FORMATION OF 4(5)-METHYLIMIDAZOLE IN PROCESSED MEAT AND IN GLUCOSE AMINO ...

183 Pages·2015·3.16 MB·English

FORMATION OF 4(5)-METHYLIMIDAZOLE IN PROCESSED MEAT AND IN GLUCOSE
AMINO ACID MODEL SYSTEM

by

FARIS A. HUSSAIN

B.S, Al-Mustansiriya University, Baghdad, Iraq, 1991
M.S., Kansas State University, Manhattan, KS, 2009

AN ABSTRACT OF A DISSERTATION

submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY

Food Science

KANSAS STATE UNIVERSITY
Manhattan, Kansas

2015
ABSTRACT

The International Agency for Research on Cancer (IARC) has classified 4(5)-
methylimidazole (4-MeI) as a possible human carcinogen. Also, the National Toxicology
Program (NTP) has concluded that 4-MeI is a probable cancer causing agent. Even though 4-MeI
has been detected in several processed foods no research has been conducted to measure levels
of 4-MeI in processed meat. A method using isobutylchloroformate (IBCF) and gas
chromatography mass-spectrometry (GC-MS) was developed to detect and quantify 4-MeI in
processed meat products. Levels of 4-MeI ranged from 0.04 to 1.01 μg/g with recovery of 94.76
to 103.94%.
Formation of 4-MeI in glucose-amino acid model system was studied. -Glucose (Glu)
D
was mixed individually in equimolar concentrations of 0.05, 0.1, or 0.15 M and equal volume
with -Alanine (Ala), -Arginine (Arg), Glycine (Gly), -Lysine (Lys), and -Serine (Ser); and
L L L L
the mixtures were heat treated at 60, 120, and 160°C for 1 h. Among all tested amino acids, Glu-
Arg produced the highest level of 4-MeI. The Glu-Lys model system showed higher browning
and lower concentrations of 4-MeI.
The effect of ascorbic acid (ASA), a strong antioxidant, was studied in the Glu-Arg
model system. Four concentrations of ASA (0.0375, 0.075, 0.15, and 0.3 M) were mixed with
0.15 M equimolar of Glu-Arg and heat treated at 160°C for 1h. ASA inhibited formation of 4-
MeI by 40.29, 69.94, 75.13, and 96.25% for the 0.0375, 0.075, 0.15, and 0.3 M concentrations.
Contrarily, ascorbic acids increased the browning in all treatments compared to control treatment
by10.67, 15.47, 18.4, and 28.8% for the 0.0375, 0.075, 0.15, and 0.3 M concentrations. Adding
ASA to processed food may reduce formation of 4-MeI and increase the browning that is a
desired attribute to processed meat products.
FORMATION OF 4(5)-METHYLIMIDAZOLE IN PROCESSED MEAT AND IN GLUCOSE
AMINO ACID MODEL SYSTEM

by

FARIS A. HUSSAIN

B.S, Al-Mustansiriya University, Baghdad, Iraq, 1991
M.S., Kansas State University, Manhattan, KS, 2009

A DISSERTATION

submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY

Food Science

KANSAS STATE UNIVERSITY
Manhattan, Kansas

2015

Approved by:
Major Professor
J. Scott Smith
ABSTRACT

The International Agency for Research on Cancer (IARC) has classified 4(5)-
methylimidazole (4-MeI) as a possible human carcinogen. Also, the National Toxicology
Program (NTP) has concluded that 4-MeI is a probable cancer causing agent. Even though 4-MeI
has been detected in several processed foods no research has been conducted to measure levels
of 4-MeI in processed meat. A method using isobutylchloroformate (IBCF) and gas
chromatography mass-spectrometry (GC-MS) was developed to detect and quantify 4-MeI in
processed meat products. Levels of 4-MeI ranged from 0.04 to 1.01 μg/g with recovery of 94.76
to 103.94%.
Formation of 4-MeI in glucose-amino acid model system was studied. -Glucose (Glu)
D
was mixed individually in equimolar concentrations of 0.05, 0.1, or 0.15 M and equal volume
with -Alanine (Ala), -Arginine (Arg), Glycine (Gly), -Lysine (Lys), and -Serine (Ser); and
L L L L
the mixtures were heat treated at 60, 120, and 160°C for 1 h. Among all tested amino acids, Glu-
Arg produced the highest level of 4-MeI. The Glu-Lys model system showed higher browning
and lower concentrations of 4-MeI.
The effect of ascorbic acid (ASA), a strong antioxidant, was studied in the Glu-Arg
model system. Four concentrations of ASA (0.0375, 0.075, 0.15, and 0.3 M) were mixed with
0.15 M equimolar of Glu-Arg and heat treated at 160°C for 1h. ASA inhibited formation of 4-
MeI by 40.29, 69.94, 75.13, and 96.25% for the 0.0375, 0.075, 0.15, and 0.3 M concentrations.
Contrarily, ascorbic acids increased the browning in all treatments compared to control treatment
by10.67, 15.47, 18.4, and 28.8% for the 0.0375, 0.075, 0.15, and 0.3 M concentrations. Adding
ASA to processed food may reduce formation of 4-MeI and increase the browning that is a
desired attribute to processed meat products.
TABLE OF CONTENTS
LIST OF FIGURES ...................................................................................................................... x 
LIST OF TABLES ...................................................................................................................... xii 
ACKNOWLEDGEMENTS ...................................................................................................... xiii 
DEDICATION............................................................................................................................ xiv 

