Formation of Acrylamide during Roasting of Coffee

Dissertationby

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 14.1 Acrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Acrylamide in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.3 Toxicity of acrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . 114.4 Acrylamide formation pathways . . . . . . . . . . . . . . . . . . . . . 13

4.4.1 The Maillard reaction . . . . . . . . . . . . . . . . . . . . . . 164.4.2 Lipid degradation . . . . . . . . . . . . . . . . . . . . . . . . . 184.4.3 Decarboxylation and deamination of asparagine . . . . . . . . 204.4.4 Other precursors for acrylamide formation (3-aminopropionamide) 21

4.5 Michael addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234.6 Coffee: plants, beans and their production . . . . . . . . . . . . . . . 24

4.6.1 Differences of Arabica and Robusta . . . . . . . . . . . . . . . 244.6.2 Harvesting and processing . . . . . . . . . . . . . . . . . . . . 264.6.3 Coffee roasting . . . . . . . . . . . . . . . . . . . . . . . . . . 27

5 Purpose of the study 29

6 Materials and Methods 316.1 Chemicals and solvent . . . . . . . . . . . . . . . . . . . . . . . . . . 316.2 Coffee samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316.3 Coffee roasting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

6.3.1 Coffee roasting in a laboratory roaster . . . . . . . . . . . . . 496.3.2 Coffee roasting in a thermostatic oven . . . . . . . . . . . . . 506.3.3 Standard condition roasting . . . . . . . . . . . . . . . . . . . 51

6.4 Typical sample preparation procedure . . . . . . . . . . . . . . . . . . 516.5 3-aminopropionamide in coffee . . . . . . . . . . . . . . . . . . . . . . 52

6.5.1 3-aminopropionamide in green coffee . . . . . . . . . . . . . . 526.5.2 3-aminopropionamide in heated coffee . . . . . . . . . . . . . . 53

6.6 Acrylamide and 3-aminopropionamide formation in a model system . 546.6.1 Preparation mixtures of asparagine with sucrose and glucose . 546.6.2 Sample preparation for optimal heating conditions estimation 556.6.3 Asparagine with ascorbic acid mixture preparation . . . . . . 55

6.7 Heating of pure asparagine . . . . . . . . . . . . . . . . . . . . . . . . 556.7.1 Acrylamide formation from pure asparagine heated at 170 °C

for 0-24 minutes . . . . . . . . . . . . . . . . . . . . . . . . . . 556.7.2 Maleimide formation at 170 °C . . . . . . . . . . . . . . . . . 566.7.3 Acrylamide formation from asparagine at high temperatures . 56

i

6.8 Heating of Amadori compound . . . . . . . . . . . . . . . . . . . . . 576.9 Derivatization methods . . . . . . . . . . . . . . . . . . . . . . . . . . 58

6.9.1 Derivatization with dansyl chloride . . . . . . . . . . . . . . . 586.9.2 Derivatization with 2-mercaptobenzoic acid . . . . . . . . . . 58

6.10 Analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596.10.1 Ion chromatography with UV detection . . . . . . . . . . . . . 596.10.2 HPLC-FLD operating conditions . . . . . . . . . . . . . . . . 606.10.3 HPLC-MS operating conditions (m/z 72, 75) . . . . . . . . . . 616.10.4 HPLC-MS operating conditions, eluent water . . . . . . . . . 626.10.5 HPLC-UV operating conditions . . . . . . . . . . . . . . . . . 636.10.6 HPLC-MS operating conditions for acrylamide derivative analysis 63

7 Results and Discussion 687.1 Coffee roasting in a laboratory roaster . . . . . . . . . . . . . . . . . 687.2 Coffee heated in a thermostatic oven . . . . . . . . . . . . . . . . . . 697.3 Standard condition roasting . . . . . . . . . . . . . . . . . . . . . . . 727.4 Acrylamide and 3-aminopropionamide formation in a model system . 74

7.4.1 Optimum time and temperature conditions for 3-aminopropionamideformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

7.4.2 Optimal heating time and temperature conditions for acrylamide 777.5 Heating of pure asparagine . . . . . . . . . . . . . . . . . . . . . . . . 817.6 3-Aminopropionamide in coffee . . . . . . . . . . . . . . . . . . . . . 827.7 Heated mixtures of asparagine with ascorbic acid . . . . . . . . . . . 837.8 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 Acrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Glycidamide formation from acrylamide . . . . . . . . . . . . . . . . 113 N -acetyl-S -(3-amino-3-oxopropyl)cysteine . . . . . . . . . . . . . . . 124 Glyceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 N -acetyl-S -(3-amino-2-hydroxy-3-oxopropyl)cysteine . . . . . . . . . 126 Acrylamide formation pathway from asparagine and dicarbonyl (adapted

from [63]) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Acrylamide formation pathways from acrolein and asparagine with sug-

ars (adapted from [62]) . . . . . . . . . . . . . . . . . . . . . . . . . 198 Acrylamide formation from asparagine by simple decarboxylation and

deamination reaction [39]. . . . . . . . . . . . . . . . . . . . . . . . . 209 Maleimide (2,5-pyroldione) . . . . . . . . . . . . . . . . . . . . . . . 2110 Fumaramic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2111 Enzymatic 3-aminopropionamide formation from asparagine (adapted

from [66]) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2212 Michael addition reaction . . . . . . . . . . . . . . . . . . . . . . . . 2313 Coffee plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2514 Arabica Zambia AA . . . . . . . . . . . . . . . . . . . . . . . . . . . 3215 Arabica Uganda Organico Biocoffee . . . . . . . . . . . . . . . . . . 3316 Arabica Tanzania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3317 Arabica Kenya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3418 Arabica Kenya AA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3519 Arabica Ethiopian Sidamo Yirgamo Grade 2 . . . . . . . . . . . . . . 3520 Arabica Indonesian Sumatra Lintong . . . . . . . . . . . . . . . . . . 3621 Arabica Indonesian Sulawesi Kalossi . . . . . . . . . . . . . . . . . . 3722 Arabica Indian Monsooned Aspinwalls Malabar AA . . . . . . . . . . 3723 Arabica Indian Plantation A . . . . . . . . . . . . . . . . . . . . . . 3824 Arabica Java WIB1 Jampit Gr1 . . . . . . . . . . . . . . . . . . . . . 3925 Arabica Nicaragua Talia Extra . . . . . . . . . . . . . . . . . . . . . 4026 Arabica Costa Rica Tarazzu . . . . . . . . . . . . . . . . . . . . . . . 4027 Arabica Guatemala SHB . . . . . . . . . . . . . . . . . . . . . . . . . 4128 Arabica Mexico Maragogype . . . . . . . . . . . . . . . . . . . . . . 4229 Arabica Mexico Altura . . . . . . . . . . . . . . . . . . . . . . . . . . 4330 Arabica Honduras SHG . . . . . . . . . . . . . . . . . . . . . . . . . 4331 Arabica Colombian Excelso . . . . . . . . . . . . . . . . . . . . . . . 4432 Arabica Santos Brazil NY 2 17/18 TOP Italian preparation . . . . . 4533 Arabica Papua New Guinea Sigri C . . . . . . . . . . . . . . . . . . . 4534 Robusta Indian Parchment . . . . . . . . . . . . . . . . . . . . . . . 4635 Robusta Indian Cherry AB . . . . . . . . . . . . . . . . . . . . . . . 4736 Vietnam Robusta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4737 Robusta Liberia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4838 Robusta Cameroon . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4939 Temperature gradient of roasting programmes 4, 6, 8 and 10. . . . . . 50

iii

40 Coffee beans roasted under different time and temperature conditions 5441 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-fructopyranose . . . . . . . . 5742 Reaction of 3-aminopropionamide to sulphonamide . . . . . . . . . . 5843 Derivatization of acrylamide with 2-mercaptobenzoic acid . . . . . . 5944 Typical chromatogram of acrylamide using ion exclusion chromatog-

raphy with UV detection . . . . . . . . . . . . . . . . . . . . . . . . 6045 Typical chromatogram of 3-aminopropionamide analysis . . . . . . . 6146 Typical chromatogram of acrylamide using MS detection . . . . . . . 6247 Typical chromatogram of acrylamide using UV detection . . . . . . . 6448 Typical chromatogram of acrylamide (6 ng/ml) after derivatization

with 2-mercaptobenzoic acid using LC-MS/MS . . . . . . . . . . . . 6549 Typical chromatogram of acrylamide in a roasted coffee sample after

derivatization with 2-mercaptobenzoic acid using LC-MS/MS . . . . 6650 Typical chromatogram of derivatized acrylamide analysis . . . . . . . 6751 Formation of acrylamide in 4 different types of coffee roasted in a

laboratory roaster . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6852 2nd Order regression curve of acrylamide content in coffee beans . . . 7053 Regression surface curve of acrylamide content in coffee beans . . . . 7154 Significant parameters in the formation of acrylamide . . . . . . . . . 7255 Acrylamide content in different coffee beans roasted under standard

conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7356 Acrylamide in heated asparagine and sucrose and asparagine and glu-

cose (molar ratio 1:0.5, 1:1 and 1:1.5) anhydrous mixtures at 130, 150and 170 � . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

57 3-Aminopropionamide in heated asparagine and sucrose and asparagineand glucose (molar ratio 1:0.5, 1:1 and 1:1.5) anhydrous mixtures at130, 150 and 170 �. . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

58 3-Aminopropionamide in heated asparagine and sucrose 1:0.5 anhy-drous mixtures at 130, 150, 170 and 190 � . . . . . . . . . . . . . . . 78

59 Acrylamide content in asparagine with glucose mixtures (molar ratio1:0.5 and 1:1) heated to different temperatures for 5 and 7 minutes . 78

60 Acrylamide formed in asparagine and glucose mixtures (1:0.5) heatedat different temperatures for 5 minutes . . . . . . . . . . . . . . . . . 79

61 Acrylamide formation in asparagine mixtures with glucose at 210 °C(molar ratio 1:0.5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

62 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 . . . . . . . . . . . 80

63 Acrylamide formation from pure asparagine heated at high temperatures 8264 Acrylamide formation in the mixtures of asparagine with ascorbic acid 8465 Acrylamide formation in asparagine and ascorbic acid mixtures heated

at 250 °C and different times . . . . . . . . . . . . . . . . . . . . . . 8466 3-Aminopropionamide formation in the mixtures of asparagine with

ascorbic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

iv

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

v

List of Tables

1 Acrylamide in foods from USA [29] . . . . . . . . . . . . . . . . . . . 22 Acrylamide in Canadian foods [30] . . . . . . . . . . . . . . . . . . . 83 Acrylamide in Australian foods [31] . . . . . . . . . . . . . . . . . . 84 Mean concentrations of acrylamide in UK food [32] . . . . . . . . . . 105 Roasting parameters of program 4, 6, 8 and 10. . . . . . . . . . . . . 506 Characteristics of green coffee beans processed by different methods. 527 Heating conditions of Amadori compound . . . . . . . . . . . . . . . 578 Concentration of acrylamide in Cameroon Robusta, heated in an ther-

mostatic oven, ng/g. . . . . . . . . . . . . . . . . . . . . . . . . . . . 699 Concentration of acrylamide in Cameroon Robusta, heated in an ther-

mostatic oven, ng/g. . . . . . . . . . . . . . . . . . . . . . . . . . . . 7110 Coffee roasted under standard conditions . . . . . . . . . . . . . . . 74

vi

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 Galceran Huguest

and her research team at University of Barcelona, Department of Analytical Chem-

istry for the cooperative work during the HEATOX project. I would like to address

particular thanks to Dra. Encarnacion 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 LATEX2ε.

This study was financed by Commission of the European Communities, spe-

cific RTD programme ”Food Quality and Safety”, FOOD-CT-2003-506820, ”Heat-

generated 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.

vii

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 3-

aminopropionamide 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 de-

tection 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 nei-

ther in green coffee beans nor in roasted ones.

Finally some experiments were done with mixtures modeling the acrylamide for-

mation 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 ascor-

bic 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-D-

fructopyranose 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 moglichen Vorstufe von Acrylamid,

gefunden. Kaffee wurde als Forschungsobjekt gewahlt, da er in großen Mengen kon-

sumiert wird und somit einen potentiell gefahrlichen Einfluß auf die menschliche

Gesundheit darstellt.

Nach der Extraktion mit Wasser und darauf folgender Festphasenextraktion wurde

Acrylamid mittels Flussigchromatographie nachgewiesen, wobei verschiedene Detek-

toren, vor allem UV und MS-Detektion, zum Einsatz kamen. Fur den Nachweis der

moglichen Acrylamid-Vorstufe 3-Aminopropionamid wurde Flussigchromatographie

mit Fluoreszenz-Detektion verwendet.

25 Sorten gruner Kaffeebohnen aus verschiedenen, uber die ganze Welt verteil-

ten Anbaugebieten wurden entweder mit einem Labor-Kaffeeroster oder im Ther-

mostatofen gerostet. Die Untersuchung zeigte, dass sich die beiden Sorten Arabica

und Robusta nicht nur in Große und Geschmacksqualitat, sondern auch im Acry-

lamidgehalt unterscheiden, und zwar unabhanging von der Anbauregion, sowie den

Bedingungen bei Ernte und Weiterverarbeitung.

Leider konnte die mogliche Acrylamid-Vorstufe 3-Aminopropionamid weder in den

grunen noch in den gerosteten Kaffeebohnen nachgewiesen werden.

