Formation of Acrylamide During Roasting of Coffee

Formation of Acrylamide during Roasting of Coﬀee

Dissertation by MSc. Kristina Bagdonaite

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

1 Acknowledgements vii

2 Summary viii

3 Zusammenfassung ix

4 Introduction 1 4.1 Acrylamide ...... 1 4.2 Acrylamideinfoods...... 2 4.3 Toxicityofacrylamide ...... 11 4.4 Acrylamideformationpathways ...... 13 4.4.1 TheMaillardreaction ...... 16 4.4.2 Lipiddegradation...... 18 4.4.3 Decarboxylation and deamination of asparagine ...... 20 4.4.4 Other precursors for acrylamide formation (3-aminopropionamide) 21 4.5 Michaeladdition ...... 23 4.6 Coffee: plants, beans and their production ...... 24 4.6.1 Differences of Arabica and Robusta ...... 24 4.6.2 Harvestingandprocessing ...... 26 4.6.3 Coffeeroasting ...... 27

5 Purpose of the study 29

6 Materials and Methods 31 6.1 Chemicalsandsolvent ...... 31 6.2 Coffeesamples...... 31 6.3 Coffeeroasting ...... 49 6.3.1 Coffeeroastinginalaboratoryroaster ...... 49 6.3.2 Coffeeroastinginathermostaticoven ...... 50 6.3.3 Standardconditionroasting ...... 51 6.4 Typical sample preparation procedure ...... 51 6.5 3-aminopropionamideincoffee ...... 52 6.5.1 3-aminopropionamide in green coffee ...... 52 6.5.2 3-aminopropionamide in heated coffee ...... 53 6.6 Acrylamide and 3-aminopropionamide formation in a model system . 54 6.6.1 Preparation mixtures of asparagine with sucrose and glucose . 54 6.6.2 Sample preparation for optimal heating conditions estimation 55 6.6.3 Asparagine with ascorbic acid mixture preparation ...... 55 6.7 Heatingofpureasparagine...... 55

6.7.1 Acrylamide formation from pure asparagine heated at 170 °C for0-24minutes...... 55

6.7.2 Maleimide formation at 170 °C ...... 56 6.7.3 Acrylamide formation from asparagine at high temperatures . 56

i 6.8 HeatingofAmadoricompound ...... 57 6.9 Derivatizationmethods...... 58 6.9.1 Derivatization with dansyl chloride ...... 58 6.9.2 Derivatization with 2-mercaptobenzoic acid ...... 58 6.10 Analyticalmethods ...... 59 6.10.1 Ion chromatography with UV detection ...... 59 6.10.2 HPLC-FLDoperatingconditions ...... 60 6.10.3 HPLC-MS operating conditions (m/z 72,75)...... 61 6.10.4 HPLC-MS operating conditions, eluent water ...... 62 6.10.5 HPLC-UVoperatingconditions ...... 63 6.10.6 HPLC-MS operating conditions for acrylamide derivative analysis 63