CHAPTER 1. REVIEW OF LITERATURE ............................................................................. 1 
INTRODUCTION .......................................................................................................................... 1 
4-MeI CHARACTERISTICS ......................................................................................................... 2 
Physical and chemicals properties .......................................................................................... 2 
PRODUCTION AND USE............................................................................................................. 3 
4-MeI FORMATION ...................................................................................................................... 4 
FACTORS AFFECTING 4-MeI FORMATION ............................................................................ 7 
Time and temperature ............................................................................................................. 7 
Moisture .................................................................................................................................. 7 
Reactant concentrations .......................................................................................................... 7 
Presence of other compounds ................................................................................................. 8 
Amino acids types ................................................................................................................... 8 
Sugar types .............................................................................................................................. 9 
pH ............................................................................................................................................ 9 
4-MeI FORMATION MECHANISMS ........................................................................................ 10 
TOXICITY .................................................................................................................................... 12 
Animal Toxicity .................................................................................................................... 12 
v
Human Toxicity .................................................................................................................... 13 
4-MeI CARCINOGENICITY ....................................................................................................... 13 
4-MeI PHARMACOKINETICS ................................................................................................... 15 
LEVELS OF 4-MeI IN FOODS ................................................................................................... 17 
EXPOSURE TO 4-MeI FROM NON-FOOD SOURCES ........................................................... 18 
LEVELS OF 4-MeI IN ANIMAL FEEDS ................................................................................... 20 
ANALYTICAL METHODS TO IDENTIFY AND QUANTIFY 4-MeI ..................................... 20 
Detection methods ................................................................................................................ 20 
Extraction methods ............................................................................................................... 21 
DERIVATIZATION OF 4-MeI .................................................................................................... 23 
CARAMEL COLORS .................................................................................................................. 25 
4-MeI LEVELS IN CARAMEL COLORS .................................................................................. 27 
4-MeI REGULATIONS IN FOODS AND CARAMEL COLORS ............................................. 29 
4-MeI RISK ASSESMENT BY REGLUATION AGENCIES .................................................... 29 
REDUCTION OF 4-MeI IN CARAMEL COLORS .................................................................... 30 
SUMMARY .................................................................................................................................. 32 
REFERENCES ............................................................................................................................. 33 
CHAPTER 2. DETECTION AND QUANTIFICATION OF 4(5)-METHYLIMIDAZOLE
IN COOKED MEAT ........................................................................................................... 46 
ABSTRACT .................................................................................................................................. 46 
INTRODUCTION ........................................................................................................................ 47 
MATERIALS AND METHODS .................................................................................................. 49 
Chemicals and reagents ......................................................................................................... 49 
vi
Sampling ............................................................................................................................... 50 
Sample Preparation ........................................................................................................... 50 
Sample derivatization........................................................................................................ 51 
Samples fat determination................................................................................................. 51 
GC-MS system ...................................................................................................................... 52 
CALCULATION OF 4-MeI LEVELS ......................................................................................... 53 
STATISTICAL ANALYSIS ........................................................................................................ 53 
RESULTS AND DISCUSSION ................................................................................................... 54 
GC-MS conditions for identification and quantification of 4-MeI ....................................... 54 
METHOD VALIDATION ............................................................................................................ 61 
4-MeI INTAKE ............................................................................................................................. 63 
4-MeI intake level through cooked meat consumption ......................................................... 63 
CONCLUSION ............................................................................................................................. 64 
REFERENCES ............................................................................................................................. 66 
CHAPTER 3. FORMATION OF 4(5)-METHYLIMIDAZOLE IN AQUEOUS -
D
GLUCOSE-AMINO ACIDS MODEL SYSTEM ............................................................. 71 
ABSTRACT .................................................................................................................................. 71 
INTRODUCTION ........................................................................................................................ 72 
MATERIALS AND METHODS .................................................................................................. 74 
Chemicals and reagents ......................................................................................................... 74 
Model system preparation ..................................................................................................... 74 
Model system reaction procedure ......................................................................................... 74 