Zum Schluß wurden einige Experimente mit Modellsystemen durchgefuhrt, wobei

ahnliche Konzentrationen von Acrylamid-Vorstufen wie in den Kaffeebohnen, sowie

ahnliche Rostbedingungen simuliert wurden. Sowohl Acrylamid als auch seine mogliche

Vorstufe 3-Aminopropionamid konnten dabei aus Mischungen von Asparagin mit

Glukose oder Succrose erzeugt werden, weniger leicht mit Ascorbinsaure. 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-dideoxy-

D-fructopyranose wurden Acrylamid und 3-Aminopropionamid gebildet.

ix

4 Introduction

4.1 Acrylamide

Acrylamide (Figure 1), also known as 2-propenamide, acrylic amide, ethylene carbox-

amide, 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 °Cand a high boiling point of 136 °C at 3.3 kPa [2, 3]. Acrylamide is very soluble in wa-

ter, 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 con-

tains 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 elec-

trophoresis 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 prepa-

ration 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 cook-

ing 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 Product Acrylamide, ppb2002 2003 2004

Baby Food ND-121Frenchfries

not baked 20-212

baked 119-1325ready to eat 117-1030 122-1250

Potatochips

117-2762 693-2510,1077

462-1970

Natural potato chips lot 1 879continued on next page

2

Table 1 – continued from previous pageFood Product Acrylamide, ppb

2002 2003 2004Natural potato chips lot 2 433Classic potato chips 0 weeks 319Classic potato chips 1 week 432Classic potato chips 2 weeks 280Classic potato chips 3 weeks 257Classic potato chips 4 weeks 343Classic potato chips 5 weeks 425

Infant For-mula

ND

Milk based Infant Formulawith Iron (liquid) NDMilk based Infant Formulawith Iron (powdered) <10Soy Infant Formulaliquid NDpowdered ND

ProteinFoods

Chicken (not baked) 32

Chicken (baked) 35Chicken ready to eat 22Fish (not baked) ND-25Fish (baked) ND-30Grilled vegetable burgers(not baked) ND-58Grilled vegetable burgers(baked) ND-116Veja-Links (uncooked) NDVeja-Links (microwaved) NDVeja-Links (toasted) ND

Bread andbakery

Pumpernickel (not toasted) 34

Pumpernickel (toasted) 364Whole grain whole wheat(not toasted) ND 102Whole grain whole wheat(toasted) 59Rye bread 42 ND-31Wheat bread <10-130Potato bread 36 41-52White bread (not toasted) 36 <10-18 ND-30

continued on next page

3

Table 1 – continued from previous pageFood Product Acrylamide, ppb

2002 2003 2004White bread (toasted) 216Wheat bagels (not toasted) 27 12-27Wheat bagels (toasted) 57Bread crumbs with cheese 39Bread crumbs regular style 42Doughnut ND-24 ND-22Flat bread from restaurant 125Pizza crust (not baked) 33Pizza crust (baked) 24Tortillas (not fried) <10-10Tortillas (fried) 13-15Seven grain bread ND-51French bread ND-25Nut bread 47Pies <10-74 NDBanana nut muffins 29

Cereals Cheerios 61-266Corn Flakes 77 70-534Corn pops 71Frosted Flakes 52 51-163Frosted mini-wheats 78Raisin bran 156Rice krispies 47Rice cereals 33Musli 11-51Granola 20-89Toasted wheat cereals 1057, 689Fruit wheels ND-92

Othersnacks

193-1243,151

Chifles 12Soy crisps 17Ropping corn (popped) 57-181 65-446Honey Dijon mustard Juli-ennapotato stix 1168Tortilla chips 117 130-196Veggie crisps 832 1340Pretzels 58-386Cheese puffs 166

continued on next page

4

Table 1 – continued from previous pageFood Product Acrylamide, ppb

2002 2003 2004Vegetable chips 73-828

Graviesand

Beef gravy (canned) ND

seasonings Chicken gravy (canned) NDMushroom gravy mix NDTurkey gravy mix NDSoy sauce NDHickory liquid smoke 54Pecan liquid smoke 151Mesquite liquid smoke 38Flavor enhancer NDInstant bouillon ND

Nuts andnut

Salted almonds 236-249

butters Smoked almonds 339-457Cashews NDRoasted peanuts ND-28Peanut butter 64-125

Crackers 37-620 39-1540Wheat crackers 26-300Crackers reduced fat 130Cheddar goldfish 57Crispbread fiber rye 504

Chocolateproducts

Droste cocoa ND-909

Unsweetened cocoa 316Baking chocolate 93-104Milk chocolate NDChocolate sweets ND-74

Fruit andvegetables Potatoes ND

Ripe olives 226-1925 798Black olives NDOlives in brine NDGreen olives <10- 19

Cannedfruits

ND-11

and veg-etables

Apple sauce <10

Baked beans 70-83continued on next page

5

Table 1 – continued from previous pageFood Product Acrylamide, ppb

2002 2003 2004Mushrooms NDChick peas NDPumpkin 25

Frozenvegetables

<10

Fruit andvegetableproducts

French fried onions 125

Breaded mushrooms 103Dried plums 31-87Raisins <10

Cookies 36-199 334-432 69-955Wafers 61-114Coffee ground, not brewed 175-207 91-609

regular roast 37-374regular roast, decaffeinated 222-361 27-149dark roast 97-319crystals, not brewed 351Instant coffee, powdered,not brewed 188-263 266-458decaffeinated 172-539Brewed 5-11, 3-13

Other hotbeverages

Instant caffeine free hot

beverage, powdered, notbrewed

3747-5399

brewed 93Driedfoods

Soups 22-1184

Noodles 11-136Rice with sauce <10

Dairy Condensed milk NDMalted milk original 43Instant nonfat dry milk 11Buttermilk blend <10Milk ND

Miscellaneous ND-398Onion rings 13Mashed potatoes ND-<10

continued on next page

6

Table 1 – continued from previous pageFood Product Acrylamide, ppb

2002 2003 2004Gelatin dessert <10 NDNon-dairy coffee creamer <10Ground toasted corn 804Pizza rolls (frozen) 29Potato skins 393

Juice Prune 267 138-239Taco,Tostadoand Tor-tilla prod-ucts

Taco shells 29-69

Tortillas 33-794Tostado 337

Restaurantand take-out food

Asian ND-169

Southersn/Cajun ND-202Hispanic ND-129Potato pancakes 229Waffle potato fries 271

Fruits andvegetables,take-out

Grilled asparagus ND

Sweet potato chips 4080Grilled carrots 61Grilled onions 70Potatoes 114-984Roasted mixed vegetables ND

As it can be seen in the Table 1, the highest amounts of acrylamide can be de-

tected in potato products (French fries, potato chips), some cereal products, cookies,

snacks, salted nuts, crackers, coffee and other powdered hot beverages, dried soups,

even black ripe olives and prune juice. Small amounts could be detected in protein

rich foods, bread and bakery products, cereals, gravies and seasonings, chocolate

products, canned and frozen fruits and vegetables, wafers, dairy products, restaurant

and take-out foods. In baby foods, infant formula, most of protein foods, gravies and

seasonings, fresh fruits and vegetables acrylamide is not found. The acrylamide levels

7

in foods can vary depending on the year of analysis (improved methods). Rather big

differences in concentrations can be observed even among different lots of the same

product.

Table 2: Acrylamide in Canadian foods [30]

Food Product Acrylamide, ppbFrench fries 59-1900Bread 14-47

Toasted French bread 290Coffee, not brewed 8-150Cocoa products Baking chocolate 190

Milk chocolate <2-40Cocoa powder - bulk 45Instant coffee 430-1700Instant cereal beverage 4300

Others Roasted almonds 260Sweet potato chips 260Potato pancakes 210-290Roasted sunflower seeds 66Roasted soy beans 25Beer <6Boiled mashed potatoes <4Hamburger <3

Potato chips 300-3400Cereals Wheat 88-170

Rice 50-160Baby food <10-60

Biscuits 120

In Canadian food, French fries, potato chips, instant coffee contain the highest

amount of acrylamide (Table 2). Potato chips have an even higher acrylamide content

compared to chips produced in the USA.

Table 3: Acrylamide in Australian foods [31]

Food Product Acrylamide, ppbCoffee Instant, decaffeinated <3

Instant <3Brewed, decaffeinated <10-15Brewed, dark roast <10-14

continued on next page

8

Table 3 – continued from previous pageFood Product Acrylamide, ppb

Brewed, light roast <10-23Instant cereal bever-age

trace

Chocolate milk <25Cocoa powder <100Prune juice 93Chocolate bar <25

plain block <30Nuts Cashew traces

Mixed, salted tracesPeanuts, dry roasted <25

Canned soups <25-50Baked beans canned <25Spanish olives black 345

green <25Meat pie <25Fried rice <25Pizza frozen oven baked <25

microwave 5 min <25Fish crumbed and fried <25

crumbed and oven baked 52Chicken schnitzel <25Hash brown fried 440

oven baked 200-320Beef schnitzel fried and oven baked <25

Surprisingly small amounts of acrylamide are reported in Australian foods (Table

3). The highest concentration of acrylamide is declared only in prune juice, Spanish

black olives, crumbed fish and hash, fried and oven baked as well. Such a huge

difference could be the outcome of not well developed analysis methods or because

only food products that are popular in Australia were chosen and which contain the

lowest amounts of acrylamide. However, potato products, such as French fries or

potato chips are not included.

In the Table 4 data from the United Kingdom are presented. The food infor-

mation sheet [32] says, that the used food samples represent the average UK diet

and sampled foods are prepared according to normal domestic practice. The dietary

exposure estimates showed that cereal-based products and potatoes are the main

9

Table 4: Mean concentrations of acrylamide in UK food [32]Food group Acrylamide, ppbBread 12Miscellaneous cereals 57Meat products 13Fish <5Oils and fats <3Sugars and preserves 23Green vegetables <2Potatoes, made-up 53-112Canned vegetables <5Fruit products <1Beverages <1Nuts <3

source of acrylamide in this European county. Acrylamide was detected in a lot of

products including cereal, meat, sweets, preserves and vegetable products. Though

presenting the quantified results with amounts of acrylamide in everyday’s food, the

Food Standard Agency does not tell people what they should eat but recommends

them to eat all types of foods including cereal, potato products, plenty of fruits and

vegetables avoiding sugar and fatty foods.

It has been noticed, that after a while the acrylamide content in certain food

matrices is getting less. The reduction of this toxin was observed in instant coffee (67

% in one year), roasted coffee (28 % in 7 months), roasted barley (15 % in 9 months)

and cocoa (2 % in 3 months). However, there are also some products (e.g. breakfast

cereals) in which the concentration of acrylamide is not changing over a prolonged

storage period of up to one year [4].

In ground coffee there can be relatively high amounts of acrylamide up to 400

ng/g powder. As acrylamide is a very polar substance, it is not surprising that it is

also detected in large quantities in brewed coffees. In analyzed grounds after brewing

no acrylamide was detected. It seems that all acrylamide available in coffee powder is

transferred to the water where it is quite stable. No significant decrease in acrylamide

levels was observed even after 5 hours of heating [33].

10

4.3 Toxicity of acrylamide

Polymeric acrylamide is nontoxic, but a monomer is neurotoxic to both humans and

laboratory animals. Acrylamide is carcinogenic to laboratory rodents and is described

by the International Agency for Research of Cancer as a probable carcinogen to hu-

mans [5]. Furthermore, acrylamide is not mutagenic in prokaryotic (organisms, that

do not have a real nucleus in their cells) mutagenesis assays, but chronic acrylamide

intake has shown to produce tumors in both rats and mice [34]. Acrylamide’s neuro-

toxicity is characterized by ataxy, distal skeletal muscle weakness and the numbness

of the hands and feet. In the human body acrylamide is oxidized into the epoxide

glycidamide (2,3-epoxypropionamide) via an enzymatic reaction (Figure 2), possibly

involving cytochrome P450 2E1 [4].

Figure 2: Glycidamide formation from acrylamide

Both acrylamide and glycidamide can form hemoglobin adducts [35], but only

glycidamide has been shown to form adducts with DNA amino groups. This fea-

ture of glycidamide implies genotoxicity. Furthermore, high levels of acrylamide can

cause genetic mutations and cellular transformation [34]. Both acrylamide and gly-

cidamide can be detoxified in the cells by glutathione conjugation, or by hydrolysis

[5]. Furthermore, higher acrylamide hemoglobin adducts are detected in smokers,

because acrylamide is also found in tobacco smoke as a result of an incomplete com-

bustion or heating of organic matter [26]. In experiments with rats it was observed

that N -(2-carbamoylethyl)valine adducts were of the magnitude that is similar to the

background level of nonsmoking humans [36]. Furthermore, acrylamide can be ab-

sorbed through the skin, but dermal uptake is only approximately 7 % of oral uptake

[37].

Acrylamide binds to DNA directly via Michael addition reaction in vitro and in

11

vivo [34]. Acrylamide can be excreted from the human body with urine, mostly in me-

tabolized form. N -acetyl-S -(3-amino-3-oxopropyl)cysteine (Figure 3) appears to be a

major metabolite (72 %). The other possible metabolites are glyceramide (hydrolized

glycidamide, 11 %) in Figure 4, glycidamide (2.6 %), N -acetyl-S -(3-amino-2-hydroxy-

3-oxopropyl)cysteine (Figure 5) and N -acetyl-S -carbamoylethylcysteine-S -oxide (14

%) [4].

Figure 3: N -acetyl-S -(3-amino-3-oxopropyl)cysteine

Figure 4: Glyceramide

Figure 5: N -acetyl-S -(3-amino-2-hydroxy-3-oxopropyl)cysteine

Metabolism of glycidamide in humans is mostly via hydrolysis with little via

glutathione conjugation.

12

4.4 Acrylamide formation pathways

There are a few acrylamide formation mechanisms postulated in the scientific re-

ports. Zyzak et al. [38] declare a decarboxylation of the Schiff base pathway,

which is followed by imine’s heterocyclic cleavage or imine’s hydrolysis, when 3-

aminopropionamide is formed and then deaminated to acrylamide. Schieberle and

Granvogl [39] propose the simplest decarboxylation followed by deamination of as-

paragine reaction pathway. Yaylayan et al. [40] show a decarboxylation of the

Schiff base via oxazolidin-5-one intermediate, decarboxylated Amadori product and

β-elimination. Stadler et al. [41] and Yasuhara et al. [42] propose a pathway of

acrylamide formation from fat degradation products when acrylic acid and acrolein

are reacting with amino acid degradation product ammonia. It was reported, that

acrylamide can only be formed from compounds that are naturally present in raw

foods. It cannot be formed from decomposed polymers used for agricultural crops

[43, 44].

Acrylamide is found in large amounts in potato products. It is experimentally

proven that for acrylamide formation in potato products free reducing sugars, mainly

glucose and fructose, have a major influence compared to free amino acids [45, 46, 47].

In experiments the addition of fructose had the strongest effect on the increase of the

acrylamide concentration. Whereas, the addition of asparagine had a rather weak

effect. When asparagine was added in combination with fructose it gave a strongest

response.

Acrylamide is formed in the outer layer of the deep fried potato products (French

fries, crisp, chips). Cooking time and temperature, but not cooking oil type have the

greatest influence on acrylamide formation [48]. With salting or pre-drying, lowering

the pH of the product the acrylamide content during frying can be reduced [4]. For

French fries it is suggested to reduce the frying temperature towards the end of

frying in order to reduce the acrylamide content and still develop a good product

color [49, 50]. Another possibility is to coat the potato slices with proteins, e.g. from

chick peas [51].

