Formation of Acrylamide during Roasting of Coffee

Dissertation by MSc. Kristina Bagdonaite

was carried out in the period of November 2003 to March 2007 at the Institute for Food Chemistry and Technology, Graz University of Technology supervised by Ao.Univ.-Prof. Dipl.-Ing. Dr.techn. Michael Murkovic.

Contents 1 Acknowledgements

vii

2 Summary

viii

3 Zusammenfassung

ix

4 Introduction 1 4.1 Acrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4.2 Acrylamide in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 4.3 Toxicity of acrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . 11 4.4 Acrylamide formation pathways . . . . . . . . . . . . . . . . . . . . . 13 4.4.1 The Maillard reaction . . . . . . . . . . . . . . . . . . . . . . 16 4.4.2 Lipid degradation . . . . . . . . . . . . . . . . . . . . . . . . . 18 4.4.3 Decarboxylation and deamination of asparagine . . . . . . . . 20 4.4.4 Other precursors for acrylamide formation (3-aminopropionamide) 21 4.5 Michael addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.6 Coffee: plants, beans and their production . . . . . . . . . . . . . . . 24 4.6.1 Differences of Arabica and Robusta . . . . . . . . . . . . . . . 24 4.6.2 Harvesting and processing . . . . . . . . . . . . . . . . . . . . 26 4.6.3 Coffee roasting . . . . . . . . . . . . . . . . . . . . . . . . . . 27 5 Purpose of the study

29

6 Materials and Methods 6.1 Chemicals and solvent . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Coffee samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Coffee roasting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1 Coffee roasting in a laboratory roaster . . . . . . . . . . . . . 6.3.2 Coffee roasting in a thermostatic oven . . . . . . . . . . . . . 6.3.3 Standard condition roasting . . . . . . . . . . . . . . . . . . . 6.4 Typical sample preparation procedure . . . . . . . . . . . . . . . . . . 6.5 3-aminopropionamide in coffee . . . . . . . . . . . . . . . . . . . . . . 6.5.1 3-aminopropionamide in green coffee . . . . . . . . . . . . . . 6.5.2 3-aminopropionamide in heated coffee . . . . . . . . . . . . . . 6.6 Acrylamide and 3-aminopropionamide formation in a model system . 6.6.1 Preparation mixtures of asparagine with sucrose and glucose . 6.6.2 Sample preparation for optimal heating conditions estimation 6.6.3 Asparagine with ascorbic acid mixture preparation . . . . . . 6.7 Heating of pure asparagine . . . . . . . . . . . . . . . . . . . . . . . . 6.7.1 Acrylamide formation from pure asparagine heated at 170 °C for 0-24 minutes . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.2 Maleimide formation at 170 °C . . . . . . . . . . . . . . . . . 6.7.3 Acrylamide formation from asparagine at high temperatures .

31 31 31 49 49 50 51 51 52 52 53 54 54 55 55 55

i

55 56 56

6.8 Heating of Amadori compound . . . . . . . . . . . . . . . . . . . . . 6.9 Derivatization methods . . . . . . . . . . . . . . . . . . . . . . . . . . 6.9.1 Derivatization with dansyl chloride . . . . . . . . . . . . . . . 6.9.2 Derivatization with 2-mercaptobenzoic acid . . . . . . . . . . 6.10 Analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.10.1 Ion chromatography with UV detection . . . . . . . . . . . . . 6.10.2 HPLC-FLD operating conditions . . . . . . . . . . . . . . . . 6.10.3 HPLC-MS operating conditions (m/z 72, 75) . . . . . . . . . . 6.10.4 HPLC-MS operating conditions, eluent water . . . . . . . . . 6.10.5 HPLC-UV operating conditions . . . . . . . . . . . . . . . . . 6.10.6 HPLC-MS operating conditions for acrylamide derivative analysis

57 58 58 58 59 59 60 61 62 63 63

7 Results and Discussion 68 7.1 Coffee roasting in a laboratory roaster . . . . . . . . . . . . . . . . . 68 7.2 Coffee heated in a thermostatic oven . . . . . . . . . . . . . . . . . . 69 7.3 Standard condition roasting . . . . . . . . . . . . . . . . . . . . . . . 72 7.4 Acrylamide and 3-aminopropionamide formation in a model system . 74 7.4.1 Optimum time and temperature conditions for 3-aminopropionamide formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 7.4.2 Optimal heating time and temperature conditions for acrylamide 77 7.5 Heating of pure asparagine . . . . . . . . . . . . . . . . . . . . . . . . 81 7.6 3-Aminopropionamide in coffee . . . . . . . . . . . . . . . . . . . . . 82 7.7 Heated mixtures of asparagine with ascorbic acid . . . . . . . . . . . 83 7.8 Amadori compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 8 Conclusions

