic schemes of the genus Aspergillus and its teleomorphs. Biotechnology 23, 355-390.

Sanchez, L.T., Carrillo, L.L., 2010. Fumonisin consumption and damage to human health. Public Health Mexico, 52.

Sands, D.C., McIntyre, J.L., Walton, G.S., 1976. Use of activated charcoal for the removal of patulin from cider. Applied and Environmental Microbiology 32, 388- 391.

Schardl, C.L., Panaccione, D.G., Tudzynski, P., 2006. The alkaloids – biology and molecular biology, Elvesier, Oxford U.K.

232

Schimmel, T.G., Coffman, A.D., Parsons, S.J., 1998. Effect of butyrolactone I on the producing fungus, Aspergillus terreus. Applied and Environmental Microbiology 64, 3703-3712.

Schuster, E., Dunn-Coleman, N., Frisvad, J., Van Dijck, P., 2002. On the safety of Aspergillus niger–a review. Applied Microbiology and Biotechnology 59, 426-435.

Schwerdt, G., Freudinger, R., Mildenberger, S., Silbernagl, S., Gekle, M., 1999. The nephrotoxin ochratoxin A induces apoptosis in cultured human proximal tubule cells. Cell Biology and Toxicology 15, 405-415.

Scott, P.M., Yeung, J.M., Lawrence, G.A., Prelusky, D.B., 1997. Evaluation of enzyme- linked immunosorbent assay for analysis of beer for fumonisins. Food Additives & Contaminants 14, 445-452.

Scott, P.M., 1984. Effects of food processing on mycotoxins. Journal of Food Protection 47, 489.

Scott, P.M., Kanhere, S.R., Lawrence, G.A., Daley, E.F., Farber, J.M., 1995. Fermentation of wort containing added ochratoxin A and fumonisins B1 and B2. Food Additives & Contaminants 12, 31-40.

Scott, P.M., Lawrence, G.A., 1994. Stability and problems in recovery of fumonisins added to corn-based foods. Journal of AOAC International 77, 541.

Scott, P.M., Somers, E., 1968. Stability of patulin and penicillic acid in fruit juices and flour. Journal of Agriculture and Food Chemistry 483

Scott, P.M., Yeung, J.M., Lawrence, G.A., Prelusky, D.B., 1997. Evaluation of enzyme- linked immunosorbent assay for analysis of beer for fumonisins. Food Additives & Contaminants 14, 445-450.

Scudamore, K.A., Patel, S., 2000. Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom. Food Additives and Contaminants 17, 407.

Seo, J., Kim, J., Lee, Y., 1996. Isolation and characterization of two new type C fumonisins produced by Fusarium oxysporum. Journal of Natural Products 59, 1003-1005.

Serra, R., Braga, A., Venâncio, A., 2005. Mycotoxin-producing and other fungi isolated from grapes for wine production, with particular emphasis on ochratoxin A. Research in Microbiology 156, 515-521.

Sewrama, V., Mshicilelia, N., Shepharda, G.S., Marasasa, W.F.O., 2003. Fumonisin mycotoxin in hair. Biomarkers 8, 110-118.

233

Shen, H.M., Ong, C.N., Shi, C.Y., 1995. Involvement of reactive oxygen species in aflatoxin B1-induced cell injury in cultured rat hepatocytes. Toxicology 99, 115- 123.

Shephard, G.S., van der Westhuizen, L., Gatyeni, P.M., Somdyala, N.I.M., Burger, H.M., Marasas, W.F.O., 2005. Fumonisin mycotoxins in traditional xhosa maize beer in South Africa. . Journal of Agricultural and Food Chemistry 53, 9634-9637.

Siantar, D.P., Trucksess, M.W., Scott, P.M., Herman, E.M., 2008. Food Contaminants: Mycotoxins and Food Allergens., American Chemical Society, Washington, DC.

Silva, L.J.G., Lino, C.M., Pena, A., Molto, J.C., 2007. Occurrence of fumonisins B1 and B2 in Portuguese maize and maize-based foods intended for human consumption. Food Additives and Contaminants 24, 381.