13

In contrast to potatoes, the addition of glucose or sucrose to bread dough has a

very small effect [45]. An addition of free asparagine to the flour before the dough

preparation increases the acrylamide contents in baked bread drastically. Therefore

it was concluded, that free asparagine is a limiting factor for acrylamide formation

when we talk about bread and bakery products [52, 53]. A possible way to reduce

acrylamide in bakery products can be the addition of citric acid. It not only reduces

the pH and acrylamide amount in bread, but it also reduces browning, sufficiency of

leavening and affects the taste. The other possibility to reduce acrylamide in bakery

products is to reduce the oven temperature and to increase the baking time [54],

as well as reducing the raising agent ammonium bicarbonate and use ammonium

hydrogencarbonate instead [4].

As lot of experiments show that acrylamide is not formed at temperatures below

120 °C. However it is noticed, that in the heated mixtures of asparagine with sucrose,

a certain amount of water is needed to hydrolize the oligosaccharides to transient

intermediates (glucose and fructose) in order to form the acrylamide [4].

Another product, that is heated to high temperatures to improve its taste and

aroma properties, is coffee. Though coffee beans are roasted at quite high tempera-

tures (220 - 250 �), the acrylamide amounts found in the roasted beans and ground

coffee were reported to be low [6]. There are no significant differences if coffee is

decaffeinated or not. Since consumers are not eating the ground coffee beans, but

prepare a beverage, it is important to calculate acrylamide content not per gram

coffee powder, but per cup. As it was reported [33], during the brewing step of the

coffee beverage, almost all acrylamide present in the coffee powder, is transferred to

the liquid phase of the coffee drink, due to its high solubility in water. Furthermore,

the dietary intake of acrylamide from coffee in northern countries (Norway, Sweden)

can be more than 30 % [55], in Denmark 20 % [56], 36 % in Switzerland [57]. No

data from other countries is available yet.

In coffee, acrylamide is formed in high concentrations during the first minutes of

roasting, resulting in >7 mg/kg. The increase of roasting time leads to the degra-

dation of acrylamide. Kinetic models and spiking experiments with isotope labeled

14

acrylamide showed that >95 % of acrylamide is lost during roasting [3, 54]. However,

the roasting conditions have an important influence on the typical coffee aroma and

taste that are desirable to consumers. Therefore, the optimization of the roasting

conditions with respect to a reduction of the acrylamide formation and maintaining

the product quality has not been realized yet.

Recent reports have announced that acrylamide is not stable in commercial coffee

that is stored in its original container [33].

It is known that the acrylamide formation is favored under low moisture conditions

[4]. It was reported that in a model systems based on fructose the acrylamide content

was increasing with increasing water activity. It is interesting to notice, that in

most experiments fructose with asparagine mixtures were more efficient in acrylamide

formation than mixtures with glucose. Even under anhydrous conditions fructose is

highly reactive and forms higher amounts of acrylamide.

It was recently reported, that the time and temperature of heating have a direct

influence on the acrylamide formation in foods. Acrylamide can already be formed

at 120 °C at a longer time of heating in asparagine mixtures with glucose, whereas

at 160 °C the highest acrylamide concentration was obtained at shorter heating time

[4].

It was reported that the pH has an influence on the acrylamide formation. Adding

some acidifying compound into the potato matrix, at a low pH (<6), a decrease of

acrylamide formation was observed. In contrast, at high pH (∼8) the highest content

of acrylamide formed was detected in potatoes heated to 160-180 °C [58]. It is worth

to notice, that the extraction of acrylamide at pH 2-7.5 gives the same results as a

normal water extraction. But an extraction at a pH >8 gives an increase of acrylamide

recovery and reaches the maximum at about pH 12 [59]. In most scientific works

acrylamide extraction is performed at normal water extraction conditions, and it is

assumed that all the water-soluble acrylamide is bioavailable [59]. By changing the

pH the matrix of the food can be changed and the chemical available acrylamide

dissolves [60]. But as it is known, acrylamide additionally released from a chemically

bound form at the high pH is not bioavailable.

15

4.4.1 The Maillard reaction

The Maillard reaction is named after the first researcher investigating the reaction

of amino acid with carbohydrates, Louis-Camille Maillard (1978-1936). It was then

reported, that amino acids in combination with different carbohydrates react at tem-

peratures above 100 °C forming the typical dark brown color and flavor of cooked

foods [3, 61]. Due to this extremely complex reaction not only desirable color, aroma

and taste properties are gained, but also antioxidant compounds are formed [3].

L.C.Maillard found, that the aldehyde group of an aldose reacts more efficiently

with amino acids than the hydroxyl groups [61]. The melanoidins (reaction products

with a dark brown color) are soluble in the early reaction stage and become insoluble

later. It was found, that the most reactive amino acids are alanine, followed by valine,

glycine, glutamic acid, leucine, sarcosine and tyrosine. Monosaccharides react easily

with amino acids. Sucrose, a non reducing sugar, needs to be hydrolized first to form

the reactive intermediates. Pentoses react faster than hexoses.

The Maillard reaction is devised into three stages. At first, the condensation

reaction of the carbonyl group from a reducing sugar with an amino compound takes

place. In this way the Schiff base is produced. Acid-catalyzed rearrangements give

Amadori rearrangement products, which are not stable above room temperature. In

the reaction of an amino acid with ketose a deoxyaldosylamine is formed by the Heyns

rearrangement [3, 61].

In the second reaction stage, during further enolisation, deamination, dehydration

and fragmentation steps various products including furfurals, furanones, pyranones

and other sugar dehydratation and fragmentation products are formed. In this stage,

amino acids also undergo deamination and decarboxylation through Strecker degra-

dation. α-Hydroxycarbonyls and deoxyosones, intermediates of the Maillard reaction,

and dicarbonyls can act as Strecker reagents and produce Strecker aldehydes. Such

aldehydes can also be formed from a Schiff base. Dehydroascorbic acid, found in

foods, can act as Strecker reagent as well.

In the third stage of the Maillard reaction high molecular mass substances (melanoidins)

16

form during a condensation reaction between carbonyls (especially aldehydes) and

amines [3].

The Maillard reaction was shown to be one of the major pathways in acrylamide

formation both in food matrices and in model systems in the presence of asparagine

and carbohydrates (Figure 6) [38, 45, 62]. Labeling experiments confirm that the

carbon skeleton and the nitrogen of the amino group are derived from asparagine

[38].

Figure 6: Acrylamide formation pathway from asparagine and dicarbonyl (adaptedfrom [63])

For this pathway dicarbonyl compounds (e.g. from glucose) and free amino acids

(e.g. asparagine) form a Schiff base. Further reactions lead to the formation of

3-aminopropionamide and finally acrylamide [38, 63].

It was shown in some experiments that out of twenty amino acids heated with

glucose only asparagine gave significant quantities of acrylamide. In comparison,

however, glucose, fructose, galactose, lactose and sucrose produced similar quantities

of acrylamide. Surprisingly, sucrose, a non-reducing sugar, can produce almost as

17

much acrylamide as some of the reducing sugars in a reaction with asparagine . It is

suggested, that sucrose in real food matrixes can undergo hydrolysis and form glucose

and fructose [3].

During the Maillard reaction a very large number of volatile compounds are

formed. Already more than 550 compounds have been identified. Over 330 com-

pounds found in the reaction systems are volatiles. Most of them give typical flavor

description of different food products [3]. In roasted coffee melanoidins are respon-

sible for the dark color and partly for the aroma. It has been noticed, that coffee

melanoidins can react with volatile compounds and in this way modify the aroma per-

ception in the coffee beverage. Furthermore, the melanoidin spectrum is significantly

influenced by the roasting degree: the darker the beans, the more high-molecular

weight melanoidins can be detected [64].

Maillard reaction some products can be mutagenic, but not carcinogenic [3, 61].

Pyrolyzates of proteins, peptides, and amino acids, especially tryptophan and glu-

tamic acid, coffee beverages prepared in a usual way, black and green tea were re-

ported to contain some mutagenic compounds resulting from the Maillard reaction.

Moreover, mutagenicity appears only above 400 °C and even more strongly at 500-600°C as the pyrolysis temperature [61].

Some compounds showing antioxidant activity were also detected among the Mail-

lard reaction products. These compounds, preventing oxidation of lipids are found

in the melanoidin fraction [61].

4.4.2 Lipid degradation

Another possible pathway for acrylamide formation in heated foods, especially in

deep-fried food products, was suggested to be an oxidative lipid degradation, when

acrolein and acrylic acid - the precursors of acrylamide - are formed [50] and then

they can react with ammonia formed from amino acids (Figure 7) [42], e.g. aspartic

acid [41]. In [4] it is noticed, that in the model system with selected amino acids

and glucose not only aspartic acid can be described as a possible precursor of acrylic

18

acid, but also β-alanine and carnosine (dipeptide). Acrylic acid can be generated

indirectly from other amino acids such as serine and cysteine, forming pyruvic acid,

then reducing it into lactic acid and finally dehydrate into acrylic acid. Free amino

acids in foods such as asparagine, glutamine, cysteine and aspartic acid can be one

of the main sources producing ammonia under thermal treatment of foods [4].

OR

OR

OR

OAcrolein

O

OH

Acrylic acid

NH3

NH2

O

HOOC

NH2

Asparagine

+ Glucoseheat

NH2

OAcrylamide

Figure 7: Acrylamide formation pathways from acrolein and asparagine with sugars(adapted from [62])

Acrylic acid may also appear as an acrolein oxidation product, which can be

formed in the thermal degradation of lipids, either from the oxidation of fatty acids

or from the glycerol moiety [3]. Other sources of acrolein could be amino acids.

A high concentration of acrylamide was found in fried potato products. Experi-

ments showed a slight difference among potato products fried in different oils. It was

observed, that samples fried in olive oil had a higher acrylamide content. Further-

more, the addition of some antioxidants, e.g. rosemary herb, reduced the acrylamide

formation by ∼25 % [62]. In the model system, when asparagine was heated in dif-

ferent oils and fats, it was observed that lard or bovine fat, both containing low levels

of unsaturated lipids, produced lower amounts of acrylamide [4]. The results of these

experiments show, that the higher the degree of unsaturation of the lipids, the larger

the amount of acrylamide is formed. Thus it seems, that acrylamide is not formed

19

Figure 8: Acrylamide formation from asparagine by simple decarboxylation anddeamination reaction [39].

directly from acrolein present in the oil itself. Additionally, by increasing the frying

time the acrylamide content in the samples was also increasing.

The acrylamide content formed in the baked, fried food products depends on

the precursors (amino acids, sugars) amount in the raw material. It was noticed,

that using an industry standard procedure and a frying temperature of 180 °C, the

acrylamide content in potato products can be properly reduced when potatoes with

low sugar content are selected [65].

4.4.3 Decarboxylation and deamination of asparagine

Recently it was reported that acrylamide can be formed from asparagine in absence

of glucose [39]. For this reaction high temperatures (>170 °C) are needed. It was

noticed, that a direct decarboxylation followed by deamination of asparagine can

occur, where reducing sugars do not take place in the reaction (Figure 8).

Furthermore, the amounts of acrylamide were up to 100 times lower in comparison

with model system where glucose was included. This fact helps to understand why

most foods heated at high temperatures contain acrylamide: low amounts of free

asparagine can be detected in almost all kinds of food products [39].

According to Yaylayan et al. [40], the main product is maleimide (Figure 9) and

not acrylamide, when pure asparagine is heated in model system. Maybe because of

the relatively high reaction temperatures (250 and 350 �) asparagine performed fast

intramolecular cyclization and so prevented the formation of acrylamide. Addition-

ally, it was reported that maleimide can be formed also in the presence of reducing

20

sugars.

Figure 9: Maleimide (2,5-pyroldione)

Fumaramic (Figure 10)acid could be one of asparagine degradation products. Its

decarboxylation could also lead to the formation of acrylamide [39].

Figure 10: Fumaramic acid

It seems, that heating of foods at extremely high temperatures (>250 �) for

a shorter time can decrease the amount of acrylamide, because at these tempera-

tures the cyclization of asparagine is preferable and this prevents the formation of

acrylamide [40].

4.4.4 Other precursors for acrylamide formation (3-aminopropionamide)

The direct precursor of acrylamide was shown to be 3-aminopropionamide. The

Strecker alcohol of asparagine (3-hydroxypropanamide) was studied under pyrolitic

conditions [63]. Acrylamide can be easily formed from 3-aminopropionamide by a one-

step dehydration process. As it is mentioned in [63], significant amounts of acrylamide

21

Figure 11: Enzymatic 3-aminopropionamide formation from asparagine (adaptedfrom [66])

in fructose/asparagine mixtures are formed in high quantities at temperatures >180�, whereas significant amounts of acrylamide from 3-aminopropionamide can already

form at 140 °C and the maximum concentration can be achieved at 180 �. It was

also observed, that the thermal degradation of 3-aminopropionamide to acrylamide

under aqueous or low water conditions can already generate at temperatures between

100 and 180 °C [66].

When we look at the asparagine molecule, we can notice, that after we eliminate

NH3 and CO2, we get the acrylamide molecule. In raw materials enzyme decar-

boxylase might generate the biogenic amine (3-aminopropionamide) from asparagine

already at temperatures <100 °C (Figure 11). This reaction does not involve reducing

carbohydrates. The biogenic amine can be formed from the amino acid with pyri-

doxal phosphate as a cofactor. As it is known that enzymatic reactions take place

22

under physiological enzymatic conditions (high water content, warm (10-40 �) tem-

peratures), 3-aminopropionamide formation was especially observed in potatoes [66].

As 3-aminopropionamide is easily deaminated to acrylamide during the heating of

food products, e.g. potatoes, it can be explained why some potatoes during heating

produce more acrylamide though the free amino acid and sugar content in this raw

material is low. Furthermore, the last studies show, that not only potatoes, but also

milk products, e.g. fermented cheese, can contain 3-aminopropionamide and after

heating this can react to form acrylamide [39].

4.5 Michael addition

Losses of acrylamide were reported by a number of researches. This decrease in acry-

lamide amount in processed foods could be a result of evaporation or polymerization

of the monomer. However, it is more likely that acrylamide still reacts with other

components in the food matrix. Such a reaction between acrylamide and nucleophilic

groups, is called Michael addition (Figure 12). Some of such nucleophilic groups could

be amino or thiol, which could be present in free amino acids or as peptides and pro-

teins such as the sulfhydryl group of cysteine, ǫ-amino group of lysine or N-terminal

amino group of proteins.