87

9 Bibliography

90

10 Curriculum Vitae

100

A Publications in the period of dissertation

101

ii

List of Figures 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Acrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycidamide formation from acrylamide . . . . . . . . . . . . . . . . N -acetyl-S -(3-amino-3-oxopropyl)cysteine . . . . . . . . . . . . . . . Glyceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N -acetyl-S -(3-amino-2-hydroxy-3-oxopropyl)cysteine . . . . . . . . . Acrylamide formation pathway from asparagine and dicarbonyl (adapted from [63]) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acrylamide formation pathways from acrolein and asparagine with sugars (adapted from [62]) . . . . . . . . . . . . . . . . . . . . . . . . . Acrylamide formation from asparagine by simple decarboxylation and deamination reaction [39]. . . . . . . . . . . . . . . . . . . . . . . . . Maleimide (2,5-pyroldione) . . . . . . . . . . . . . . . . . . . . . . . Fumaramic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic 3-aminopropionamide formation from asparagine (adapted from [66]) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael addition reaction . . . . . . . . . . . . . . . . . . . . . . . . Coffee plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arabica Zambia AA . . . . . . . . . . . . . . . . . . . . . . . . . . . Arabica Uganda Organico Biocoffee . . . . . . . . . . . . . . . . . . Arabica Tanzania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arabica Kenya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arabica Kenya AA . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arabica Ethiopian Sidamo Yirgamo Grade 2 . . . . . . . . . . . . . . Arabica Indonesian Sumatra Lintong . . . . . . . . . . . . . . . . . . Arabica Indonesian Sulawesi Kalossi . . . . . . . . . . . . . . . . . . Arabica Indian Monsooned Aspinwalls Malabar AA . . . . . . . . . . Arabica Indian Plantation A . . . . . . . . . . . . . . . . . . . . . . Arabica Java WIB1 Jampit Gr1 . . . . . . . . . . . . . . . . . . . . . Arabica Nicaragua Talia Extra . . . . . . . . . . . . . . . . . . . . . Arabica Costa Rica Tarazzu . . . . . . . . . . . . . . . . . . . . . . . Arabica Guatemala SHB . . . . . . . . . . . . . . . . . . . . . . . . . Arabica Mexico Maragogype . . . . . . . . . . . . . . . . . . . . . . Arabica Mexico Altura . . . . . . . . . . . . . . . . . . . . . . . . . . Arabica Honduras SHG . . . . . . . . . . . . . . . . . . . . . . . . . Arabica Colombian Excelso . . . . . . . . . . . . . . . . . . . . . . . Arabica Santos Brazil NY 2 17/18 TOP Italian preparation . . . . . Arabica Papua New Guinea Sigri C . . . . . . . . . . . . . . . . . . . Robusta Indian Parchment . . . . . . . . . . . . . . . . . . . . . . . Robusta Indian Cherry AB . . . . . . . . . . . . . . . . . . . . . . . Vietnam Robusta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robusta Liberia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robusta Cameroon . . . . . . . . . . . . . . . . . . . . . . . . . . . . Temperature gradient of roasting programmes 4, 6, 8 and 10. . . . . . iii

1 11 12 12 12 17 19 20 21 21 22 23 25 32 33 33 34 35 35 36 37 37 38 39 40 40 41 42 43 43 44 45 45 46 47 47 48 49 50