7 Results and Discussion 68 7.1 Coffeeroastinginalaboratoryroaster ...... 68 7.2 Coffeeheatedinathermostaticoven ...... 69 7.3 Standardconditionroasting ...... 72 7.4 Acrylamide and 3-aminopropionamide formation in a model system . 74 7.4.1 Optimum time and temperature conditions for 3-aminopropionamide formation ...... 77 7.4.2 Optimal heating time and temperature conditions for acrylamide 77 7.5 Heatingofpureasparagine...... 81 7.6 3-Aminopropionamideincoffee ...... 82 7.7 Heated mixtures of asparagine with ascorbic acid ...... 83 7.8 Amadoricompound...... 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 ...... 1 2 Glycidamideformationfromacrylamide ...... 11 3 N -acetyl-S-(3-amino-3-oxopropyl)cysteine ...... 12 4 Glyceramide ...... 12 5 N -acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine ...... 12 6 Acrylamide formation pathway from asparagine and dicarbonyl (adapted from[63]) ...... 17 7 Acrylamide formation pathways from acrolein and asparagine with sugars(adaptedfrom[62]) ...... 19 8 Acrylamide formation from asparagine by simple decarboxylation and deaminationreaction [39]...... 20 9 Maleimide(2,5-pyroldione) ...... 21 10 Fumaramicacid ...... 21 11 Enzymatic 3-aminopropionamide formation from asparagine (adapted from[66]) ...... 22 12 Michaeladditionreaction ...... 23 13 Coﬀeeplant ...... 25 14 Arabica ZambiaAA ...... 32 15 Arabica UgandaOrganicoBiocoﬀee ...... 33 16 Arabica Tanzania ...... 33 17 Arabica Kenya ...... 34 18 Arabica KenyaAA ...... 35 19 Arabica EthiopianSidamoYirgamoGrade2 ...... 35 20 Arabica IndonesianSumatraLintong ...... 36 21 Arabica Indonesian Sulawesi Kalossi ...... 37 22 Arabica Indian Monsooned Aspinwalls Malabar AA ...... 37 23 Arabica IndianPlantationA ...... 38 24 Arabica JavaWIB1JampitGr1 ...... 39 25 Arabica NicaraguaTaliaExtra ...... 40 26 Arabica CostaRicaTarazzu ...... 40 27 Arabica GuatemalaSHB ...... 41 28 Arabica MexicoMaragogype ...... 42 29 Arabica MexicoAltura ...... 43 30 Arabica HondurasSHG ...... 43 31 Arabica ColombianExcelso ...... 44 32 Arabica Santos Brazil NY 2 17/18 TOP Italian preparation . . . . . 45 33 Arabica PapuaNewGuineaSigriC ...... 45 34 Robusta IndianParchment ...... 46 35 Robusta IndianCherryAB ...... 47 36 Vietnam Robusta ...... 47 37 Robusta Liberia ...... 48 38 Robusta Cameroon ...... 49 39 Temperature gradient of roasting programmes 4, 6, 8 and 10...... 50

iii 40 Coffee beans roasted under different time and temperature conditions 54 41 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-fructopyranose ...... 57 42 Reaction of 3-aminopropionamide to sulphonamide ...... 58 43 Derivatization of acrylamide with 2-mercaptobenzoic acid ...... 59 44 Typical chromatogram of acrylamide using ion exclusion chromatog- raphywithUVdetection ...... 60 45 Typical chromatogram of 3-aminopropionamide analysis ...... 61 46 Typical chromatogram of acrylamide using MS detection ...... 62 47 Typical chromatogram of acrylamide using UV detection ...... 64 48 Typical chromatogram of acrylamide (6 ng/ml) after derivatization with 2-mercaptobenzoic acid using LC-MS/MS ...... 65 49 Typical chromatogram of acrylamide in a roasted coffee sample after derivatization with 2-mercaptobenzoic acid using LC-MS/MS .... 66 50 Typical chromatogram of derivatized acrylamide analysis...... 67 51 Formation of acrylamide in 4 different types of coffee roasted in a laboratoryroaster ...... 68 52 2nd Order regression curve of acrylamide content in coffee beans ... 70 53 Regression surface curve of acrylamide content in coffee beans .... 71 54 Significant parameters in the formation of acrylamide ...... 72 55 Acrylamide content in different coffee beans roasted under standard conditions ...... 73 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 ...... 75 57 3-Aminopropionamide in heated asparagine and sucrose and asparagine and glucose (molar ratio 1:0.5, 1:1 and 1:1.5) anhydrous mixtures at

130, 150 and 170 ...... 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 ratio 1:0.5 and 1:1) heated to diﬀerent temperatures for 5 and 7 minutes . 78 60 Acrylamide formed in asparagine and glucose mixtures (1:0.5) heated atdiﬀerenttemperaturesfor5minutes ...... 79

