OCHRATOXIN Aâ•š: Evaluation of Methodologies for Determination of Ochratoxin A in Food

147 Pages·2017·2.58 MB·English

by  Lakshmi Gompa

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Dissertations, Theses, & Student Research in
Food Science and Technology Department
Food Science and Technology
5-2013
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Lakshmi Gompa
University of Nebraska-Lincoln, [email protected]
Follow this and additional works at: https://digitalcommons.unl.edu/foodscidiss
Gompa, Lakshmi, ""OCHRATOXIN A”: Evaluation of Methodologies for Determination of Ochratoxin A in
Food Commodities, Contamination Levels in Different Products Available in the US Market and Evaluation
of Fungal Microbiota Associated with Some of the Products" (2013). Dissertations, Theses, & Student
Research in Food Science and Technology. 33.
https://digitalcommons.unl.edu/foodscidiss/33
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“OCHRATOXIN A”
EVALUATION OF METHODOLOGIES FOR DETERMINATION OF
OCHRATOXIN A IN FOOD COMMODITIES, CONTAMINATION LEVELS IN
DIFFERENT PRODUCTS AVAILABLE IN THE US MARKET AND
EVALUATION OF FUNGAL MICROBIOTA ASSOCIATED WITH SOME OF
THE PRODUCTS

by

Lakshmi Gompa

A THESIS

Presented to the Faculty of

The Graduate College at the University of Nebraska

In Partial Fulfillment of Requirements

For the Degree of Master of Science

Major: Food Science and Technology

Under the Supervision of Professor Andreia Bianchini

Lincoln, Nebraska

May 2013
“OCHRATOXIN A”

EVALUATION OF METHODOLOGIES FOR DETERMINATION OF OCHRATOXIN A IN
FOOD COMMODITIES, CONTAMINATION LEVELS IN DIFFERENT PRODUCTS
AVAILABLE IN THE US MARKET AND EVALUATION OF FUNGAL MICROBIOTA
ASSOCIATED WITH SOME OF THE PRODUCTS
Lakshmi Gompa, M.S.
University of Nebraska, 2013
Adviser: Professor Andreia Bianchini
Abstract: Ochratoxin A (OTA) is one of the important mycotoxins that contaminate a wide range
of food commodities available in the market. In this study, methodologies for analyzing OTA in
commodities such as, roasted coffee, cocoa and meat were evaluated. The methods with the best
recovery rates were used to analyze the incidence of OTA in these food products. Among
different samples analyzed, predominantly 35% of the cocoa samples and 3% of meat samples
were contaminated with OTA. Decaffeinated coffee samples showed the highest incidence of
contamination (16.7%). Also as a part of this study, Veratox TM (Neogen, MI) ELISA test kit was
validated for quantification of OTA in meat using a modified extraction method. Results for
recovery, repeatability, cross-reactivity and robustness and linearity showed that the method was
suitable for the analysis. Incidence of OTA levels in dried fruits was evaluated and among them
raisins and dates showed high levels of incidence of OTA (100% and 70%, respectively). Fungal
microbiota was also isolated from dried fruits was characterized and different strains of A. niger
and A. tubingensis were identified.All the characterized isolates were tested for OTA production
and 36.8% among them were OTA producers. Most of the OTA producers were identified as
Aspergillus niger or Aspergillus tubingensis
iii

DEDICATION
From the depth of my heart, I thank my mom and dad for the wonderful life given to me.
This thesis is dedicated to my beloved dad G.V Ramana, certainly the greatest person I
have met in my life.
To my loving husband, for the encouragement and support all through this journey. I
couldn’t make this happen without you.
To all my well-wishers for their kind words and personal time that have shared with me,
at times when I’m down.

Thank you
iv

ACKNOWLEDGEMENTS
First, I would like to thank my advisors, Dr. Andreia Bianchini and
Dr. Jayne Stratton, who have given me a wonderful opportunity to work with them. I
really thank them for their support and guidance. It was a blessing to work with them.

I would also like to thank my committee member, Dr. Randy Wehling and Dr. Rolando
Flores for their guidance all through my academic year. I appreciate all the suggestions
and advices while in the duration as graduate committee.

I also thank Dr. Heather Hallen-Adams and Dr. Devin Rose for their support and
suggestions during my thesis work. Thanks to Bismarck, Maria Carmen and Carmen
Cano for all their time and friendship. I would also like to thank the faculty members and
students of Food microbiology lab for their help and friendship.
v

TABLE OF CONTENTS
Page

Chapter 1: LITERATURE REVIEW ............................................................................14
1.1 Introduction ............................................................................................................15
1.2 Biosynthesis ...........................................................................................................16
1.3 Toxicity ..................................................................................................................18
1.4 Regulatory limits imposed on Ochratoxin A .........................................................21
1.5 Occurrence in food commodities ...........................................................................22
1.5.1 Ochratoxin A in coffee ..............................................................................23
1.5.2 Ochratoxin A in cocoa ..............................................................................24
1.5.3 Ochratoxin A in meat ................................................................................25
1.5.4 Ochratoxin A in dried fruits ......................................................................26
1.6 Ochratoxin A detection and quantification ...........................................................27
1.6.1 Methods used for analysis of coffee samples ............................................33
1.6.2 Methods used for analysis of cocoa samples ............................................35
1.6.3 Methods used for analysis of meat samples ...............................................36
1.6.4 Methods used for analysis of dried fruits ...................................................36
1.7 Identification of fungal microbiota ........................................................................37
vi