40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

57

58 59 60 61 62 63 64 65 66

Coffee beans roasted under different time and temperature conditions 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-fructopyranose . . . . . . . . Reaction of 3-aminopropionamide to sulphonamide . . . . . . . . . . Derivatization of acrylamide with 2-mercaptobenzoic acid . . . . . . Typical chromatogram of acrylamide using ion exclusion chromatography with UV detection . . . . . . . . . . . . . . . . . . . . . . . . Typical chromatogram of 3-aminopropionamide analysis . . . . . . . Typical chromatogram of acrylamide using MS detection . . . . . . . Typical chromatogram of acrylamide using UV detection . . . . . . . Typical chromatogram of acrylamide (6 ng/ml) after derivatization with 2-mercaptobenzoic acid using LC-MS/MS . . . . . . . . . . . . Typical chromatogram of acrylamide in a roasted coffee sample after derivatization with 2-mercaptobenzoic acid using LC-MS/MS . . . . Typical chromatogram of derivatized acrylamide analysis . . . . . . . Formation of acrylamide in 4 different types of coffee roasted in a laboratory roaster . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2nd Order regression curve of acrylamide content in coffee beans . . . Regression surface curve of acrylamide content in coffee beans . . . . Significant parameters in the formation of acrylamide . . . . . . . . . Acrylamide content in different coffee beans roasted under standard conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acrylamide in heated asparagine and sucrose and asparagine and glucose (molar ratio 1:0.5, 1:1 and 1:1.5) anhydrous mixtures at 130, 150 and 170 ‰ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-Aminopropionamide in heated asparagine and sucrose and asparagine and glucose (molar ratio 1:0.5, 1:1 and 1:1.5) anhydrous mixtures at 130, 150 and 170 ‰. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-Aminopropionamide in heated asparagine and sucrose 1:0.5 anhydrous mixtures at 130, 150, 170 and 190 ‰ . . . . . . . . . . . . . . . Acrylamide content in asparagine with glucose mixtures (molar ratio 1:0.5 and 1:1) heated to different temperatures for 5 and 7 minutes . Acrylamide formed in asparagine and glucose mixtures (1:0.5) heated at different temperatures for 5 minutes . . . . . . . . . . . . . . . . . Acrylamide formation in asparagine mixtures with glucose at 210 °C (molar ratio 1:0.5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acrylamide content (µg/g) in asparagine mixtures with glucose (1:0.5) heated at 250 °C for 1, 3, 4, 5, 7 and 10 minutes . . . . . . . . . . . Acrylamide formation from pure asparagine heated at high temperatures Acrylamide formation in the mixtures of asparagine with ascorbic acid Acrylamide formation in asparagine and ascorbic acid mixtures heated at 250 °C and different times . . . . . . . . . . . . . . . . . . . . . . 3-Aminopropionamide formation in the mixtures of asparagine with ascorbic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iv

54 57 58 59 60 61 62 64 65 66 67 68 70 71 72 73

75

76 78 78 79 80 80 82 84 84 85

67

Acrylamide and 3-aminopropionamide formation from 1-N -(asparaginyl)5-azido-1,5-dideoxy-D-fructopyranose . . . . . . . . . . . . . . . . . . 86

v

List of Tables 1 2 3 4 5 6 7 8 9 10

Acrylamide in foods from USA [29] . . . . . . . . . . . . . . . . . . . Acrylamide in Canadian foods [30] . . . . . . . . . . . . . . . . . . . Acrylamide in Australian foods [31] . . . . . . . . . . . . . . . . . . Mean concentrations of acrylamide in UK food [32] . . . . . . . . . . Roasting parameters of program 4, 6, 8 and 10. . . . . . . . . . . . . Characteristics of green coffee beans processed by different methods. Heating conditions of Amadori compound . . . . . . . . . . . . . . . Concentration of acrylamide in Cameroon Robusta, heated in an thermostatic oven, ng/g. . . . . . . . . . . . . . . . . . . . . . . . . . . . Concentration of acrylamide in Cameroon Robusta, heated in an thermostatic oven, ng/g. . . . . . . . . . . . . . . . . . . . . . . . . . . . Coffee roasted under standard conditions . . . . . . . . . . . . . . .

vi

2 8 8 10 50 52 57 69 71 74

1

Acknowledgements

I am very thankful to my supervisor Ao.Univ.-Prof. Dipl.-Ing. Dr.techn. Michael Murkovic for his great help in the laboratory, for his cooperative work and wise suggestions during the period of research and summary of the results. I would like to express my great thanks to the colleagues of the Institute for the Food Chemistry and Technology at Graz University of Technology for their helpful hand, suggestions and enjoyable working atmosphere. Especially I would like to thank Ing. Sigrid Draxl for her patience, dedicated work and honest help. My special thanks I would like to dedicate to Dra. Maria Teresa Galcer´an Huguest and her research team at University of Barcelona, Department of Analytical Chemistry for the cooperative work during the HEATOX project. I would like to address particular thanks to Dra. Encarnaci´on Moyano Morcillo and Sra. Elisabet Bermudo Rubio for the help developing the analytical method for acrylamide detection. I would like to thank Dipl.-Ing. Dr.techn. Univ.-Doz. Tanja Maria Wrodnigg from the Institute for Organic Chemistry at Graz University of Technology for giving me the chemicals for our experiments. I would like to address my special thanks to my boyfriend Dipl.-Ing. Bernhard Schraußer for his devoted patience by helping me to understand the tricks of the most complicated typesetting system called LATEX 2ε .