Sims, C.A., Morris, J.R., 1985. pH effects on the color of wine from two grape species. Arkansas Farm Research 34, 9.

Sims, C.A., Morris, J.R., 1984. Effects of pH, sulfur dioxide, storage time, and temperature on the color and stability of red muscadine grape wine. American Journal of Enology and Viticulture 35, 35.

Sistrunk, W.A., Morris, J.R., 1982. Influence of cultivar, extraction and storage temperature, and time on quality of muscadine grape juice. Journal of the American Society of Horticultural Sciences 107, 1110.

Skaug, M.A., Helland, I., Solvoll, K., Saugstad, O.D., 2001. Presence of ochratoxin A in human milk in relation to dietary intake. Food Additive Contamination Part A 18, 321.

Smith, G.W., Constable, P.D., Tumbleson, M.E., Rottinghaus, G.E., Haschek, W.M., 1999. Sequence of cardiovascular changes leading to pulmonary edema in swine fed culture material containing fumonisin. American Journal of Veterinary Research 60, 1292-1300.

Smith, J., Thakur, R., 1996. Occurrence and fate of fumonisins in beef. , In L. Jackson (Ed.), Fumonisins in Food. Plenum Press, New York, pp. 39-55.

Smith, J.E., Moss, M.O., 1985. Mycotoxins. Formation, analysis and significance. John Wiley and Sons, Toronto, Canada.

Spiegel, S., Milstien, S., 2002. Sphingosine 1-Phosphate, a key cell signaling molecule. The Journal of Biological Chemistry 277, 25851-25854.

Stack, M.E., 1998. Analysis of fumonisin B1 and its hydrolysis product in tortillas. Journal of AOAC International 81, 737-740.

234

Staunton, J., Weissman, K.J., 2001. Polyketide biosynthesis: a millennium review. Natural Product Reports 18, 380.

Stevens, V.L., Tang, J., 1997. Fumonisin B1-induced sphingolipid depletion inhibits vitamin uptake via the glycosylphosphatidylinositol-anchored folate receptor. Journal of Biological Chemistry 272, 18020-18025.

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.

Stringer, M., 2005. Food safety objectives e role in microbiological food safety management. Food Control 16, 775-794.

Susca, A., Moretti, A., Stea, G., Villani, A., Haidukowski, M., Logrieco, A.F., Munkvold, G.P., 2014. Comparison of species composition and fumonisin production in Aspergillussection Nigri populations in maize kernels from USA and Italy. International Journal of Food Microbiology 188, 75-82.

Susca, A., Moretti, A., Stea, G., Villani, A., Haidukowski, M., Logrieco, A., Munkvold, G., 2014. Comparison of species composition and fumonisin production in Aspergillus section Nigri populations in maize kernels from USA and Italy. International Journal of Food Microbiology 188, 75.

Susca, A., Proctor, R., Mulè, G., Stea, G., Ritieni, A., Logrieco, A., Moretti, A., 2010. Correlation of mycotoxin fumonisin B2 production and presence of the fumonisin biosynthetic gene fum8 in Aspergillus niger from grape. Journal of Agricultural and Food Chemistry 58, 9266.

Suzuki, T., Takeda, M., Tanabe, H., 1971. A new mycotoxin produced by Aspergillus clavatus. Chemical and Pharmaceutical Bulletin 19, 1786-1788.

Sydenham, E.W., Shephard, G.S., Thiel, P.G., Marasas, W.F.O., Stockenstro, M. S., 1991. Fumonisins contamination of commercial corn-based human foodstuffs. Journal of Agriculture and Food Chemistry 39, 2014.

Talcott, S.T., Lee, J.H., 2002. Ellagic acid and flavonoid antioxidant content of muscadine wine and juice. Journal of Agricultural and Food Chemistry 50, 3186- 3192.