Figure 12: Michael addition reaction

The Michael addition reaction may be reversible and in certain circumstances it

can lead to the release of acrylamide [3].

23

4.6 Coffee: plants, beans and their production

4.6.1 Differences of Arabica and Robusta

Coffee plants belong to the Rubiacea family, which includes more than 500 genera and

∼8000 species [67]. There are at least 66 species of the genus Coffea L.. Economically

important sorts are Coffea arabica L., Arabica coffee, which represents three quarters

of the world coffee productions, and Coffea canephora Pierre, Robusta coffee, which

makes only one quarter of the world coffee production. Both species are grown

in tropical and subtropical regions: in Central and South America mainly Arabica

is produced, and Africa and South Eastern Asia countries are the main Robusta

production region.

The Arabica and Robusta coffees have different characteristics. The plant (Figure

13), the Arabica coffee tree is 2.5 to 4.5 meters high, is usually cultivated in highlands.

It is resistant to lower humidity conditions and can survive at colder temperatures.

The plant of the Robusta tree is 4.5 to 6.5 meters high, is cultivated more in lowlands.

It tolerates warmer temperatures and higher humidity and is more sensitive to the

cold. At cupping Arabica beans have a good bitter/acid balance and chocolaty to

flowery aroma while the Robusta coffee beans are famous for more bitter taste and

a woody to earthy aroma. That has an influence on the price of the green beans:

Robusta’s price is 20-25 % lower than Arabica’s. In addition, Arabica coffees contain

less caffeine than Robusta (1.2 and 2.2 %, respectively).

The coffee tree in blossom period forms clusters of two to nineteen white flowers.

Fruits are 1.5 cm in diameter and are called ”cherries”, which ripen for seven to nine

months (Arabica coffees) or even nine to eleven months (Robusta coffees). The cherry

has a red or yellow exocarp (skin) when ripe with a gelatinous-pectic mesocarp (the

pulp) of 0.5 to 2 mm thickness, rich in sugars and water, glued over the endocarp

(the parchment), enclosing each seed. The pectins present in the pulp may work as

an energy storage. There are usually two seeds (beans) per cherry, sometimes there

can be only one seed, which is called peaberry. Seeds can vary in size, shape and

24

Figure 13: Coffee plant

density according to growing conditions and genotype.

The cell structure of the beans is characterized by very thick cell walls, which

make raw beans extremely hard.

Amino acids are present in green coffee beans both free (5 % of the total) and

bound to proteins. The free amino acid level depends on maturation. According to

Murkovic et al. [68], the main free amino acid detectable in green coffee beans is

alanine, followed by asparagine. Furthermore, more free amino acids are found in

Robusta green coffee beans.

Carbohydrates are present in green coffee beans both as insoluble and as soluble

polysaccharides, with some arabinose, oligosaccharides, mainly sucrose and traces of

reducing sugars [68, 69]. More sucrose is detected in Arabica (up to 90 mg/g) than in

Robusta beans, but Robusta beans contain more reducing sugars e.g. fructose [68]. In

addition, the glucose content in green coffee beans may influence the cup sweetness.

There are only 0.2-0.3 % of lipids in the green coffee beans present in a waxy layer

surrounding and protecting the beans.

25

4.6.2 Harvesting and processing

Harvesting is done in one of two ways, either by stripping or by picking. In the case

of harvesting by stripping, all cherries (immature, ripe, overripe (raisins), dry) are

stripped together with leaves from the branches at the same time, either by hand or

mechanically. When the picking or finger-picking method is used, only ripe cherries

are hand-picked, and collected in baskets or heavy pieces of cloth laid underneath the

trees. Sometimes techniques that combine stripping and picking can be used, when

only ripe cherries are picked and the other - overripe, green, dry - are collected very

late in the season. Another harvesting method, that is sometimes used, leaves the

cherries on the trees until they are dried and then they are harvested all at the same

time late in the season by shaking the trees.

The crop processing must start as soon as possible after harvesting the cherries

in order to avoid the fermentation and off-flavor formation. The cherries can be

processed either by dry or wet method. Near the equator, only a wet process is

possible because of the rainy climate condition throughout the year. In areas, with

little rainfall and where enough sunshine is ensured, the dry process method must

be used. Subtropical areas, where the rainy and dry season are well defined, both

methods (dry or wet) can be used.

Beans processed with the dry method are sun-dried on patios. After the cherries

are brought in from the fields, they are first cleaned from impurities by compressed

air and by washing. By using water the leaves, wood sticks and soil particles can be

separated from the beans. Also cherries can be separated into two categories: heavier

ripe cherries and lighter overripe ones. After twelve to twenty days of drying the

beans finally contain 12 % humidity and are ready for roasting. Coffees processed by

this method are so-called natural coffees, giving the cupping a full body and a mild

aroma.

The wet method can only be used with finger picked cherries and when there

are only a few or no not yet ripe cherries in the harvest. The coffee beans are

separated by removing their skin mechanically and the mucilage is washed away after

26

the fermentation step.

In the beginning the cherries are washed in large water tanks, where heavy (ripe)

and light (immature) cherries are separated. After the cherries move through the

depulping machine, which removes the skin and some adhering pulp, they remain

covered with a layer of 0.5-2 mm thick mucilage. Due to naturally present microor-

ganisms, the beans ferment now for 18-36 hours, depending on ambient temperature,

wether they are stored in brick or concrete tanks, or wether they are immersed in

water (so-called wet fermentation) or not (so-called dry fermentation). Fermentation

ends when the parchment loses the slimy layer of the mucilage. After the beans are

washed and dried to a humidity level of ∼12 % they are ready for roasting. When

beans are processed by the wet method, they are called washed coffees, resulting in

a highly aromatic cup with a fine body and an acid live aroma.

The third method, that can be used, is a semi-dry process [70]. It is a mixture

between dry and wet processing methods where the fermentation step is omitted.

The beans, when this method is used, are washed and depulped. The remaining

mucilage is not fermented, and the beans are dried in the sun. After the beans are

dried to ∼12 % of humidity, the mucilage is mechanically removed and the beans are

transported to a roasting facility.

Usually the beans are electronically sorted before the roasting to avoid that defect,

fungal contaminated beans or other beans with unwanted properties which could have

an undesirable effect on the cup quality, are roasted as well. It was observed, that

so-called black and sour beans roast to a smaller degree, thus reducing the beverage

quality [71]. Interestingly to notice that natural coffees regularly have more defects

compared to the washed ones, but the natural coffee beans are easier to sort out [67].

4.6.3 Coffee roasting

The roasting process takes place at quite high temperatures: It can range from 240°C up to 300 °C for industrial roasting [72]. For laboratory bean roasting 24 minutes

at 220-230 °C could be estimated as optimal conditions for the acceptable sensory

27

properties for the coffee beverage [73].

Roasting induces several visible changes in color, texture, density and size [74, 75,

76]. Also the beans lose up to 8 % in dry weight, mainly because of the loss in sugars

taking place in the Maillard reaction and pyrolysis [77]. It was observed, that coffee

beverages show some antioxidant properties [78, 79]. Especially green coffee beans

are rich in antioxidative compounds (e.g. chlorogenic acid), which during roasting

are almost lost [80]. The roasting process for the coffee beans has some advantages:

not only the pleasant taste and aroma are formed, but also the natural contaminant

ochratoxin A can be eliminated [81, 82].

The roasting process can be divided into three phases. The initial is a drying phase

when the moisture is eliminated from the coffee beans, keeping the bean temperature

at around 100 �. This phase lasts only a few seconds since the green coffee beans

contain no more than 12 % of humidity.

The second phase is the actual roasting step. In this phase at ∼170 °C complex

exothermic pyrolytic reactions take place. Large quantities of CO2, water and volatile

substances are released. At temperatures >120 °C the Maillard or non-enzymatic

browning reactions take place, which are followed by Streckers degradation at ∼160°C. Sugar and minor lipid degradation reactions also take place at the temperatures

close to 180-200 �. And of course at such high roasting temperatures intermediate

decomposition products interact as well.

The third phase is the cooling phase. The beans are rapidly cooled using cold air

or water as a cooling agent. Coffee beans can be roasted to a different roasting degree

which depends on the consumers taste. It is known that light roasting is prefered

in northern countries whereas dark or very dark roasting is preferred in southern

regions.

28

5 Purpose of the study

Because acrylamide is very small polar molecule, and coffee in comparison is a com-

plex enough matrix, methods of extraction and clean-up, followed by analytical ones

were established.

In order to understand the differences between the coffee types better, the follow-

ing successive steps had to be taken:� Coffee was roasted to different roasting degrees, using a small scale laboratory

roaster.� The influence on acrylamide formation in coffee roasted under standard roasting

conditions was studied.� Coffee was roasted at different time and temperature conditions in order to

observe the acrylamide formation kinetics.� Extraction and a liquid chromatography with fluorescence detection analytical

method was established in order to determinate 3-aminopropionamide in green

and roasted coffee beans

With a model system imitating similar to acrylamide formation reaction condi-

tions the following steps were taken:� The influence of time and temperature, molar differences on acrylamide and 3-

aminopropionamide formation kinetics in the asparagine mixtures with sucrose,

glucose was observed.� Optimal formation conditions for acrylamide and 3-aminopropionamide in the

anhydrous mixtures of asparagine with glucose were defined.� Pure asparagine was heated in order to determinate acrylamide or other possible

compounds that have formed.

29

� Asparagine mixtures with ascorbic acid were heated in order to observe other

acrylamide formation theories.� Amadori compound was heated in order to review an acrylamide formation

pathway during the Maillard reaction.

30

6 Materials and Methods

6.1 Chemicals and solvent

Water was distilled twice and further purified using a water purification system

Simplicity 185 (Millipore, Billerica, MA, USA). 3-Aminopropionamide hydrochlo-

ride 97 % was purchased from ABCR (Karlsruhe, Germany). Other solvents and

chemicals like HCl, dansyl chloride approx. 95% TLC, 2-mercaptobenzoic acid, L-

ascorbic acid were purchased from Sigma-Aldrich Chemie Gmbh (Steinheim, Ger-

many), and NaHCO3, NaOH, acrylamide >99% L-asparagine anhydrous, D-(+)-

glucose anhydrous, D(+)-sucrose, glycine, diethyl ether were purchased from Fluka

(Buchs, Switzerland). 13C3-Labeled acrylamide 99+% purity was obtained from

MERCK-Schuchardt (Hohenbrunn, Germany).

Acetonitrile, methanol both HPLC grade, acetone and n-hexane were purchased

from LGC Promochem (Wesel, Germany), formic acid 98-100% purity from Riedel-de

Haen (Seelze, Germany).

6.2 Coffee samples

There were altogether twenty five types of green coffee beans roasted and analysed

for acrylamide or 3-aminopropionamide. Five sorts of Robusta and twenty sorts of

Arabica coffees are described onward.

Six different types of Arabica beans from Africa, namely Zambia AA in Figure 14,

Uganda Organico Biocoffee in Figure 15, Tanzania in Figure 16, Kenya in Figure 17,

Kenya AA in Figure 18 and Ethiopian Sidamo Yirgamo Grade 2 in Figure 19 were

used.

The average size of Zambia coffee beans was 9.96 mm; the average weight of ten

31

Figure 14: Arabica Zambia AA

beans was 1.8 g. The beans had a green color with smooth surface, they were not

fouled by any insects, no stones or other impurities were found. However, a few bro-

ken beans were found. The Zambia coffee beans were washed - processed according

to the wet method. The quality of these beans could be described as very good.

The average size of Arabica Uganda Organico Biocoffee coffee beans was 9.58 mm;

the average weight of ten beans 1.6 g. The beans had a green color with smooth sur-

face, they were not fouled by any insects, no stones or other impurities were found,

and no broken beans were found. The Uganda Organico Biocoffee coffee beans were

washed - processed according to the wet method. The quality of these coffee beans

could be described as above average.

The average size of Arabica Tanzania coffee beans was 8.36 mm, the average

weight of ten beans was 1.6 g. The beans had a yellowish brown color, some of the

beans were fouled by insects, some were broken. However, no stones or other impu-

rities were found. The quality of Tanzania coffee beans could be described as below

average, though they were washed - processed according to the wet method.

32

Figure 15: Arabica Uganda Organico Biocoffee

Figure 16: Arabica Tanzania

33

Figure 17: Arabica Kenya

The average size of Arabica Kenya coffee beans was 9.76 mm; the average weight

of ten beans was 1.7 g. The beans had a green color, some of the beans were fouled

by insects. However, no stones or other impurities were found. The quality of Ara-

bica Kenya coffee beans could be described as average, though they were washed -

processed according to the wet method.

The average size of Arabica Kenya AA coffee beans was 8.81 mm; the average

weight of ten beans was 1.5 g. The beans were processed by the wet method, had a

green color, no beans were fouled by insects, no stones or other impurities could be

seen. The Arabica Kenya AA coffee beans could be described of having an excellent

quality.

The average size of Arabica Ethiopian Sidamo Yirgamo Grade 2 coffee beans was

9.07 mm; the average weight of ten beans was 1.6 g. The wet processing of the beans

and letting then in the sun let the Arabica Ethiopian Sidamo Yirgamo Grade 2 coffee

have an excellent quality.

34

Figure 18: Arabica Kenya AA

Figure 19: Arabica Ethiopian Sidamo Yirgamo Grade 2

In our experiments we used five types of Arabica from Asia. They were Indone-

sian Sumatra Lintong in Figure 20, Indonesian Sulawesi Kalossi in Figure 21, Indian

Monsooned Aspinwalls Malabar AA in Figure 22, Indian Plantation A in Figure 23

35

Figure 20: Arabica Indonesian Sumatra Lintong

and Java WIB1 Jampit Gr1 in Figure 24.

The average size of Arabica Indonesian Sumatra Lintong coffee beans was 9.98

mm; the average weight of ten beans was 1.7 g. The beans were processed by a

dry-process method and dried in the sun, had a green color, no beans were fouled by

insects, no stones or other impurities could be seen. However, some beans were bro-

ken and had a brownish color. Despite these disadvantages, the Arabica Indonesian

Sumatra Lintong coffee beans could be described of having an excellent quality.

The average size of Arabica Indonesian Sulawesi Kalossi coffee beans was 9.45

mm; the average weight of ten beans was 1.8 g. The beans were processed by a

semiwashed-process method, had a green color, no beans were fouled by insects, no

stones or other impurities could be detected. Some beans were broken and had a

brownish color. However, the Arabica Indonesian Sulawesi Kalossi coffee beans could

be described of having an excellent quality.