Chapter 2: MATERIALS AND METHODS .................................................................42
2.1 Evaluation of HPLC methods for analysis of ochratoxin A ..................................43
2.1.1 Coffee and cocoa........................................................................................43
2.1.1.1 Spiking levels for coffee and cocoa ...............................................43
2.1.1.2 Ochratoxin A determination .........................................................44
2.1.2 Meat ...........................................................................................................46
2.1.2.1 Spiking levels for meat .................................................................46
2.1.2.2 Procedure ......................................................................................47
2.1.3 Limit of detection and limit of quantification ............................................47
2.2 Ochratoxin A quantification on market samples ...................................................49
2.2.1 Coffee .........................................................................................................49
2.2.2 Cocoa .........................................................................................................50
2.2.3 Meat ...........................................................................................................51
2.3 Validation of ELISA test kits for analyzing Ochratoxin A in meat .......................51
2.3.1 Recovery experiment ................................................................................52
2.3.2 Linearity ....................................................................................................52
2.3.3 Cross-Reactivity ........................................................................................53
2.3.4 Robustness ................................................................................................53
2.3.5 Repeatability .............................................................................................53
2.3.6 Statistical Evaluation ................................................................................54
2.4 Detection of ochratoxin A in dry fruits using ELISA test kits .............................54
2.4.1 Sampling of dried fruits ............................................................................55
vii

2.4.2 Extraction ..................................................................................................55
2.4.3 ELISA test protocol ..................................................................................56
2.5 Isolation and identification of fungi from dried fruits ..........................................56
2.5.1 Fungal incidence .......................................................................................56
2.5.2 Isolation of fungal microbiota from dried fruits .......................................57
2.5.3 Fungal preparation for DNA extraction ....................................................57
2.5.4 Characterization of fungal isolates using PCR and genome sequencing ...58
2.5.5 Phylogenetic analysis of fungal isolates ...................................................59
2.5.6 Evaluation of potential for ochratoxin A production by the
fungi isolated form dried fruits .................................................................59
Chapter 3: RESULTS AND DISCUSSION ..................................................................60
3.1 Evaluation of HPLC methods for analysis of ochratoxin A ..................................61
3.1.1 Coffee .........................................................................................................61
3.1.2 Cocoa .........................................................................................................63
3.1.3 Meat ..........................................................................................................65
3.1.4 Limit of detection (LOD) and limit of quantification (LOQ) ....................67
3.2 Ochratoxin A quantification in market samples ...................................................67
3.2.1 Coffee .........................................................................................................67
3.2.2 Cocoa .........................................................................................................70
3.2.3 Meat ..........................................................................................................72
3.3 Validation of ELISA test kit for quantification of OTA in meat samples ............73
3.3.1 Recovery experiments ...............................................................................73
3.3.2 Linearity ....................................................................................................75
viii

3.3.3 Cross reactivity .........................................................................................75
3.3.4 Robustness ................................................................................................77
3.3.5 Repeatability .............................................................................................78
3.4 Detection of ochratoxin A in dried fruits using ELISA test kits............................79
3.5 Isolation and identification of fungi from dried fruits ..........................................81
3.5.1 Fungal incidence .......................................................................................81
3.5.2 Isolation of fungal microbiota from dried fruits .......................................83
3.5.3 Characterization of fungal isolates using PCR and genomic sequencing ..83
3.5.4 Phylogenetic analysis of fungal isolates ...................................................86
3.5.5 Evaluation of potential for ochratoxin A production by fungi
isolated from dried fruits ...........................................................................91
Chapter 4: CONCLUSIONS AND FUTURE RESEARCH ........................................96
4.1 Conclusions ............................................................................................................97
4.2 Future Research ....................................................................................................99
ix

APPENDIX .....................................................................................................................101
A Statistical analysis for the recovery experiments in coffee ..................................102
B Statistical analysis for the recovery experiments in cocoa……………………..105
C Statistical analysis for the recovery experiments of OTA in meat using ELISA
test kit ..................................................................................................................108
D Statistical analysis for the cross-reactivity experiments using ELISA test kit ....112
E Statistical analysis for the robustness experiments using ELISA test kit ...........116
F Statistical analysis for the repeatability experiments using ELISA test kit .........120
1) Low spiking level .....................................................................................120
2) High spiking level ....................................................................................122
G Statistical analysis for comparison conventional and organic dried fruits
between OTA levels in conventional and organic dried fruits ...........................126

H Identification of each isolate obtained from dried fruits
according to information on GenBank ................................................................131
REFERENCES………………………………………………………………………...134