Taylor, K. (2016). The 4 biggest ways American beverage consumption will change in 2016. Retrieved 2/2/16, from http://www.businessinsider.com/4-big-beverage- industry-trends-in-2016-2015-12

Threlfall, R., 2002. Preliminary findings – Nutraceutical content of juices from several muscadine varieties. Initiative for Future Agriculture and Food Systems Project Coordination Meeting, Biloxi, MS.

235

Tian, Y., Griffiths, D., 1993. N-alkyl pyridiniums as inhibitors of Complex 1. Biochemical Society Transactions 22, 71S.

Torres, M.R., Sanchis, V., Ramos, A.J., 1998. Occurrence of fumonisins in Spanish beers analyzed by an enzyme-linked immunosorbent assay method. International Journal of Food Microbiology 39, 139-143.

Tucker, S.L., Talbot, N.J., 2001. Surface attachment and pre-penetration stage development by plant pathogenic fungi. Annual Review of Phytopathology 39, 385-417.

Tudzynski, P., Tenberge, K.B., Oeser, B., 1995. Claviceps purpurea., In K. Kohmoto, Singh, U.S., Singh, R.P. (Eds.), Pathogenesis and Host Specificity in Plant Diseases: Histopathological, Biochemical, Genetic and Molecular Bases. Elsevier Science Ltd., Tarrytown, New York, pp. 161-187.

Turner, W.B., 1978. Fungal Metabolites. Academic Press, London, U.K.

Turner, W.B., Alderidge, D.C., 1983. Fungal Metabolites II, Academic Press, London, U.K.

Ueno, Y., 1977. Mode of action of trichothecenes. Pure Applied Chemistry 49, 1737- 1745.

United States Food and Drug Administration. 2001. Guidance for industry: Fumonisin levels in human foods and animal feeds; final guidance. Retrieved 2/8/13, from http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceD ocuments/ChemicalContaminantsandPesticides/ucm109231.htm/)

United States Food and Drug Administration. 2004. Guidance for Industry: Juice HACCP Hazards and Controls Guidance First Edition; Final Guidance. Retrieved 1/15/13, from http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInf ormation/Juice/ucm072557.htm van Waes, J.G., Voss, K.A., Stevens, V.L., Speer, M.C., Riley, R.T., 2009. Maternal fumonisin exposure as a risk factor for neural tube defects. In S.L. Taylor (Ed.), Advances in Food and Nutrition Research. Elvesier, Oxford UK, pp. 145.

Varga, J., Juhasz, A., Kevei, F., Kozakiewicz, Z., 2004. Molecular diversity of agriculturally important Aspergillus species. European Journal of Plant Pathology 110, 627-640.

Varga, J., Kozakiewicz, Z., 2006. Ochratoxin A in grapes and grape-derived products. Trends in Food Science and Technology 17, 72-81.

236

Vasanthaiah, H., Thangadurai, D., Basha, S.M., Biradar, D.P., Kambirnada, D., Louime, C., 2011. Muscadiniana. In C. Kole (Ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits. Springer-Verlag, New York.

Vesonder, R.F., Ciegler, A., Jensen, A.H., Rohwedder, W.K., Wiesleer, D., 1976. Co- identity of the refusal and emetic principle from Fusarium-infected corn. Applied and Environmental Microbiology 31, 280.

Viljoen, D.l., Buccimazza, S., Dunne, T., Molteno, C., 1995. The prevalence and prevention of neural tube defects in Cape Town. South African Medical Journal 85, 630-632.

Visconti, A., Doko, M.B., Bottalico, C., Schurer, B., Boenke, A., 1994. Stability of fumonisins (FB1 and FB2) in solution. Food Additives & Contaminants 11, 427- 431.

Visconti, A., Perrone, G., Cozzi, G., Solfrizzo, M., 2008. Managing ochratoxin A risk in the grape-wine food chain. Food Additives & Contaminants: Part A 25, 193-202.

Visconti, A., Doko, M., Bottalico, C., Schurer, B., Boenke, A., 1994. Stability of fumonisins (FB1 and FB2) in solution. Food Additives & Contaminants 11, 427- 431.