Arabica Indian Monsooned Aspinwalls Malabar AA was the only ’exotic’ type of

36

Figure 21: Arabica Indonesian Sulawesi Kalossi

Figure 22: Arabica Indian Monsooned Aspinwalls Malabar AA

37

Figure 23: Arabica Indian Plantation A

coffee. ’Monsooned’ denotes that the coffee beans are dried in the sun, but exposed to

high humidity and rain. The average size of these beans was 10.61 mm; the average

weight of ten beans was 1.6 g. The beans were processed by a wet-process method,

had a yellowish brown color, a few beans were fouled by insects, no stones or other

impurities could be noticed. The Arabica Indian Monsooned Aspinwalls Malabar AA

coffee beans were of an excellent quality.

The average size of Arabica Indian Plantation A coffee beans was 9.72 mm; the

average weight of ten beans was 1.7 g. The beans were processed by a wet-process

method, had a green color, no beans were fouled by insects, no stones or other impu-

rities could be seen. The Arabica Indian Plantation A coffee beans could be described

of having a very good quality.

The average size of Arabica Java WIB1 Jampit Gr1 coffee beans was 9.49 mm; the

average weight of ten beans was 1.6 g. The beans were processed by a wet-process

method, had a green color, no beans were fouled by insects, no stones or other impu-

rities could be detected. The Arabica Java WIB1 Jampit Gr1 coffee beans could be

38

Figure 24: Arabica Java WIB1 Jampit Gr1

described of having a very good quality.

The majority of the analysed coffee samples came from Middle and South Amer-

ica. The green coffee beans were Nicaragua Talia Extra in Figure 25, Costa Rica

Tarazzu in Figure 26, Guatemala SHB in Figure 27, Mexico Maragogype in Figure

28, Mexico Altura in Figure 29, Honduras SHG in Figure 30, Colombian Excelso in

Figure 31 and Santos Brazil NY 2 17/18 TOP Italian preparation in Figure 32.

The average size of Arabica Nicaragua Talia Extra coffee beans was 9.38 mm; the

average weight of ten beans was 1.8 g. The beans were processed by a wet-process

method, had a green color, no beans were fouled by insects, no stones or other impu-

rities could be noticed and no broken beans were seen. The ArabicaNicaragua Talia

Extra green coffee beans had an excellent quality.

The average size of Arabica Costa Rica Tarazzu coffee beans was 9.11 mm; the

average weight of ten beans was 1.7 g. The beans were processed by a wet-process

39

Figure 25: Arabica Nicaragua Talia Extra

Figure 26: Arabica Costa Rica Tarazzu

40

Figure 27: Arabica Guatemala SHB

method, had a green color, no beans were fouled by insects, no stones or other im-

purities could be noticed, but a few broken beans were detected. Despite this disad-

vantage the Arabica Costa Rica Tarazzu green coffee beans had of an excellent quality.

The average size of Arabica Guatemala SHB coffee beans was 8.62 mm; the av-

erage weight of ten beans was 1.5 g. The beans were processed by a wet-process

method, had a green color, a few beans were fouled by insects, no stones or other

impurities could be noticed, but some broken beans were detected. However, the

Arabica Guatemala SHB green coffee beans had an excellent quality.

The Arabica Mexico Maragogype coffee beans were the biggest in size and heavi-

est in weight compared to other coffee samples. The average size of Arabica Mexico

Maragogype coffee beans was 12.28 mm; the average weight of ten beans was 2.6 g.

The beans were processed by a wet-process method, had a green color, no beans were

fouled by insects, no stones or other impurities could be noticed, but a few broken

beans were seen. Despite small disadvantages, the Arabica Mexico Maragogype green

coffee beans could be described as having an excellent quality.

41

Figure 28: Arabica Mexico Maragogype

The average size of Arabica Mexico Altura coffee beans was 9.48 mm; the average

weight of ten beans was 1.8 g. The beans had a green color, no beans were fouled by

insects, no stones or other impurities could be noticed. However, the Arabica Mexico

Altura green coffee beans had a very good quality.

The average size of Arabica Honduras SHG coffee beans was 9.42 mm; the average

weight of ten beans was 1.6 g. The beans were processed by a wet-process method,

had a green color, no beans were fouled by insects, no stones or other impurities

could be noticed. Nevertheless, the Arabica Honduras SHG green coffee beans could

be described of having an above average quality.

The average size of Arabica Colombian Excelso coffee beans was 9.57 mm; the

average weight of ten beans was 1.5 g. The beans were processed by a wet-process

method, had a green color, no beans were fouled by insects, no stones or other im-

purities could be noticed. The Arabica Colombian Excelso green coffee beans had an

above average quality.

42

Figure 29: Arabica Mexico Altura

Figure 30: Arabica Honduras SHG

43

Figure 31: Arabica Colombian Excelso

The average size of Arabica Santos Brazil NY 2 17/18 TOP Italian preparation

coffee beans was 9.29 mm; the average weight of ten beans was 1.6 g. The beans

were processed by a wet-process method, had a green color, no beans were fouled

by insects, no stones or other impurities could be noticed. Nevertheless, the Arabica

Santos Brazil green coffee beans had an above average quality.

We have analysed only one type of Arabica coffee form Oceania - Papua New

Guinea Sigri C in Figure 33. The average size of Arabica Papua New Guinea Sigri

C coffee beans was 9.79 mm; the average weight of ten beans was 1.7 g. The beans

were processed by a wet-process method, had a green color, no beans were fouled

by insects, no stones or other impurities could be noticed. Despite this, the Arabica

Papua New Guinea Sigri C green coffee beans could be described as having an above

average quality.

Robusta coffees bean used in our experiments were notedly of lower quality. We

44

Figure 32: Arabica Santos Brazil NY 2 17/18 TOP Italian preparation

Figure 33: Arabica Papua New Guinea Sigri C

45

Figure 34: Robusta Indian Parchment

had analysed Robusta coffee beans from Africa, Asia and Middle and South America.

The largest part of analysed Robustas came from Asia. They were described as

Indian Parchment in Figure 34, Indian Cherry AB in Figure 35 and Vietnam in Fig-

ure 36.

The average size of Robusta Indian Parchment coffee beans was 7.58 mm; the aver-

age weight of ten beans was 1.4 g. The beans were processed by a wet-process method

and dried in the sun, had a greenish color, a few beans were fouled by insects, no

stones or other impurities could be noticed, but we could detect a few broken beans.

The Robusta Indian Parchment green coffee beans had an above average quality.

The average size of Robusta Indian Cherry AB coffee beans was 9.20 mm; the

average weight of ten beans was 1.6 g. The beans were processed by a dry-process

method, had a light green color, a few beans were fouled by insects, no stones or other

impurities could be noticed, but we could detect a few broken beans. The Robusta

Indian Cherry AB green coffee beans had an average quality.

46

Figure 35: Robusta Indian Cherry AB

Figure 36: Vietnam Robusta

47

Figure 37: Robusta Liberia

The average size of Robusta Vietnam coffee beans was 8.22 mm; the average

weight of ten beans was 1.4 g. The beans were processed by a dry-process method,

had a brownish color, a few beans were fouled by insects, we found damaged, dark

beans, some were broken, no stones or other impurities could be noticed. The Robusta

Vietnam green coffee beans could be described as having a below average quality.

The only Robusta coffee from Africa we analysed was described as Robusta Liberia

showed in Figure 37. The average size of Robusta Liberia coffee beans was 8.29 mm;

the average weight of ten beans was 1.3 g. The beans were processed by a dry-process

method, had a brownish color, some beans were fouled by insects, we found damaged,

broken, dark beans, some stones could be noticed. The Robusta Liberia green coffee

beans had a below average quality.

The only Robusta coffee from Middle and South America we analysed was Robusta

Cameroon showed in Figure 38. The average size of Robusta Cameroon coffee beans

was 8.65 mm; the average weight of ten beans was 1.4 g. The beans were processed

by a dry-process method and dried in the sun, had a brownish color, some beans

were fouled by insects, a large number of beans were broken, some stones and other

48

Figure 38: Robusta Cameroon

impurities could be noticed. Robusta Cameroon green coffee beans had a below

average quality.

6.3 Coffee roasting

6.3.1 Coffee roasting in a laboratory roaster

80.0 g of each type of green coffee beans were roasted in a laboratory coffee roaster

according to program 4, 6, 8 and 10. The beans were heated for 300, 450, 720 and

870 seconds. Cooling always took 300 seconds. The temperature during roasting did

not go over 250 °C (Table 5).

Temperature gradient for program 4, 6, 8 and 10 and each coffee is shown in

Figure 39.

After the roasting and cooling process was completed, the coffee beans were

ground in a laboratory grinder and prepared for HPLC ion exclusion chromatography

49

Table 5: Roasting parameters of program 4, 6, 8 and 10.Programme Roasting time, sec Highest detected temperature, °C

4 360 2376 450 2438 720 24710 870 246

Figure 39: Temperature gradient of roasting programmes 4, 6, 8 and 10.

with UV detection analysis using a typical sample preparation procedure.

6.3.2 Coffee roasting in a thermostatic oven

10.0 g of green Arabica and Robusta types of coffee beans were roasted in a thermo-

static oven at different time and temperature conditions. Before roasting, the glass

dishes were preheated in an oven for 10 minutes. After roasting the samples were

immediately put on ice for 15 minutes. The cooled coffee was ground in a coffee

50

grinder and the samples were prepared for HPLC-UV or HPLC-MS analysis using

typical sample preparation procedure.

6.3.3 Standard condition roasting

In this experiment the 19 types of green coffee beans processed by different methods

(Table 6) from different regions of the world (from Africa (n = 5), Asia (n = 6),

Oceania (n = 2), South America (n = 1), Central America (n = 5) were roasted

and analysed for acrylamide. Coffee beans were roasted in an oven under standard

conditions. Coffee samples of 10 g each were heated at 240 °C for 7 minutes in glass

dishes. The glass dishes were preheated for 10 minutes; after heating the dishes with

coffee samples were cooled for 15 minutes on ice. Then coffee beans were ground and

prepared for analysis by LC-MS using a typical sample preparation procedure.

6.4 Typical sample preparation procedure

After roasting and cooling, coffee was ground immediately in a coffee grinder. 5.0

± 0.1 g of coffee were taken to the 250 ml flask and the fat was removed by an

extraction with 25 ml of n-hexane for 10 minutes each. The procedure was performed

in duplicate. The residues of n-hexane were removed under a stream of nitrogen

(purity 5.0). For extraction of acrylamide 10 ml of water were added to 500 ± 0.1 mg

of defatted coffee in a centrifuge tube. After the extraction for 30 seconds the samples

were centrifuged at 4000 U/min for 30 minutes and filtered (Acrodisc 0.45 µm, Pall

Gelman Laboratory, Ann Arbor, MI, USA). Then the supernatant was purified by

SPE Bond Elut Accucat (Varian, Middleburg, The Netherlands). The SPE cartridge

(200 mg) was conditioned with 3 ml methanol and 3 ml water. As acrylamide is not

retained in the first 1 ml of eluate, the first 1 ml of the eluting solution was discarded

and the rest 2 ml were collected. Until HPLC analysis the samples were stored at 4°C .

51

Table 6: Characteristics of green coffee beans processed by different methods.Type Place of origin Processed1

Arabica Africa Tansania wEthiopian Sidamo YirgamoGrade 2

w+s

Zambia AA wKenia wKenia A/A w

Asia Indian Monsooned AspinwallsMalabar AA

w+m

Indian Plantation A wIndonesian Sumatra Lintong d+sIndonesian Sulawesi Kalossi sw

Central America Nicaragua Talia Extra wGuatemala SHB wMexico Maragogype wMexico AlturaCosta Rica Tarazzu w

South America Honduras s.h.g wOceania Papua New Guinea Sigri C w

Java WIB1 Jampit Gr1 wRobusta Asia Indian Parchment w+s

Vietnam d1 w - wet-processed: washed coffees, d - dry-processed: natural coffees, s -

sundried, m - monsooned (dried in the sun, but exposed to high humidityand rain), sw - semiwashed.

For LC-MS analysis the samples were prepared similarly with adding 100 µl of

13C3-labeled acrylamide (10 µg/ml) as an internal standard before water extraction

was performed.

6.5 3-aminopropionamide in coffee

6.5.1 3-aminopropionamide in green coffee

For the analysis of 3-aminopropionamide in green coffee beans three types of coffee

were chosen: Indian Cherry AB Robusta (Coffea canephora, dry-processed), Liberia

52

Robusta (Coffea canephora, dry-processed), Honduras SHG Arabica (Coffea arabica,

washed).

First, green coffee beans were ground with an analytical mill and second with

a ball mill in order to get a fine powder. 100 to 500 mg of the fine powder were

balanced into 14 ml centrifuge tube, 7 ml of 0.1 M HCl were added and mixed. After

10 minutes of ultrasonic treatment and centrifugation at 4000 rpm for 30 minutes 5

ml of aliquot were transferred to a 10 ml flask. The pH was adjusted to ∼7 with

0.25 M NaHCO3. Then the flask was filled with water to the mark. Supernatant

was filtered and derivatized with dansyl chloride. Analysis for 3-aminopropionamide

was performed by HPLC with fluorescence detection. The standard solutions of

3-aminopropionamide in 0.25 M NaHCO3 (200, 400 and 600 ng/ml) were used as

external standards.

6.5.2 3-aminopropionamide in heated coffee

First of all 10.0 g of Liberia Robusta (Coffea canephora, dry-processed) green beans

were roasted in an oven at 200, 220 and 240 °C for 5, 10 or 15 minutes (Figure 40).

Secondly Liberia Robusta (Coffea canephora, dry-processed) and Indian Plantation A

Arabica (Coffea arabica, washed) were heated at 150 and 170 °C for 7 minutes. Before

roasting, the glass dishes were preheated in an oven for 10 minutes. After roasting the

samples were immediately put on ice for 15 minutes. After cooling the heated coffee

beans, they were ground in an analytical mill and prepared for HPLC-FL analysis

according to the same procedure used for green coffee.

53

Figure 40: Coffee beans roasted under different time and temperature conditions

6.6 Acrylamide and 3-aminopropionamide formation in a model

system

6.6.1 Preparation mixtures of asparagine with sucrose and glucose

Asparagine and sugars in molar ratio of 1:0.5, 1:1 or 1:1.5 were dissolved in 20 ml of

purified water in a round flask. Samples were frozen (-20 °C) overnight and freeze-

dried to get a fine dry powder. Approximately 50 mg of the freeze-dried asparagine,

sucrose or glucose mixtures were heated in 4 ml vials at 130, 150, 170 and 190 °C for

1 to 30 minutes. After heating the samples were cooled for 40 seconds in the air (20°C) and 15 more minutes on ice. The heated samples were dissolved in 3 ml of 0.25

M NaHCO3 (pH ∼8). After the aliquot had been sonicated for 10 minutes 500 µl of

the solution were centrifuged for 5 minutes, diluted if necessary 10 times and then

derivatized with dansyl chloride or analysed for acrylamide by LC-UV.