61 Acrylamide formation in asparagine mixtures with glucose at 210 °C (molarratio1:0.5) ...... 80 62 Acrylamide content (µg/g) in asparagine mixtures with glucose (1:0.5)

heated at 250 °Cfor1,3,4,5,7and10minutes ...... 80 63 Acrylamide formation from pure asparagine heated at high temperatures 82 64 Acrylamide formation in the mixtures of asparagine with ascorbic acid 84 65 Acrylamide formation in asparagine and ascorbic acid mixtures heated

at 250 °Canddiﬀerenttimes ...... 84 66 3-Aminopropionamide formation in the mixtures of asparagine with ascorbicacid ...... 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 AcrylamideinfoodsfromUSA[29] ...... 2 2 AcrylamideinCanadianfoods[30] ...... 8 3 AcrylamideinAustralianfoods[31] ...... 8 4 Mean concentrations of acrylamide in UK food [32] ...... 10 5 Roastingparametersofprogram4,6,8and10...... 50 6 Characteristics of green coffee beans processed by different methods. 52 7 Heating conditions of Amadori compound ...... 57 8 Concentration of acrylamide in Cameroon Robusta, heated in an ther- mostaticoven,ng/g...... 69 9 Concentration of acrylamide in Cameroon Robusta, heated in an ther- mostaticoven,ng/g...... 71 10 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 Galcer´an 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. Encarnaci´on Moyano Morcillo and Sra. Elisabet Bermudo Rubio for the help developing the analytical method for acrylamide detection.

I would like to thank Dipl.-Ing. Dr.techn. Univ.-Doz. Tanja Maria Wrodnigg from the Institute for Organic Chemistry at Graz University of Technology for giving me the chemicals for our experiments.

I would like to address my special thanks to my boyfriend Dipl.-Ing. Bernhard Schraußer for his devoted patience by helping me to understand the tricks of the most complicated typesetting system called 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 detection instruments - mainly UV and mass spectroscopy. Moreover, for the analysis of the possible acrylamide precursor 3-aminopropionamide liquid chromatography with fluorescence detection was used. 25 green coffees of different origin were roasted either in a laboratory coffee roaster or thermostatic oven. The study showed, that Arabica and Robusta coffee beans differ not only in size, cup quality, but also in acrylamide content independent on the region of growth, harvesting and processing conditions. The probable precursor of acrylamide 3-aminopropionamide was not detected neither in green coffee beans nor in roasted ones. Finally some experiments were done with mixtures modeling the acrylamide formation during the roasting of coffee. Acrylamide and its possible precursor 3-aminopropionamide were easily formed in mixtures of asparagine with glucose or sucrose. Less 3-aminopropionamide was formed in mixtures of asparagine with ascorbic acid. Even some acrylamide was formed when pure asparagine was heated at high temperatures. The heating of the Amadori product 1-N -(asparaginyl)-5-azido-1,5-dideoxy-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 möglichen Vorstufe von Acrylamid, gefunden. Kaffee wurde als Forschungsobjekt gewählt, da er in großen Mengen kon- sumiert wird und somit einen potentiell gefährlichen Einfluß auf die menschliche Gesundheit darstellt. Nach der Extraktion mit Wasser und darauf folgender Festphasenextraktion wurde Acrylamid mittels Flüssigchromatographie nachgewiesen, wobei verschiedene Detek- toren, vor allem UV und MS-Detektion, zum Einsatz kamen. Für den Nachweis der möglichen Acrylamid-Vorstufe 3-Aminopropionamid wurde Flüssigchromatographie mit Fluoreszenz-Detektion verwendet. 25 Sorten grüner Kaffeebohnen aus verschiedenen, über die ganze Welt verteil- ten Anbaugebieten wurden entweder mit einem Labor-Kaffeeröster oder im Ther- mostatofen geröstet. Die Untersuchung zeigte, dass sich die beiden Sorten Arabica und Robusta nicht nur in Größe und Geschmacksqualität, sondern auch im Acry- lamidgehalt unterscheiden, und zwar unabhänging von der Anbauregion, sowie den Bedingungen bei Ernte und Weiterverarbeitung. Leider konnte die mögliche Acrylamid-Vorstufe 3-Aminopropionamid weder in den grünen noch in den gerösteten Kaffeebohnen nachgewiesen werden. Zum Schluß wurden einige Experimente mit Modellsystemen durchgeführt, wobei ähnliche Konzentrationen von Acrylamid-Vorstufen wie in den Kaffeebohnen, sowie ähnliche Röstbedingungen simuliert wurden. Sowohl Acrylamid als auch seine mögliche Vorstufe 3-Aminopropionamid konnten dabei aus Mischungen von Asparagin mit Glukose oder Succrose erzeugt werden, weniger leicht mit Ascorbinsäure. 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 °C