Voss, K.A., Smith, G.W., Haschek, W.M., 2007. Fumonisins: Toxicokinetics, mechanism of action and toxicity. Animal Feed Science and Technology, 299-325.

Voss, K.A., Bacon, C.W., Norred, W.P., Chapin, R.E., Champerlain, W.J., Plattner, R.D., Meredith, F.I., 1996. Studies on the reproductive effects of fusarium moniliforme culture material in rat and the biodistribution of [14C] fumonisin B1 rats and mice. Natural toxins 4, 24-33.

Voss, K.A., Gelineau-Van Waes, J.B., Riley, R.T., 2006. Fumonisins: current research trends in developmental toxicology. Mycotoxin Research 22, 61-69.

Wang, C.C.C., Chiang, Y.M., Kuo, P.L., Chang, J.K., Hsu, Y.L., 2008. Norsolorinic acid inhibits proliferation of T24 human bladder cancer cells by arresting the cell cycle at the G0/G1 phase and inducing a Fas/membrane-bound Fas ligand-mediated apoptotic pathway. Clinical and Experimental Phamacology and Physiology 35, 1301-1308.

Wang, E., Riley, R.E., Meredith, F.I., Merrill, A.H., 1999. Fumonisin B1 consumption by rats causes reversible, dose-dependent increases in urinary sphinganine and sphingosine. Journal of Nutrition 129, 214-220.

Wang, J.S., Groopman, J.D., 1999. DNA damage by mycotoxins. Mutation Research 424, 167-181.

237

Wangikar, P.B., Swivedi, P., Neeraj, S., 2004. Effect in rat of simultaneous prenatal exposure to OTA and Aflatoxin B1 maternal toxicity and fetal malformations. Birth defects research. Birth Defects Research 71, 343.

Wannemacher, R.W., Wiener, S.L., 1997. Tricothecene mycotoxins. In F.R. Sidell, E.T. Takafuji, D.R. Franz (Eds.), Medical Aspects of Chemical and Biological Warfare. U.S. Army TMM Publications Borden Institute Walter Reed Army Medical Center, Washington, DC, pp. 655.

Weed, D.L., 2005. Weight of evidence: A review of concept and methods. Risk Analysis 25, 1545-1557.

Weibking, T.S., Ledoux, D.R., Bermudez, A.J., Turk, J.R., Rottinghaus, G.E., Wang, E., Merrill Jr., A.H., 1993. Effects of feeding Fusarium moniliforme culture material, containing known levels of fumonisin B1, on the young broiler chick. Poultry Science, 456-466.

Westhuizen, L., Shepard, G.S., Scussel, V.M., Costa, L.L., Vismer, H.F., Rheeder, J.P., 2003. Fumonisin contamination and Fusarium incidence in corn from Santa Catarina, Brazil. Journal of Agricultural and Food Chemistry 51, 5574.

Widstrom, N.W., 1996. The aflatoxin problem with corn grain. In D. Sparks (Ed.), Advances in Agronomy. Academic Press, New York, pp. 219-280.

Wilson, D.M., Abramson, D., 1992. Mycotoxins. In D.B. Sauer (Ed.), Storage of Cereal Grains and Their Products. American Association of Cereal Chemists, Inc., St. Paul, Minnesota, pp. 341-391.

Withanage, G.S., Murata, H., Koyama, T., Ishiwata, I., 2001. Agonistic and antagonistic effects of zearalenone, an etrogenic mycotoxin, on SKN, HHUA, and HepG2 human cancer cell lines. Veterinary and Human Toxicology 43, 6-10.

Woehlecke, H., Osada, H., Herrmann, A., Lage, H., 2003. Reversal of breast cancer resistance protein-mediated drug resistance by tryprostatin A. International Journal of Cancer 107, 721-728.

Wong, W.L., Dimitroulakos, J., Minden, M., Penn, L., 2002. HMG-CoA reductase inhibitors and the malignant cell: the statin family of drugs as triggers of tumor- specific apoptosis. Leukemia 16, 508-519.