54

6.6.2 Sample preparation for optimal heating conditions estimation

Approximately 50 mg of the freeze-dried asparagine with glucose (molar ratio 1:0.5

and 1:1) powder were taken into 4 ml HPLC heated to a provided temperature for

a certain time. After heating the vials were cooled for 40 seconds in the air and

another 15 minutes on ice. After that, the heated each mixture was dissolved in 3 ml

of purified water, it was sonicated for 10 minutes, centrifuged for 5 minutes. Before

the HPLC-UV analysis the aliquot was diluted with purified water 10 times.

6.6.3 Asparagine with ascorbic acid mixture preparation

Asparagine and ascorbic acid in molar ratio of 1:0.5, 1:1 or 1:1.5 were dissolved in

20 ml of purified water in a round flask. Samples were frozen (-20 �) overnight and

freeze-dried to get a homogenious powder. Approximately 50 mg of the freeze-dried

asparagine and ascorbic acid mixtures were heated in 4 ml vials at 150, 170, 190,

210, 230 and 250 °C for 1 to 15 minutes. After heating the samples were cooled

for 40 seconds in the air (20 �) and 15 more minutes on ice. The heated samples

were dissolved in 3 ml of purified water. After the aliquots had been sonicated for

10 minutes 100 µl of the solution were added to 900 µl of 0.25 M NaHCO3 and then

taken for 3-aminopropionamide analysis. The rest of aliquot was derivatized with

2-mercaptobenzoic acid and analysed for acrylamide by using LC-MS.

6.7 Heating of pure asparagine

6.7.1 Acrylamide formation from pure asparagine heated at 170 °C for0-24 minutes

Pure asparagine was heated in 2 ml HPLC vials in a heating block. Vials were

put at once all together (screw caps not totally closed). After heating for a certain

amount of time (0, 3, 6, 9, 12, 15, 18, 21, 24 minutes), each vial was taken out

55

of the heating block, tightly closed and kept in the air for 40 seconds, and put on

ice for another 15 minutes. The heated asparagine samples were dissolved in 1.5

ml purified water, transferred to the 2 ml centrifuge tubes and centrifuged for 5

minutes. The samples were prepared for HPLC-FLD (3-aminopropionamide) and

LC-MS (acrylamide) analysis.

For HPLC-MS analysis 50 µl of the centrifuged sample were purified by SPE SAM

OASIS (Waters, Milford, MA, USA). The cartridges were preconditioned with 2 mL

MeOH and 2 ml H2O. After SPE purification 13C3-labeled acrylamide (final conc.

100 ng/ml) was added.

6.7.2 Maleimide formation at 170 °CPure asparagine was heated for 20 minutes at 170 °C in a 2 ml HPLC vial. Then it

was dissolved in 1.5 ml of purified water. The aliquot was centrifuged and analysed by

HPLC-MS. Analysis was carried out with SIM mode, we were looking for acetamide

(m/z 60), propanamide (m/z 74), acrylamide (m/z 72), maleimide (m/z 98) and

succinimide (m/z 100).

6.7.3 Acrylamide formation from asparagine at high temperatures

50 mg of anhydrous asparagine were heated in 4 ml vials at 210, 230 and 250 °C for

2, 5 and 10 minutes. After heating, the samples were cooled for 40 seconds in the

air and another 15 minutes on ice. After the heated asparagine was dissolved in 3

ml of purified water and sonicated for 10 minutes, the aliquots were derivatized with

2-mercaptobenzoic acid and analysed for acrylamide by HPLC-MS.

56

6.8 Heating of Amadori compound

Three Amadori compound, 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-fructopyranose

(Figure 41) was kindly provided by Dipl.-Ing. Dr.techn. Univ.-Doz. Tanja Maria

Wrodnigg from the Institute for Organic Chemistry at Graz University of Technol-

ogy. The compound was heated in 2 ml HPLC vials at different time and temper-

ature conditions (Table 7). After heating the vials were cooled for 40 seconds in

the air and another 15 minutes on ice. After the substances were dissolved in 0.5

ml of purified water and sonicated for 10 minutes, 100 µl of the aliquot was taken

for 3-aminopropionamide analysis (derivatized with dansyl chloride and analysed by

HPLC-FLD) and the rest was derivatized with 2-mercaptobenzoic acid in order to be

able to analyse for acrylamide.

Table 7: Heating conditions of Amadori compoundCompound Heating temperature, � Heating time, min1-N -(asparaginyl)-5-azido- 170 21,5-dideoxy-D-fructopyranose 190 2

210 2

Figure 41: 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-fructopyranose

57

6.9 Derivatization methods

6.9.1 Derivatization with dansyl chloride

Our samples, in order to be able to detect them by using high performance liq-

uid chromatography with fluorescence detection, were derivatized with dansyl chlo-

ride ([5-(dimethylamino]-naphthalene-1-sulfonylchloride) [83]. A derivatization of 3-

aminopropionamide was proceeded according to the method mentioned by [84, 85, 86]

involving some modifications. The reaction scheme is shown in Figure 42.

Figure 42: Reaction of 3-aminopropionamide to sulphonamide

100 µl of the dansyl chloride solution (5mg/ml in acetone) were added to 100 µl

of sample in a test tube. The mixture was thoroughly mixed and left in the dark

overnight. In order to eliminate excessive dansyl chloride, 20 µl of a glycine solution

(100 mg/ml) were added after the reaction and left for another 15 minutes at ambient

temperature. The sample was then extracted twice with 1 ml of diethyl ether. The

combined extracts were dried under a stream of nitrogen (purity 5.0) and the residue

was re-dissolved in 700 µl of acetonitrile. The analysis was performed with LC-FLD.

6.9.2 Derivatization with 2-mercaptobenzoic acid

3 ml of the supernatant (after ultrasonic treatment for 10 minutes) were collected in 4

ml vials, pH ∼8 was adjusted by adding 10 µl 1 M NaOH. 200 µl of 2-mercaptobenzoic

acid (154 mg in 10 ml of 1M NaOH) were added and the mixture had to be stirred

for 3 hours constantly by shaking. The derivatized solutions were diluted 1:10 with

58

Figure 43: Derivatization of acrylamide with 2-mercaptobenzoic acid

purified water and then analysed by LC-MS. The reaction mechanism is shown in

Figure 43.

6.10 Analytical methods

6.10.1 Ion chromatography with UV detection

HPLC analysis was carried out on model HP1100 consisting of a temperature con-

trolled autosampler, a quaternary pump, a vacuum degasser, VW detector HP1050,

data system, analytical ion exclusion column Ion Pac ICE-AS1 (250 × 9 mm). Mobile

phase 30 % acetonitrile in 3 mM aqueous formic acid solution (isocratic); flow rate

0.400 ml/min; injection volume 10 µl; column temperature 20 °C; autosampler tem-

perature 8 °C; VWD wavelength 202 nm. The concentration of acrylamide in each

sample was calculated from a linear calibration (standard solutions of acrylamide

were in a range of concentrations 5-1000 ng/ml). The limit of detection (LOD) of

5 ng/ml, the limit of quantification (LOQ) of 17 ng/ml and RSD of 4.85 % were

determined by using the software ValiData V1.01.

59

Figure 44: Typical chromatogram of acrylamide using ion exclusion chromatographywith UV detection

The typical chromatogram of the acrylamide solution and the coffee sample is

given in Figure 44.

6.10.2 HPLC-FLD operating conditions

For HPLC analysis 10 µl of the sample were injected into a HP 1100 liquid chromato-

graph (Hewlett Packard, Waldbron, Germany) with a fluorescence detector, equipped

with cooling autosampler set at 6 °C, quaternary pump, degasser. The HPLC column

MERCK Lichrosphere 60 Select B 125 × 4 mm i.d., 5 µm particle size (MERCK,

Darmstadt, Germany) with a precolumn was used. Analysis was performed at ambi-

ent temperature. As a mobile phase 10 % of acetonitrile and 90 % water was used.

The gradient elution programme was held to 30 % of acetonitrile at 2 minutes, in-

creased to 70 % at 12 minutes and held until the end of the run (20 minutes) with a

flow rate of 1 ml/min. Post time was set to 6 minutes. Dansyl chloride derivatives

60

were detected with a fluorescence detector, set at λEx= 320 nm and λEm

= 500 nm.

Figure 45: Typical chromatogram of 3-aminopropionamide analysis

Quantification was done by external calibration. Standard solutions of

3-aminopropionamide in 0.25 M NaHCO3 (50 to 1000 ng/ml) were used. The limit of

detection (LOD) was determined as 17 ng/ml, the limit of quantification (LOQ) as

30 ng/ml, RSD of 3.2% software supported using Validata (version 1.01). The typical

chromatogram of the standard 3-APA (1000 ng/ml) and the sample (asparagine and

sucrose mixture heated to 150 °C for 7 min) is given in Figure 45.

6.10.3 HPLC-MS operating conditions (m/z 72, 75)

For LC-MS analysis an Agilent HP 1100-MS equipped with vacuum degasser, quar-

ternary pump, autosampler, temperature-contolled column oven, diode array detector

was used which was coupled to a MS equipped with electrospray source. For analysis

a Synergi 4 µ Polar-RP 80A analytical column, 150 × 4.6 mm (Phenomenex) with

precolumn (Phenomenex) was used. As a mobile phase we used a mixture of water,

61

acetonitrile and formic acid 98.6/0.2/0.2. Column temperature was 25 °C, flow rate

1 ml/min, injection volume 20 µl. Detection was performed at a wavelength of 210

nm and MS positive (API-ES) mode, SIM at m/z 72 and 75, fragmentor voltage 50

V, drying gas temperature 350 °C, capillary voltage 3.5 kV. The limit of detection

(LOD) was determined as 4 ng/ml, the limit of quantification (LOQ) as 15 ng/ml,

RSD of 4% using software Validata (version 1.01).

Figure 46: Typical chromatogram of acrylamide using MS detection

The typical chromatogram of the acrylamide analysis in coffee is given in Figure

46.

6.10.4 HPLC-MS operating conditions, eluent water

For LC-MS analysis an Agilent HP 1100-MS equipped with vacuum degasser, quar-

ternary pump, autosampler, temperature-controlled column oven was used. A Diode

Array Detector was coupled to a mass spectrometer equipped with electrospray

source. For analysis Synergi 4 µ Polar-RP 80A, 150 × 4.60 mm i.d. (Phenomenex)

62

analytical column with precolumn 1 × 4.6 mm (Phenomenex) was used. As a mobile

phase water was used. The column temperature was maintained at 20 °C, flow rate

1 ml/min, post time was set to 6 minutes, injection volume 10 µl. Detection was

performed at a wavelength of 210 nm and MS positive (API-ES) mode, SIM at m/z

72 and 75. The limit of detection (LOD) was determined as 10 ng/ml, the limit of

quantification (LOQ) as 33 ng/ml, RSD of 2.2%.

6.10.5 HPLC-UV operating conditions

For LC-UV analysis an Agilent HP 1100-MS equipped with vacuum degasser, quar-

ternary pump, autosampler, temperature-controlled column oven, Diode Array De-

tector was used. For analysis Synergi 4 µ Polar-RP 80A, 150 × 4.60 mm i.d. (Phe-

nomenex) analytical column with precolumn 1 × 4.6 mm (Phenomenex) was used.

As a mobile phase water was used. The column temperature was maintained at 20°C, flow rate 1 ml/min, post time was set to 5 minutes, injection volume 10 µl. De-

tection was performed at a wavelength of 210 nm. The acrylamide concentration of

the samples was calculated by external calibration. For this purpose the acrylamide

aqueous standard solutions in the range of 10-10 000 ng/ml were prepared. The limit

of detection (LOD) was determined as 58 ng/ml, the limit of quantification (LOQ)

as 105 ng/ml, RSD of 0.7%.

The typical chromatogram of the acrylamide analysis is given in Figure 47.

6.10.6 HPLC-MS operating conditions for acrylamide derivative analysis

The analysis of acrylamide derivatives was developed according to the method re-

cently described in the literature [16]. Since we could not apply this method di-

rectly in our laboratory, the method was modified and improved with help of Dra.

M.T.Galceran and her research team at University of Barcelona, Department of An-

alytical Chemistry.

For this purpose derivatized acrylamide standard solutions in the range of 0.6-622

63

Figure 47: Typical chromatogram of acrylamide using UV detection

ng/ml and 62.5 ng/ml of d3-acrylamide were prepared. The coffee beans were roasted

at 240 °C for 5, 10 and 15 minutes, prepared according to the method described in 6.4

and derivatized with 2-mercaptobenzoic acid according to the method described in

6.9.2. Before the analysis by LC-MS/MS the solutions were diluted 1:50 with purified

water.

For LC-MS/MS an Agilent HP 1100 equipped with vacuum degasser, quarternary

pump, autosampler coupled to a PE Sciex API 3000 (Applied Biosystems, Foster City,

CA, USA) equipped with an electrospray (ES) as an ionization source and a triple

quadrupole as an analyzer was used. The optimal ionization source working param-

eters were: nebulizer gas 10 a.u.; curtain gas 12 a.u.; vaporizer temperature 400 °C;

electro spray voltage (ion spray voltage) 5.5 kV; declustering potential 50 V. The

data acquisition was performed using selected reaction monitoring (SRM), using as

precursor ion the protonated molecule [M + H]+ and monitoring two product ions,

the most abundant product ion for quantitative purposes and another one for confir-

mation. The transitions precursor→product ion used for acrylamide derivative were

64

m/z 226→191 (quantitative analysis), 191→163 (confirmatory analysis) and while

for deutereted acrylamide derivative were m/z 229→211, 229→193 and 229→194.

The chromatographic separation of acrylamide was carried out by reverse-phase

liquid chromatography using a Waters Symetry C8 150 × 2.1 mm i.d. 5 µm particle

size analytical column, the eluent composition was 30 % acetonitrile and 70 % acetic

acid (0.1 %) aqueous solution. Flow rate 0.3 ml/min was performed in isocratic

elution. The sample injection volume was 10 µl.