and a high boiling point of 136 °C at 3.3 kPa [2, 3]. Acrylamide is very soluble in water, alcohols, acetone, acetonitrile, slightly soluble in ethyl acetate, dichlormethane, diethyl ether. It is insoluble in hexane and other alkanes and alkenes. Low, but significant volatility of acrylamide was observed. It has no significant UV-absorption above 220 nm and does not fluoresce.

Figure 1: Acrylamide

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

1 [13, 15, 16, 17, 18, 19, 20] or liquid chromatography [7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23], and even by capillary zone electrophoresis [24]. Using liquid chromatography with single or tandem mass spectrometry it can be analyzed directly [7, 8, 11, 12, 18, 21, 22, 23, 25] or derivatized [16], also UV detection gives satisfactory results [9].

4.2 Acrylamide in foods

As Tareke et al. in 2002 [26] reported, acrylamide hemoglobin adduct background levels originate from foods of a normal diet. Acrylamide was detected in all kind of foods including meat, bread and potato products prepared at high temperatures

(>160 ) [26, 27]. Relatively small amounts can be found in boiled and microwaved

(where temperatures can reach up to 260 °C [28]) foods, but not fresh ones. Even roasted tea leaves and roasted barley grains contain acrylamide in the concentration up to 570 and 320 ng/g respectively. Acrylamide is formed in high-carbohydrate foods during frying, baking, roasting and extrusion. In commercially processed foods as well as in home-cooked meals the acrylamide content tends to increase with cooking time and temperature. The surface color of the products correlates highly with acrylamide levels in food: the darker the surface, the more acrylamide it contains [4]. Some data on acrylamide in food are presented in Tables 1, 2, 3, 4.

Table 1: Acrylamide in foods from USA [29]

Food Product Acrylamide, ppb 2002 2003 2004 BabyFood ND-121 French notbaked 20-212 fries baked 119-1325 readytoeat 117-1030 122-1250 Potato 117-2762 693-2510, 462-1970 chips 1077 Natural potato chips lot 1 879 continued on next page

2 Table 1 – continued from previous page Food Product Acrylamide, ppb 2002 2003 2004 Natural potato chips lot 2 433 Classic potato chips 0 weeks 319 Classic potato chips 1 week 432 Classic potato chips 2 weeks 280 Classic potato chips 3 weeks 257 Classic potato chips 4 weeks 343 Classic potato chips 5 weeks 425 Infant For- ND mula Milk based Infant Formula with Iron (liquid) ND Milk based Infant Formula with Iron (powdered) <10 Soy Infant Formula liquid ND powdered ND Protein Chicken (not baked) 32 Foods Chicken (baked) 35 Chicken ready to eat 22 Fish (not baked) ND-25 Fish (baked) ND-30 Grilled vegetable burgers (not baked) ND-58 Grilled vegetable burgers (baked) ND-116 Veja-Links (uncooked) ND Veja-Links (microwaved) ND Veja-Links (toasted) ND Bread and Pumpernickel (not toasted) 34 bakery Pumpernickel (toasted) 364 Whole grain whole wheat (nottoasted) ND 102 Whole grain whole wheat (toasted) 59 Ryebread 42 ND-31 Wheat bread <10-130 Potatobread 36 41-52 White bread (not toasted) 36 <10-18 ND-30 continued on next page