World Health Organization, 2002. Evaluation of certain mycotoxins in food. Geneva, 906.

Wösten, H.A.B., Scholtmeijer, K., de Vries, R.P., 2007. Hyperproduction of enzymes by fungi. In J. Dijksterhuis, R.A. Samson (Eds.), Food Mycology. A multifaceted approach to fungi and food. CRC Press, Boca Raton, FL, pp. 183-196.

238

Wu, J., Tan, Y., Wang, Y., Xu, R., 2011. Occurrence of ochratoxin A in wine and beer samples from China. Food Additives and Contaminants: Part B 4, 52-56.

Xiao, H., Srinivasa, M., Marquardt, R.R., Li, S., Vodela, J.K., Frohlich, A.A., Kemppainen, B.W., 1996. Toxicity of ochratoxin A, its opened lactone form, and several of its analogs: structure-activity relationships. Toxicology and Applied Pharmacology 137, 182-192.

Xu, J., Caro-Diaz, E.J., Lacoske, M.H., Hung, C.I., Jamora, C., Theodorakis, E.A., 2012. Fusarisetin A: scalable total synthesis and related studies. Chemical Science 3, 3378.

Yamada, T., Muroga, Y., Jinno, M., Kajimoto, T., Usami, Y., Numata, A., Tanaka, R., 2011. New class azaphilone produced by a marine fish-derived Chaetomium globosum. The stereochemistry and biological activities. Bioorganic Medicinal Chemistry 19, 4113.

Yeung, J.M., Wang, H.Y., Prelusky, D.B., 1996. Fumonisin B1 induces protein kinase C translocation via direct interaction with diacylglycerol binding site. Toxicology and Applied Pharmacology, 178-184.

Zhao, S., Smith, K.S., Deveau, A.M., Dieckhaus, C.M., Johnson, M.A., Macdonald, T.L., Cook, J.M., 2002. Biological activity of the tryprostatins and their diastereomers on human carcinoma cell lines. Journal of Medicinal Chemistry 45, 1559-1562.

Zimmerli, B., Dick, R., 1996. Ochratoxin A in table wine and grape juice: occurrence and risk assessment. Food Additive Contamination Part A 6, 655-668.

Zimmerli, B., Dick, R., 1995. Determination of ochratoxin A at the ppt level in human blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column cleanup: methodology and Swiss data. Journal of Chromatography B: Biomedical Sciences and Applications 666, 85.

Zinedine, A., Brera, C., Elakhdari, S., Catano, C., Debegnach, F., Angelini, S., Santis, B.D., Faid, M., Benlemlih, M., Minardi, V., Miraglia, M., 2006. Natural occurrence of mycotoxins in cereals and spices commercialized in Morocco. Food Control 17, 868.

Zinedine, A., Soriano, J.M., Moltó, J.C., Mañes, J., 2007. Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: An oestrogenic mycotoxin. Food and Chemical Toxicology 45, 1-18.

Zoecklein, B., Fugelsang, K.C., Gump, B.H., Nury, F.S., 1999. Wine Analysis and Production. Aspen Publishers, New York.

239

BIOGRAPHICAL SKETCH

Devin Lewis was born and raised in Houston, Texas. At a very young age he was intrigued by the sights and sounds of his mother and grandmother toiling in the kitchen.

This led him to pursue a Bachelor of Science in culinary arts from Johnson & Wales

University, Miami, FL. After graduating Summa Cum Laude in May 2002, Devin began honing his culinary skills as he worked with several notable chefs. While working in the kitchen, Devin became interested in chemical aspects of food and product development.

Devin further explored his new found interest by attending a Research Chef’s

Association Convention. Shortly thereafter, Devin decided to pursue his master’s degree at the University of Florida, majoring in food science. After graduating in August

2010, Devin was given the opportunity to become a McKnight Doctoral Fellow and pursue a Ph.D in food science. Devin used his Ph.D project to focus on the evaluation of risk associated with fungal toxins in Florida wines. Following his graduation in August,

2016, Devin hopes to pursue a career in food chemistry and food product research and development.

240