Model system pH measurements .......................................................................................... 75 
vii
Model system water activity measurements ......................................................................... 76 
Model system browning measurements ................................................................................ 76 
4-MeI sample measurements ................................................................................................ 77 
Derivatization .................................................................................................................... 77 
GC-MS system operation conditions ................................................................................ 77 
STATISTICAL ANALYSIS ........................................................................................................ 78 
RESULTS AND DISCUSSION ................................................................................................... 80 
Change in the pH and effects of pH ...................................................................................... 80 
Effect of temperature ............................................................................................................ 84 
Water activity ........................................................................................................................ 85 
4-MeI levels .......................................................................................................................... 87 
Reactant concentrations ........................................................................................................ 93 
Browning ............................................................................................................................... 93 
CONCLUSION ............................................................................................................................. 99 
REFERENCES ........................................................................................................................... 100 
CHAPTER 4. INHIBITION OF 4(5)-METHYLIMIDAZOLE FORMATION IN AN
AQUEOUS D-GLUCOSE L-ARGININE MODEL SYSTEM ........................................ 105 
ABSTRACT ................................................................................................................................ 105 
INTRODUCTION ...................................................................................................................... 106 
MATERIALS AND METHODS ................................................................................................ 110 
Chemicals and reagents ....................................................................................................... 110 
Model system preparation ................................................................................................... 111 
Model system reaction procedure ....................................................................................... 111 
viii
pH measurements of the model system .............................................................................. 112 
Model system water activity measurement ......................................................................... 112 
Model system browning measurement ............................................................................... 113 
4-MeI sample measurement ................................................................................................ 113 
Derivatization .................................................................................................................. 113 
GC-MS system operation conditions .............................................................................. 114 
Determining volatile compounds in Glu-Arg and Glu-Arg-ASA treatments ..................... 115 
STATISTICAL ANALYSIS ...................................................................................................... 115 
RESULTS AND DISCUSSION ................................................................................................. 116 
Effect of ASA on 4-MeI formation ..................................................................................... 117 
Effect of ASA on the browning color development ........................................................... 120 
Other factors affecting formation of 4-MeI and browning ................................................. 122 
Effect of ASA on aroma formation ..................................................................................... 124 
Converting ASA concentrations to percentages ................................................................. 124 
CONCLUSION ........................................................................................................................... 125 
REFERENCES ........................................................................................................................... 129 
SUMMARY ............................................................................................................................... 134 
APPENDIX ................................................................................................................................ 135 
ix
LIST OF FIGURES
Figure 1. Tautomerism structures of 4-MeI molecule. .................................................................. 3 
Figure 2. Formation of 4-MeI from zinc hydroxide....................................................................... 5 
Figure 3. Formation mechanisms of 4-MeI. .................................................................................. 6 
Figure 4. Debus-Radziszewski 4-MeI formation mechanism. ..................................................... 11 
Figure 5. 4-MeI metabolism......................................................................................................... 16 
Figure 6. Oxidative metabolism of 4-MeI and formation of reactive intermediates. .................. 17 
Figure 7. Reaction of 4-MeI with isobutylchloroformate. ........................................................... 25 
Figure 8. Inhibition of 4-MeI formation. ..................................................................................... 32 
Figure 9. Chemical structure of 4-MeI-d6. .................................................................................. 51 
Figure 10. Mass spectrum of relative ion m/z fragments abundance for tautomeric derivatives
(A) 4-MeIa, and (B) 4-MeIb obtained in SCAN mode of the GC-MS. ................................ 56 
Figure 11. Mass spectrum of relative ion m/z fragments abundance for (A) 4-MeI-d6a, and (B)
4-MeI-d6b obtained in scan mode of the GC-MS................................................................. 57 
Figure 12. Reconstructed GC-MS chromatogram of beef gravy in merge format for ion m/z 182
for 4-MeI (0.06 mg/kg) and ion m/z 187 for 4-MeI-d6 (0.1 mg/kg). .................................... 58 
Figure 13. Average microgram levels of 4-MeI levels per serving size in different cooked meat
products.. ............................................................................................................................... 64 
Figure 14. Heating vessel used in model system thermal treatment. ........................................... 76 
Figure 15. Effect of pH on the deprotonated Gly amino group. .................................................. 81 
Figure 16. GC-MS chromatogram in SIM mode for m/z 81, 82, 109, and 182 ions showing two
compounds (4-MeIa and 4-MeIb) after derivatization with isobutylchloroformate for Glu-
Arg sample with 0.1 M concentration and heat treatment at 160°C for 1h. ......................... 88 
x