This study was financed by Commission of the European Communities, specific RTD programme ”Food Quality and Safety”, FOOD-CT-2003-506820, ”Heatgenerated food toxicants Identification, characterisation and risk minimisation”. It does not necessarily reflect its views and in no way anticipates the Commission’s future policy in this area.

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2

Summary

Within this thesis methods for extraction and analysis of acrylamide were successfully established. Also the detection method for a probable precursor of acrylamide 3aminopropionamide was developed. Coffee as a research object was chosen because of its high consumption and therefore possible hazardous influence on human health. After the extraction with water and proceeded solid phase extraction clean-up procedure, acrylamide was analysed using liquid chromatography with different detection instruments - mainly UV and mass spectroscopy. Moreover, for the analysis of the possible acrylamide precursor 3-aminopropionamide liquid chromatography with fluorescence detection was used. 25 green coffees of different origin were roasted either in a laboratory coffee roaster or thermostatic oven. The study showed, that Arabica and Robusta coffee beans differ not only in size, cup quality, but also in acrylamide content independent on the region of growth, harvesting and processing conditions. The probable precursor of acrylamide 3-aminopropionamide was not detected neither in green coffee beans nor in roasted ones. Finally some experiments were done with mixtures modeling the acrylamide formation during the roasting of coffee. Acrylamide and its possible precursor 3-aminopropionamide were easily formed in mixtures of asparagine with glucose or sucrose. Less 3-aminopropionamide was formed in mixtures of asparagine with ascorbic acid. Even some acrylamide was formed when pure asparagine was heated at high temperatures. The heating of the Amadori product 1-N -(asparaginyl)-5-azido-1,5-dideoxy-Dfructopyranose resulted in the formation of acrylamide and 3-aminopropionamide.

viii

3

Zusammenfassung

Im Rahmen dieser Arbeit wurden verschiedene Methoden zur Extraktion und Analyse von Acrylamid erfolgreich entwickelt und angewandt. Außerdem wurde eine Methode zum Nachweis von 3-Aminopropionamid, einer m¨oglichen Vorstufe von Acrylamid, gefunden. Kaffee wurde als Forschungsobjekt gew¨ahlt, da er in großen Mengen konsumiert wird und somit einen potentiell gef¨ahrlichen Einfluß auf die menschliche Gesundheit darstellt. Nach der Extraktion mit Wasser und darauf folgender Festphasenextraktion wurde Acrylamid mittels Fl¨ ussigchromatographie nachgewiesen, wobei verschiedene Detektoren, vor allem UV und MS-Detektion, zum Einsatz kamen. F¨ ur den Nachweis der m¨oglichen Acrylamid-Vorstufe 3-Aminopropionamid wurde Fl¨ ussigchromatographie mit Fluoreszenz-Detektion verwendet. 25 Sorten gr¨ uner Kaffeebohnen aus verschiedenen, u ¨ ber die ganze Welt verteilten Anbaugebieten wurden entweder mit einem Labor-Kaffeer¨oster oder im Thermostatofen ger¨ostet. Die Untersuchung zeigte, dass sich die beiden Sorten Arabica und Robusta nicht nur in Gr¨oße und Geschmacksqualit¨at, sondern auch im Acrylamidgehalt unterscheiden, und zwar unabh¨anging von der Anbauregion, sowie den Bedingungen bei Ernte und Weiterverarbeitung. Leider konnte die m¨ogliche Acrylamid-Vorstufe 3-Aminopropionamid weder in den gr¨ unen noch in den ger¨osteten Kaffeebohnen nachgewiesen werden. Zum Schluß wurden einige Experimente mit Modellsystemen durchgef¨ uhrt, wobei ¨ahnliche Konzentrationen von Acrylamid-Vorstufen wie in den Kaffeebohnen, sowie ¨ahnliche R¨ostbedingungen simuliert wurden. Sowohl Acrylamid als auch seine m¨ogliche Vorstufe 3-Aminopropionamid konnten dabei aus Mischungen von Asparagin mit Glukose oder Succrose erzeugt werden, weniger leicht mit Ascorbins¨aure. Sogar beim Erhitzen von reinem Asparagin auf hohe Temperaturen wurde Acrylamid gebildet. In Experimenten mit dem Amadori Produkt 1-N -(asparaginyl)-5-azido-1,5-dideoxyD-fructopyranose wurden Acrylamid und 3-Aminopropionamid gebildet.