3 Table 1 – continued from previous page Food Product Acrylamide, ppb 2002 2003 2004 White bread (toasted) 216 Wheat bagels (not toasted) 27 12-27 Wheat bagels (toasted) 57 Bread crumbs with cheese 39 Bread crumbs regular style 42 Doughnut ND-24 ND-22 Flat bread from restaurant 125 Pizza crust (not baked) 33 Pizza crust (baked) 24 Tortillas (not fried) <10-10 Tortillas (fried) 13-15 Seven grain bread ND-51 French bread ND-25 Nut bread 47 Pies <10-74 ND Banana nut muffins 29 Cereals Cheerios 61-266 CornFlakes 77 70-534 Cornpops 71 FrostedFlakes 52 51-163 Frosted mini-wheats 78 Raisinbran 156 Rice krispies 47 Rice cereals 33 Müsli 11-51 Granola 20-89 Toasted wheat cereals 1057, 689 Fruit wheels ND-92 Other 193-1243, snacks 151 Chifles 12 Soy crisps 17 Roppingcorn(popped) 57-181 65-446 Honey Dijon mustard Juli- enna potatostix 1168 Tortilla chips 117 130-196 Veggie crisps 832 1340 Pretzels 58-386 Cheese puffs 166 continued on next page

4 Table 1 – continued from previous page Food Product Acrylamide, ppb 2002 2003 2004 Vegetable chips 73-828 Gravies Beef gravy (canned) ND and seasonings Chicken gravy (canned) ND Mushroom gravy mix ND Turkey gravy mix ND Soy sauce ND Hickory liquid smoke 54 Pecan liquid smoke 151 Mesquite liquid smoke 38 Flavor enhancer ND Instant bouillon ND Nuts and Salted almonds 236-249 nut butters Smoked almonds 339-457 Cashews ND Roastedpeanuts ND-28 Peanut butter 64-125 Crackers 37-620 39-1540 Wheat crackers 26-300 Crackers reduced fat 130 Cheddar goldﬁsh 57 Crispbread ﬁber rye 504 Chocolate Drostecocoa ND-909 products Unsweetened cocoa 316 Baking chocolate 93-104 Milk chocolate ND Chocolate sweets ND-74 Fruit and vegetables Potatoes ND Ripeolives 226-1925 798 Black olives ND Olives in brine ND Green olives <10- 19 Canned ND-11 fruits and veg- Apple sauce <10 etables Baked beans 70-83 continued on next page

5 Table 1 – continued from previous page Food Product Acrylamide, ppb 2002 2003 2004 Mushrooms ND Chick peas ND Pumpkin 25 Frozen <10 vegetables Fruit and vegetable French fried onions 125 products Breaded mushrooms 103 Dried plums 31-87 Raisins <10 Cookies 36-199 334-432 69-955 Wafers 61-114 Coffee ground,notbrewed 175-207 91-609 regularroast 37-374 regularroast,decaffeinated 222-361 27-149 darkroast 97-319 crystals, not brewed 351 Instant coffee, powdered, notbrewed 188-263 266-458 decaffeinated 172-539 Brewed 5-11,3-13

Other hot Instant caﬀeine free hot beverages beverage, powdered, not 3747-5399 brewed brewed 93 Dried Soups 22-1184 foods Noodles 11-136 Rice with sauce <10 Dairy Condensed milk ND Malted milk original 43 Instant nonfat dry milk 11 Buttermilk blend <10 Milk ND Miscellaneous ND-398 Onion rings 13 Mashed potatoes ND-<10 continued on next page

6 Table 1 – continued from previous page Food Product Acrylamide, ppb 2002 2003 2004 Gelatin dessert <10 ND Non-dairy coﬀee creamer <10 Groundtoastedcorn 804 Pizza rolls (frozen) 29 Potato skins 393 Juice Prune 267 138-239 Taco, Taco shells 29-69 Tostado and Tor- tilla products Tortillas 33-794 Tostado 337 Restaurant Asian ND-169 and take- out food Southersn/Cajun ND-202 Hispanic ND-129 Potato pancakes 229 Waﬄe potato fries 271 Fruits and Grilled asparagus ND vegetables, take-out Sweet potato chips 4080 Grilled carrots 61 Grilled onions 70 Potatoes 114-984 Roasted mixed vegetables ND