Calibration standard solutions of derivatized acrylamide were prepared and anal-

ysed by LC-MS/MS (Figure 48). The chromatogram of the derivatized acrylamide

in roasted coffee is given in Figure 49.

An important signal suppression was observed due to the matrix effect in the

ionization process. To improve the signal it was necessary to dilute the sample with

purified water 1:50.

Figure 48: Typical chromatogram of acrylamide (6 ng/ml) after derivatization with2-mercaptobenzoic acid using LC-MS/MS

For LC-MS analysis in our laboratory an Agilent HP 1100 equipped with vacuum

degasser, quarternary pump, autosampler and MSD was used. The chromatographic

65

Figure 49: Typical chromatogram of acrylamide in a roasted coffee sample afterderivatization with 2-mercaptobenzoic acid using LC-MS/MS

separation of acrylamide was carried out using a Phenyl-Hexyl 150 × 3 mm i.d. 3

µm particle size analytical column, the eluent composition was 30 % acetonitrile and

70 % acetic acid (0.1 %) aqueous solution. The analytical column temperature was

25 °C. Flow rate 0.4 ml/min was performed in isocratic elution. The sample injection

volume was 3 µl. Detection was performed at a MS positive (API-ES) mode, SIM at

m/z 226 (for acrylamide) and 248 (for acrylamide and Na+ adducts).

The acrylamide concentration of the samples was calculated by external calibra-

tion. For this purpose the acrylamide aqueous standard solutions in the range of

10-5000 ng/ml were prepared and derivatized. The limit of detection (LOD) was

determined as 157 ng/ml, the limit of quantification (LOQ) as 270 ng/ml, RSD of

5.6% using software Validata (version 1.01).

The typical chromatogram of the derivatized acrylamide analysis is given in Figure

50 .

66

Figure 50: Typical chromatogram of derivatized acrylamide analysis

67

7 Results and Discussion

7.1 Coffee roasting in a laboratory roaster

Four different types of coffee were used for this experiment: Cameroon Robusta (low

quality), Santos Brazil NY 2 17/18 TOP Italian preparation, Arabica (low quality),

Colombian Excelso Centrals mild, Arabica and Uganda Organico Biocoffee, Arabica

(high quality). Cameroon Robusta and Santos Brazil Arabica were roasted according

to programmes 4, 6, 8 and 10. Both Colombian Excelso and Uganda Organico Bio-

coffee were roasted using programme 6. 80 g of each coffee green beans were taken to

the laboratory roaster. 5.0 g of each roasted coffee were taken for acrylamide analysis

performed by liquid ion exclusion chromatography, and the rest of coffee was put in

amber glass bottles, flushed with nitrogen and stored in the freezer (-20 �). Green

coffee beans (50.0 g) were prepared for storage in the same way.

Figure 51: Formation of acrylamide in 4 different types of coffee roasted in a labora-tory roaster

Roasting programme 6 is usually used for common drinking coffee. During roast-

68

ing the coffee the temperature in a laboratory roaster is high, but normally not above

250 �. Our experiment showed significant difference of acrylamide concentration in

different coffee beans roasted in a laboratory roaster. Cameroon Robusta showed a

significantly larger amount of acrylamide formed during roasting (Figure 51), while

in other types of coffee beans - Arabica, both washed and not washed - concentration

of acrylamide detected was 7.6 (Santos Brazil) to 10.5 (Colombian Excelso) times

less. Furthermore, Cameroon Robusta coffee beans roasted according to programme

4 and 8 had a higher acrylamide concentration compared to Santos Brazil.

Commercially available coffee (Lavazza 100 % Arabica, Torino, Italy) was analysed

as well. Additional standards of acrylamide solutions (50 and 100 ng/ml) were used

for this analysis, and the concentration of this food toxin was calculated from the

calibration curve. We have detected 191 ng/g acrylamide in analysed coffee powder.

7.2 Coffee heated in a thermostatic oven

Cameroon Robusta coffee beans were chosen for this experiment. 10.0 g of beans

were roasted in an oven at 180, 200, 220 °C for 1, 3 and 5 minutes and at 220, 240,

260 °C for 5, 10 or 15 minutes. Before roasting, the glass dishes were preheated in

an oven for 10 minutes. After roasting the samples were immediately put on ice for

15 minutes. The cooled coffee was ground in a coffee grinder and the samples were

prepared for ion exclusion with UV detection chromatographic analysis.

Table 8: Concentration of acrylamide in Cameroon Robusta, heated in an thermo-static oven, ng/g.

Temperature � Time, min5 10 15

220 463 250 0488 158 0

240 191107110

260 239 0 0

69

Figure 52: 2nd Order regression curve of acrylamide content in coffee beans

In our experiments we detected the highest concentration of acrylamide in beans

roasted at 240 °C for 5 minutes (Table 8). Also in coffee beans, roasted at 220 and

260 °C for 5 minutes we detected higher amounts of acrylamide than in those roasted

at 10 or 15 minutes (Figure 52). With the increase of roasting time we noticed a

acrylamide decrease at all temperatures. Figure 52 shows how acrylamide is formed in

coffee beans heated at temperatures between 220 and 260 �. The largest acrylamide

amount forms during the first five minutes and lower temperature (220 �). Heating

at higher temperatures and for longer (5, 10 minutes) time acrylamide concentration

in the coffee beans is decreasing. The acrylamide formation and elimination processes

are faster at higher temperatures than at lower ones.

In the experiment, when coffee beans were roasted at 180, 200 and 220 °C for

1, 3 and 5 minutes (Table 9) the highest acrylamide amount was detected in beans,

heated to 220 °C for 5 minutes. With the increase of the roasting time at all temper-

atures (180, 200, 220 �) we noticed an increase of acrylamide formation. At higher

temperatures (220 �) this increase was more rapid. We observed the acrylamide

increase in the samples, roasted for longer time, e.g. 5 minutes. With the increase

of temperature we noticed a rapid acrylamide increase in the samples. It seems, that

the highest acrylamide amount forms in the first 5 minutes of coffee roasting and

later it decreases. The higher the roasting temperature and the longer roasting time,

70

Figure 53: Regression surface curve of acrylamide content in coffee beans

the faster the acrylamide degradation is in coffee samples.

In our roasting experiments we analysed time and temperature influence on the

acrylamide formation. According to our results (Figure 53) both time and tempera-

ture conditions are significant for the formation of the toxicant.

In the presented coffee roasting experiments we could analyse time and tempera-

ture parameter influence on the acrylamide formation. According to our results we

can affirm that both time and temperature conditions have influence on the forma-

tion of the toxicant. It seems that acrylamide is formed in coffee during the first 5

heating minutes. After that we noticed an acrylamide decrease in our samples. The

higher the temperature and the longer the time of the roasting process, the faster

acrylamide degradation can be observed in the coffee.

Table 9: Concentration of acrylamide in Cameroon Robusta, heated in an thermo-static oven, ng/g.

Temperature, � Time, min1 3 5

180 31.5 28.5 39.7200 n.d. 41.7 42.9220 29.4 40.2 122

Figure 53 shows how acrylamide is formed in coffee beans heated at temperatures

71

Figure 54: Significant parameters in the formation of acrylamide

between 220 and 260 �. The largest acrylamide amount forms in first five minutes

and lower temperature (220 �). Heating at higher temperatures and for longer (5,

10 minutes) time acrylamide concentration in the coffee beans is decreasing. The

acrylamide formation and elimination processes are faster at higher temperatures

than at lower ones.

Figure 54 shows the significance of parameters in formation of acrylamide. In

our experiments both time and temperature were significant for the formation of

acrylamide.

7.3 Standard condition roasting

In this experiment experiment 19 types of green coffee beans from different regions

of the world (from Africa (n = 5), Asia (n = 6), Oceania (n = 2), South America

(n = 1), Central America (n = 5) were roasted and analysed for acrylamide. Green

coffee beans are described in Table 6. The green coffee beans were heated in an oven

under standard conditions. Coffee samples of 10 g each were heated at 240 °C for 7

minutes in glass dishes. Glass dishes were preheated for 10 minutes; after heating the

dishes with coffee samples were cooled for 15 minutes on ice. Then the beans were

ground and prepared for analysis by LC-MS.

72

Figure 55: Acrylamide content in different coffee beans roasted under standard con-ditions

When we analysed 19 coffees from different parts of the world, roasted under stan-

dard roasting conditions (240 °C for 7 minutes), we noticed a significant difference

between Robusta and Arabica coffees (Figure 55). Exact values of this experiment are

shown in Table 10. Most of our studied Arabicas were washed and rather high qual-

ity with exception for coffees from Tanzania and one coffee from Kenya. Our results

showed no big difference in acrylamide amount formed in Arabica coffees, whereas

acrylamide amounts in Robusta coffees were higher than in all tested Arabicas. Ac-

cording to our results it seems, that the growth area of coffee beans did not have a

significant influence in acrylamide formation. In coffee beans from Asia we noticed

lower acrylamide amounts in dry-processed and semi-washed beans, whereas washed

Arabica beans and especially monsooned ones had higher acrylamide amounts. In-

donesian coffees had similar acrylamide content, though they were processed by dif-

ferent methods. Also in Robusta dry-processed coffee beans we noticed a lower AA

amount than in washed ones.

73

Table 10: Coffee roasted under standard conditionsCoffee Acrylamide formed, ng/gRobusta Indian Parchment 762Robusta Vietnam 653Indian Monsooned Aspinwalls Malabar AA 575Indian Plantation A 401Indonesian Sumatra Lintong 301Indonesian Sulawesi Kalossi 306Tansania Arabica 354Ethiopian Sidamo Yirgamo Grade 2 425Zambia AA 331Kenia washed 531Kenia A/A 299Nicaragua Talia Extra 433Guatemala SHB 374Mexico Maragogype 363Mexico Altura 380Costa Rica Tarazzu 310Honduras 307Papua New Guinea Sigri C 352Java WIB1 Jampit Gr1 357

It seems, that neither place of origin nor processing method has significant influ-

ence on the acrylamide formation. We can conclude, that medium roasted coffee has

the highest amount of acrylamide and it can be reduced by roasting the coffee beans

to a darker color, allowing the coffee to roast for longer time.

7.4 Acrylamide and 3-aminopropionamide formation in a modelsystem

As it was recently reported 3-aminopropionamide seems to be an important precursor

for acrylamide formation. In our study we prepared asparagine and sugars (sucrose,

glucose) mixtures of different molar ratios, heated and analysed them for acrylamide

and 3-aminopropionamide.

Asparagine mixtures with sugars were heated at 130, 150 and 170 °C for 7 minutes.

In this experiment we could detect both 3-aminopropionamide and acrylamide in high

amounts (Figure 57 and Figure 56).

74

Figure 56: 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 �

Acrylamide amount in heated asparagine mixtures with sucrose or glucose was

detected much higher than 3-aminopropionamide amount (Figure 56). In asparagine

mixture with sucrose acrylamide was detected already at 150 �. With an increase

of temperature we noticed an increase of acrylamide. Furthermore, the highest con-

centration of acrylamide was detected in heated asparagine samples with sucrose or

glucose at 170 �.

Asparagine mixture with glucose had a higher capacity already at lower tem-

peratures (130 �) to form 3-aminopropionamide and acrylamide. We detected the

highest amount of 3-aminopropionamide in the mixtures heated at 150 �. It seems,

that the molar ratio of asparagine to glucose did not have a significant influence on

the 3-aminopropionamide formation. Whereas in the asparagine mixtures with su-

crose at high temperatures (170 �) we noticed a decrease in 3-aminopropionamide

formation when the sucrose amount in the mixture was increasing. At 150 °C we no-

ticed the opposite effect. We did neither detect 3-aminopropionamide nor acrylamide

in the asparagine and sucrose mixtures heated at 130 °C. This temperature is too low

75

Figure 57: 3-Aminopropionamide in heated asparagine and sucrose and asparagineand glucose (molar ratio 1:0.5, 1:1 and 1:1.5) anhydrous mixtures at 130, 150 and170 �.

for sucrose to form necessary reaction products. In this experiment we also noticed

an acrylamide increase with increasing the heating temperature. Furthermore, the

amount of acrylamide formed in the mixture was similar to 3-aminopropionamide

amount, especially in the mixtures heated at 170 �.

3-aminopropionamide was detected in heated anhydrous mixtures of asparagine

with either glucose or sucrose. The model reaction was carried out in a temperature

range from 130 to 170 �. In the model reaction with glucose 3-aminopropionamide

was formed starting at 130 �, whereas with sucrose 3-aminopropionamide was only

found in the reaction carried out at 170 °C (Figure 57).

As we know, for the formation of 3-aminopropionamide reducing sugars such as

glucose or fructose are needed. As sucrose fragments into glucose and fructose at the

temperature above 170 �, 3-aminopropionamide was not detected at temperatures

below 170 �. Furthermore, in heated mixtures with an increasing molar ratio of

76

asparagine to glucose at 130, 150 and 170 °C and sucrose at 170 °C the formation of

3-aminopropionamide decreased. In addition, in the asparagine samples with glucose,

heated to 150 °C the highest concentration of 3-aminopropionamide was detected.

7.4.1 Optimum time and temperature conditions for 3-aminopropionamideformation

We noticed, that the highest amount of 3-aminopropionamide was formed in as-

paragine and sucrose mixture 1:0.5. The mixture was heated to 130, 150, 170 and 190°C at 7 minutes. The data showed, that the highest amount of 3-aminopropionamide

was detected in the mixture, heated to 170 °C (Figure 58). At this temperature we

heated the asparagine and sucrose mixture for 1, 3, 7, 10, 15 and 20 minutes. At 7

minutes of heating we have detected the maximum amount of 3-aminopropionamide.

It seems, that 3-aminopropionamide is rapidly forming to the highest possible amount

in the first 5-7 minutes and after that it is eliminated by probable transformation into

acrylamide.

7.4.2 Optimal heating time and temperature conditions for acrylamide

We have performed a similar experiment as described in 7.4.1 to estimate the opti-

mum time and temperature conditions for acrylamide formation. For this purpose as-

paragine and glucose mixtures (molar ratio 1:0.5 and 1:1) were prepared. Asparagine

and sugar were dissolved in 60 ml of purified water and freeze-dried. Approximately

50 mg of the fine powder were taken for one sample. The asparagine and glucose

freeze-dried mixtures were heated at 150, 170, 180, 190 and 200 °C for 5 and 7 min-

utes (Figure 59). According to the results shown, the mixture of asparagine and

glucose (1:0.5) was chosen for the next experiments in order to determine optimal

time and temperature heating conditions. As it can be seen in asparagine and glucose

mixture (1:0.5) the acrylamide amount formed during the heating was much higher.