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4

Introduction

4.1

Acrylamide

Acrylamide (Figure 1), also known as 2-propenamide, acrylic amide, ethylene carboxamide, propenoic acid amide, vinyl amide, propenamide, acrylamide monomer [1], is a very polar molecule with a molecular weight of 71, a melting point of 84.5 ± 0.3 °C and a high boiling point of 136 °C at 3.3 kPa [2, 3]. Acrylamide is very soluble in water, alcohols, acetone, acetonitrile, slightly soluble in ethyl acetate, dichlormethane, diethyl ether. It is insoluble in hexane and other alkanes and alkenes. Low, but significant volatility of acrylamide was observed. It has no significant UV-absorption above 220 nm and does not fluoresce.

Figure 1: Acrylamide

The amide group is protonated by medium and strong acids. Acrylamide contains a reactive electrophilic double bond and a reactive amide group. It exhibits both weak acidic and basic properties [4]. Acrylamide can be produced industrially for the synthesis of polyacrylamide. Polyacrylamide can be used in waste water treatment as a flocculent, in soil stabilization, in grout for repairing sewers, in the cosmetics, paper and textile industries [4, 5]. Polymerized acrylamide is also widely used in electrophoresis for protein separation. Acrylamide is described as a neurotoxin, genotoxin and is probably carcinogenic to humans [5, 6]. Some analytical methods for acrylamide have been reported. The sample preparation mainly consists of water extraction and a solid phase extraction procedure for the sample clean-up [7, 8, 9, 10, 11, 12, 13, 14]. Acrylamide can be analyzed by gas

1

[13, 15, 16, 17, 18, 19, 20] or liquid chromatography [7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23], and even by capillary zone electrophoresis [24]. Using liquid chromatography with single or tandem mass spectrometry it can be analyzed directly [7, 8, 11, 12, 18, 21, 22, 23, 25] or derivatized [16], also UV detection gives satisfactory results [9].

4.2

Acrylamide in foods

As Tareke et al. in 2002 [26] reported, acrylamide hemoglobin adduct background levels originate from foods of a normal diet. Acrylamide was detected in all kind of foods including meat, bread and potato products prepared at high temperatures (>160 ‰) [26, 27]. Relatively small amounts can be found in boiled and microwaved (where temperatures can reach up to 260 °C [28]) foods, but not fresh ones. Even roasted tea leaves and roasted barley grains contain acrylamide in the concentration up to 570 and 320 ng/g respectively. Acrylamide is formed in high-carbohydrate foods during frying, baking, roasting and extrusion. In commercially processed foods as well as in home-cooked meals the acrylamide content tends to increase with cooking time and temperature. The surface color of the products correlates highly with acrylamide levels in food: the darker the surface, the more acrylamide it contains [4]. Some data on acrylamide in food are presented in Tables 1, 2, 3, 4.

Table 1: Acrylamide in foods from USA [29] Food Baby Food French fries

Product 2002 ND-121 20-212

not baked baked ready to eat

Acrylamide, ppb 2003 2004

119-1325 117-1030 117-2762

Potato chips

Natural potato chips lot 1 879 continued on next page 2

693-2510, 1077

122-1250 462-1970

Table 1 – continued from previous page Food Product Acrylamide, ppb 2002 2003 2004 Natural potato chips lot 2 433 Classic potato chips 0 weeks 319 Classic potato chips 1 week 432 Classic potato chips 2 weeks 280 Classic potato chips 3 weeks 257 Classic potato chips 4 weeks 343 Classic potato chips 5 weeks 425 Infant ForND mula Milk based Infant Formula with Iron (liquid) ND Milk based Infant Formula with Iron (powdered)