As it can be seen in the Table 1, the highest amounts of acrylamide can be detected 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, ppb French fries 59-1900 Bread 14-47 Toasted French bread 290 Coffee, not brewed 8-150 Cocoa products Baking chocolate 190 Milk chocolate <2-40 Cocoa powder - bulk 45 Instantcoffee 430-1700 Instant cereal beverage 4300 Others Roasted almonds 260 Sweet potato chips 260 Potatopancakes 210-290 Roasted sunflower seeds 66 Roasted soy beans 25 Beer <6 Boiled mashed potatoes <4 Hamburger <3 Potato chips 300-3400 Cereals Wheat 88-170 Rice 50-160 Baby 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, ppb Coffee Instant, decaffeinated <3 Instant <3 Brewed, decaffeinated <10-15 Brewed, dark roast <10-14 continued on next page

8 Table 3 – continued from previous page Food Product Acrylamide, ppb Brewed, light roast <10-23 Instant cereal bever- trace age Chocolate milk <25 Cocoa powder <100 Prune juice 93 Chocolate bar <25 plain block <30 Nuts Cashew traces Mixed, salted traces Peanuts, dry roasted <25 Canned soups <25-50 Baked beans canned <25 Spanish olives black 345 green <25 Meat pie <25 Fried rice <25 Pizza frozen oven baked <25 microwave 5 min <25 Fish crumbed and fried <25 crumbed and oven baked 52 Chicken schnitzel <25 Hash brown fried 440 oven baked 200-320 Beef 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 information 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, ppb Bread 12 Miscellaneous cereals 57 Meat products 13 Fish <5 Oils and fats <3 Sugars and preserves 23 Green vegetables <2 Potatoes, made-up 53-112 Canned vegetables <5 Fruit products <1 Beverages <1 Nuts <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 humans [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 glycidamide 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 reports. 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 asparagine 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 degradation 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 degradation. 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 (adapted from [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 compounds 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 glutamic acid, coffee beverages prepared in a usual way, black and green tea were reported 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].

Acrylic acid OR Acrolein O O OR OR OH

NH3 O Acrylamide

NH2 NH2 O

+ Glucose HOOC NH2 heat Asparagine

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 different 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 and deamination 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 temperatures 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 (adapted from [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 decarboxylase 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 pyridoxal phosphate as a cofactor. As it is known that enzymatic reactions take place

22 under physiological enzymatic conditions (high water content, warm (10-40 ) temperatures), 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 acrylamide 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 proteins 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 complex 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 following 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 conditions 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 13 (Buchs, Switzerland). C3-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 broken 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 surface, 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 impurities 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 broken 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 impurities 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 impurities 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 impurities 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 impurities 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 average 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 impurities 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 average 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 4 360 237 6 450 243 8 720 247 10 870 246

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 thermostatic 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 w Ethiopian Sidamo Yirgamo w+s Grade 2 Zambia AA w Kenia w Kenia A/A w Asia Indian Monsooned Aspinwalls w+m Malabar AA Indian Plantation A w Indonesian Sumatra Lintong d+s Indonesian Sulawesi Kalossi sw Central America Nicaragua Talia Extra w Guatemala SHB w Mexico Maragogype w Mexico Altura Costa Rica Tarazzu w South America Honduras s.h.g w Oceania Papua New Guinea Sigri C w Java WIB1 Jampit Gr1 w Robusta Asia Indian Parchment w+s Vietnam d 1 w - wet-processed: washed coffees, d - dry-processed: natural coffees, s - sundried, m - monsooned (dried in the sun, but exposed to high humidity and rain), sw - semiwashed.

For LC-MS analysis the samples were prepared similarly with adding 100 µl of 13 C3-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 for 0-24 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 13 MeOH and 2 ml H2O. After SPE purification C3-labeled acrylamide (final conc. 100 ng/ml) was added.