In the next step, asparagine and glucose (1:0.5) freeze-dried mixture was heated

77

Figure 58: 3-Aminopropionamide in heated asparagine and sucrose 1:0.5 anhydrousmixtures at 130, 150, 170 and 190 �

Figure 59: Acrylamide content in asparagine with glucose mixtures (molar ratio 1:0.5and 1:1) heated to different temperatures for 5 and 7 minutes

78

Figure 60: Acrylamide formed in asparagine and glucose mixtures (1:0.5) heated atdifferent temperatures for 5 minutes

for 5 minutes at different temperatures: 190, 200, 210, 220, 230, 240, 250 and 260°C (Figure 60). As it is shown in the Figure 60, we cannot distinguish the optimal

heating temperature because of a lack of obvious extreme value. But because of the

lack of sample for analysis, two heating temperatures (210 and 250 �) were chosen

for comparison.

Figure 61 shows the acrylamide amount in asparagine and glucose mixture (1:0.5)

heated to 210 °C for 1, 3, 5, 7, 10, 15, 20 and 30 minutes. The highest concentration

of acrylamide was observed in the sample, heated for 7 minutes. After 15 minutes

the acrylamide amount in the samples was below the limit of detection.

In Figure 62 values of acrylamide formation in the asparagine and glucose mixture

heated at 250 °C are shown. It seems that acrylamide at such a high temperature

forms extremely quickly. In this experiment the highest acrylamide concentration

was observed already after 1 minute of heating.

In our experiments with a model system (asparagine mixtures with sucrose and

glucose) we observed acrylamide and its potential precursors 3-aminopropionamide

79

Figure 61: Acrylamide formation in asparagine mixtures with glucose at 210 °C(molar ratio 1:0.5)

Figure 62: 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

80

formation. It seems, that 3-aminopropionamide needs lower temperatures to obtain

a maximum amount, whereas acrylamide can reach a maximum concentration in the

mixture heated to rather high temperature (250 �). Both substances are rapidly

formed in the first minutes of heating and with increasing of the heating time the

amount of both 3-aminopropionamide and acrylamide is decreasing. However, acry-

lamide formation is connected to a heating temperature value: the higher the heating

temperature, the shorter time is needed to achieve a maximum acrylamide concen-

tration in the mixture. Furthermore, in the asparagine and glucose mixtures both

3-aminopropionamide and acrylamide can be formed already in the mixtures heated

to 130 �.

7.5 Heating of pure asparagine

Heating of asparagine to 170 °C for up to 24 minutes did not result in a formation of

neither acrylamide nor 3-aminopropionamide. In the sample, heated at 170 °C for 20

minutes maleimide (2,5-pyroldione) was detected. (Figure 9). The same results were

obtained by Yaylayan et al. [40], who suggested that carbohydrates or carbohydrate

degradation products are necessary for acrylamide formation from asparagine. The

acrylamide formation pathway from decarboxylation of Schiff base which leads to the

decarboxylation of Amadori products is preferred by most scientists, because as the

experimental data show when asparagine is pyrolyzed in the absence of carbohydrates,

maleimide formes avoiding therefore acrylamide formation.

Surprisingly, we could detect acrylamide in pure asparagine samples, heated at

high temperatures (210, 230 and 250 °C) for 2, 5 and 10 minutes (Figure 63). We have

detected this toxin in the samples, heated at 230 °C for 5 and 10 minutes (with the

increase of heating time, acrylamide concentration was increasing) and in the samples

heated at 250 °C for 2 and 5 minutes. At 250 °C heated samples had more acrylamide

formed than the ones heated at 230 °C. However, the concentrations detected in this

experiment were extremely small (only 2-6 µg/g) in comparison to other previous

experiments, when asparagine was reacting with carbohydrates or ascorbic acid.

81

Figure 63: Acrylamide formation from pure asparagine heated at high temperatures

This experiment can prove, that acrylamide can be formed when pure asparagine

is heated at high temperatures by simple decarboxylation and deamination reaction

(Figure 8). However, the amount of acrylamide formed is very small and this reaction

can not be accepted as a main pathway of acrylamide formation.

7.6 3-Aminopropionamide in coffee

No 3-aminopropionamide was found in green coffee beans. 3-aminopropionamide

can form in raw food stuffs, when enzymatic reaction takes place. According to

Schieberle [66], 3-aminopropionamide can form in a biochemical pathway, when the

decarboxylation of asparagine to 3-aminopropionamide takes place with pyridoxal

phosphate as co-factor. In this reaction the enzyme decarboxylase is needed (Figure

11). In our study we could not detect 3-aminopropionamide in green coffee. Maybe

82

because of disadvantageous conditions of the matrix, where humidity is no more than

6 % and enzymatic reactions do not take place.

We could not detect 3-aminopropionamide in coffee beans roasted at 150, 170,

200, 220, 240 °C for different times neither.

7.7 Heated mixtures of asparagine with ascorbic acid

It was recently delivered that ascorbic acid also known as vitamin C can significantly

reduce acrylamide formation in French fries [87]. Since it is known that vitamin

C can be found in all fresh vegetables including potatoes, it could be considered

as a natural inhibitor for acrylamide formation. In our experiments we tried to

investigate if asparagine and ascorbic acid alone under non-aqueous conditions can

be a substratum for acrylamide formation.

We chose different molar ratios of asparagine to ascorbic acid and heated the

mixtures at temperatures up to 150-250 °C (Figure 64).

Our experiment showed, that at lower temperatures (150, 170 �) there was no

acrylamide detected in the mixtures. Only at 190 °C when the asparagine and vita-

min C molar ratio was 1:1.5 after 5 minutes of heating we detected up to 20 µg/g

acrylamide. With increasing the heating temperature acrylamide concentration was

increasing (heating time 5 minutes) and at 250 °C it reached 50 µg/g. However, after

10 minutes of heating acrylamide concentration was decreasing. In comparison to

higher ascorbic acid amount in the mixture, when asparagine molar ratio to ascorbic

acid was 1:0.5 we observed much lower amounts of acrylamide formed: not more than

26 µg/g. Interesting results brought us the mixture of asparagine and ascorbic acid,

when molar ratio was 1:1. We could detect acrylamide only in the mixture heated

at 230 °C for 5 minutes. In general it is obvious, that acrylamide in asparagine and

ascorbic acid mixtures is formed at relatively high temperatures, short 5 minutes-

time (Figure 65) and in small amounts.

However, acrylamide formation is not intense in the mixture of asparagine and

ascorbic acid. When asparagine reacts with glucose, the acrylamide concentration is

83

Figure 64: Acrylamide formation in the mixtures of asparagine with ascorbic acid

Figure 65: Acrylamide formation in asparagine and ascorbic acid mixtures heated at250 °C and different times

84

usually 20 times higher.

We have also analysed asparagine and ascorbic acid anhydrous mixtures for 3-

aminopropionamide. At 170 °C 10 minutes heating was the first detection point for

this substance (Figure 66). At a molar ratio asparagine to vitamin C of 1:0.5 5 minutes

of heating time at higher than 170 °C we detected relatively high concentrations of

3-aminopropionamide: up to 350 µg/g. With increasing the heating temperature we

noticed a decrease in 3-aminopropionamide amounts formed.

Figure 66: 3-Aminopropionamide formation in the mixtures of asparagine with ascor-bic acid

7.8 Amadori compound

The Amadori compound 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-fructopyranose con-

sists of asparagine and monosaccharide residue. When we analysed it for acrylamide

85

and 3-aminopropionamide after heating, we detected, that both substances were

formed. However, the results show (Figure 67) that 3-aminopropionamide formed

in the first 2 minutes at 170, 190 and 210 °C was two times higher than acrylamide

formed. Moreover, with increasing the heating temperature the amount of acrylamide

and 3-aminopropionamide was increasing.

Figure 67: Acrylamide and 3-aminopropionamide formation from 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-fructopyranose

These results are in a strong agreement with other findings form model systems

where glucosylamine of asparagine is degrades to acrylamide via generated interme-

diate 3-aminopropionamide [39, 40, 41].

86

8 Conclusions

Within the scope of this dissertation extraction, clean-up and analytical methods for

analysis of acrylamide and 3-aminopropionamide were established.

As the simplest 3-aminopropionamide derivatized with dansyl chloride analysis

method the liquid chromatography with fluorescence detection was used. For acry-

lamide analysis in model systems a liquid chromatography with either UV or mass

spectroscopy was used. For the mass spectroscopy analysis method for acrylamide

derivatives with 2-mercaptobenzoic acid showed to be quite sensitive and rather ef-

ficient. Unfortunately, this method is not suitable for the coffee matrix because of

acrylamide low levels in it. For acrylamide measurements in coffee samples in routine

analysis the ion exchange chromatography with UV detection showed to be the most

efficient.

After the extraction, clean-up and analysis methods were established, the follow-

ing could be concluded� Arabica and Robusta coffee beans differ in acrylamide amounts formed. When

coffee was roasted in a laboratory roaster to common degrees for consumer, as

well as in the thermostatic oven under standard roasting conditions, Robusta

showed to have the highest amounts of acrylamide. It seems, that asparagine

is a limiting factor for acrylamide formation in coffee, because Robusta coffees

also contain higher amounts of asparagine than Arabicas.� The highest acrylamide amounts in coffee are formed at the very beginning

of the roasting process. After five minutes of roasting at temperatures higher

than 220 °C the amount of acrylamide is decreasing with increasing the roasting

time. Furthermore, acrylamide forms in lower amounts at higher temperatures

because of the faster elimination process.� The method for 3-aminopropionamide analysis was established. Unfortunately,

we could not detect 3-aminopropionamide neither in raw coffee beans nor in

87

roasted ones. In raw coffee beans the enzymatic conditions are unsatisfying

and probably enzymatic reactions do not take place. In the roasted coffee it is

difficult to detect 3-aminopropionamide because of possible low amounts of this

substance or its quite fast degradation into acrylamide. It also can be, that the

extraction method is not suitable for the coffee beans.

The experiments with model systems following conclusions could be made:� In the asparagine mixtures with glucose and sucrose it was observed that a

higher capacity of glucose form 3-aminopropionamide and acrylamide already

at the temperature of 130 °C, whereas in the mixtures with sucrose needs higher

temperatures to degrade into reactive compounds, acrylamide formation was ob-

served first only at 150 °C. Furthermore, the amounts of 3-aminopropionamide

and acrylamide formed were alike.� After some experiments were carried out with the model systems, we observed,

that asparagine mixtures with sucrose in the molar ratio 1:0.5 heated 170 °Cfor 7 minutes of heating produce the highest amounts of 3-aminopropionamide,

whereas optimal conditions for acrylamide formation were asparagine and glu-

cose mixture 1:0.5 heated at 250 °C for 1 minute.� After pure asparagine was heated at temperatures >200 °C, surprisingly acry-

lamide formation was detected. However, the amounts of this neurotoxin were

extremely low. Heated asparagine to 170 °C performs fast intramolecular cy-

clization forming maleimide and so prevents the formation of acrylamide.� As we expected in the asparagine mixtures with ascorbic acid heated at quite

high temperatures 3-aminopropionamide and acrylamide were detected. It was

noticeable, that for this reaction in order to form acrylamide and its precursor

higher temperatures and longer heating time are needed.� Heating of the Amadori compound 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-

fructopyranose showed that it was able to form 3-aminopropionamide and acry-

88

lamide. This is in agreement with other studies where it was said that as-

paragine is used as a skeleton for acrylamide’s formation.

89

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10 Curriculum Vitae

Personal information

Surname/First name: Bagdonaite KristinaDate of birth: 10.07.1979Place of birth: Radviliskis, LithuaniaNationality: LithuanianAddress: 4/7/39, Gaussgasse, Graz, A-8010, AustriaOffice: Institute for Food Chemistry and Technology, Graz Uni-

versity of Technology, Petersgasse 12/II, A-8010, GrazTelephone: +43(0)316 873-6969E-mail: kristina.bagdonaite@tugraz.at

Education

since 2003 Graz University of Technology, (Graz, Austria), PhD student2001-2003 Food Chemistry and Technology, Kaunas University of Technology,

(Kaunas, Lithuania), Master of Science in Chemical Engineering(with honours)

1997-2001 Food Product Technology, Kaunas University of Technology, (Kau-nas, Lithuania), Bachelor of Science in Chemical Engineering

1986-1997 Geguziai Secondary School (Siauliai, Lithuania)

Internship

04.2006 Short Term Scientific Mission at University of Barcelona, Depart-ment of Analytical Chemistry

10.2002-03.2003

SOCRATES/Erasmus student at Graz University of Technology,Department for Food Chemistry and Technology

Further Training

since11.2005

Member of the Sensoric Panel at the Department of FoodChemistry and Technology, Graz University of Technology

2002 SOCRATES short course on Identification of volatile andaroma active compounds in food

Languages

Mothertongue

Lithuanian

Other lan-guages

English, Russian, German (good writing and speaking skills)

Graz, June 2007 MSc. Kristina Bagdonaite

100

A Publications in the period of dissertation

Conference talks

1. Formation of Acrylamide During Roasting of Coffee. Osterreichische Lebens-

mittelchemikertage 2004, 11-12 May 2004, Bregenz, Austria

2. Factors Affecting the Formation Of Acrylamide in Coffee. Chemical reactions

in Foods V, 29th September-1st October 2004, Prague, Czech Rebublic

Publications

1. Bagdonaite K., Murkovic M., 2004, Factors affecting the formation of acry-

lamide in coffee. Czech J. Food Sci. 22, 22-24.

2. Bagdonaite K., Viklund G., Skog K., Murkovic M., 2006, Analysis of

3-aminopropionamide: a potential precursor of acrylamide. J. Biochem. Bio-

phys. Meth. 69, 215-221.

Posters

1. Bagdonaite K., Viklund G., Skog K., Murkovic M., Analysis of

3-aminopropionamide: a potential precursor of acrylamide. 8th Symposium on

Instrumental Analysis, 25-28 September 2005, Graz, Austria

2. Bagdonaite K., Viklund G., Skog K., Murkovic M., 3-Aminopropionamide - a

possible intermediate in the pathway leading to acrylamide. 1st International

Symposium of the Human Nutrition and Metabolism Research and Training

Centre, 24-26 October 2005, Graz, Austria

3. Bagdonaite K., Murkovic M., Acrylamide formation in coffee. Oesterreichische

Lebensmittelchemikertage 2006, 12-14 September, 2006, Vienna, Austria

101