6.7.2 Maleimide formation at 170 °C

Pure 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 temperature 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 compound

Compound Heating temperature, Heating time, min 1-N -(asparaginyl)-5-azido- 170 2 1,5-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 liquid chromatography with ﬂuorescence detection, were derivatized with dansyl chloride ([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 µl1MNaOH. 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 controlled 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 chromatography with 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 ambient 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, quarternary 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, quarternary 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, quarternary 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 recently described in the literature [16]. Since we could not apply this method directly in our laboratory, the method was modiﬁed and improved with help of Dra. M.T.Galcer´an 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 analysed 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 with 2-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 after derivatization 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 calibration. 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 laboratory 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 thermostatic oven, ng/g.

Temperature Time, min 5 10 15 220 463 250 0 488 158 0 240 191 107 110 260 239 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 temperature conditions are significant for the formation of the toxicant. In the presented coffee roasting experiments we could analyse time and temperature parameter influence on the acrylamide formation. According to our results we can affirm that both time and temperature conditions have influence on the formation 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 thermostatic oven, ng/g.

Temperature, Time, min 1 3 5 180 31.5 28.5 39.7 200 n.d. 41.7 42.9 220 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 conditions

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 quality 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 different 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 conditions Coﬀee Acrylamide formed, ng/g Robusta Indian Parchment 762 Robusta Vietnam 653 Indian Monsooned Aspinwalls Malabar AA 575 Indian Plantation A 401 Indonesian Sumatra Lintong 301 Indonesian Sulawesi Kalossi 306 Tansania Arabica 354 Ethiopian Sidamo Yirgamo Grade 2 425 Zambia AA 331 Kenia washed 531 Kenia A/A 299 Nicaragua Talia Extra 433 Guatemala SHB 374 Mexico Maragogype 363 Mexico Altura 380 Costa Rica Tarazzu 310 Honduras 307 Papua New Guinea Sigri C 352 Java WIB1 Jampit Gr1 357

It seems, that neither place of origin nor processing method has significant influence 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 model system

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 concentration 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 noticed 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 asparagine and glucose (molar ratio 1:0.5, 1:1 and 1:1.5) anhydrous mixtures at 130, 150 and

170 . 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-aminopropionamide formation

We noticed, that the highest amount of 3-aminopropionamide was formed in asparagine 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 optimum time and temperature conditions for acrylamide formation. For this purpose asparagine 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 minutes (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 anhydrous

mixtures at 130, 150, 170 and 190

Figure 59: Acrylamide content in asparagine with glucose mixtures (molar ratio 1:0.5 and 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 at diﬀerent 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, acrylamide formation is connected to a heating temperature value: the higher the heating temperature, the shorter time is needed to achieve a maximum acrylamide concentration 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 vitamin 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 at

250 °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 ascorbic acid

7.8 Amadori compound

The Amadori compound 1-N -(asparaginyl)-5-azido-1,5-dideoxy-D-fructopyranose consists 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 intermediate 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 acrylamide 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 efficient. 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 following 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 °C for 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 acrylamide formation was detected. However, the amounts of this neurotoxin were

extremely low. Heated asparagine to 170 °C performs fast intramolecular cyclization 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 asparagine is used as a skeleton for acrylamide’s formation.

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99 10 Curriculum Vitae

Personal information Surname/First name: Bagdonaite Kristina Dateofbirth: 10.07.1979 Place of birth: Radviliskis, Lithuania Nationality: Lithuanian Address: 4/7/39, Gaussgasse, Graz, A-8010, Austria Oﬃce: Institute for Food Chemistry and Technology, Graz Uni- versity of Technology, Petersgasse 12/II, A-8010, Graz Telephone: +43(0)316873-6969 E-mail: [email protected]

Education since 2003 Graz University of Technology, (Graz, Austria), PhD student 2001-2003 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- SOCRATES/Erasmus student at Graz University of Technology, 03.2003 Department for Food Chemistry and Technology Further Training since Member of the Sensoric Panel at the Department of Food 11.2005 Chemistry and Technology, Graz University of Technology 2002 SOCRATES short course on Identiﬁcation of volatile and aroma active compounds in food Languages Mother Lithuanian tongue Other lan- English, Russian, German (good writing and speaking skills) guages

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 acrylamide 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