Non solo caffeina: il viaggio di un chimico dentro a un chicco di caffè

Non solo caffeina: il viaggio di un chimico dentro a un chicco di caffè Stefano Mammi Dipartimento di Scienze Chimiche Università di Padova stefano.mammi@unipd.it Composition of Green Coffee Coffee is not just caffeine Complex mixture Plant Derived, Roasted Many unique, biologically active compounds that alter metabolism Independentof caffeine, coffee has acute effects on blood pressure, cognition, glucose homeostasis and insulin sensitivity 1

The perfect espresso Dose 7.0 ± 0.5 g Water temperature 90 ± 2 C Extraction pressure 9.0 ± 0.5 atm Percolation time 30 ± 5 s Volume 25 ± 5 ml No other drink manages to convey such a pleasant, unforgettable taste and flavour at the moment of drinking, and for 30 minutes thereafter, as a good espresso does Ernesto Illy (1925 2008) 2

The perfect espresso thick layer of foam on top of the beverage it adds to the visual appeal of the cup opaque underlying liquid, due to a dispersed phase of very small oil droplets in emulsion it increases viscosity (the «body» sensation) it enhances flavour (lipids are aroma carriers) it prolongs after-taste (oil/water persistence) ARABICA COFFEE GROWING AREAS MAJOR PRODUCERS: BRAZIL, COLOMBIA, MEXICO, ETHIOPIA, INDIA, CENTRAL AMERICAN COUNTRIES, ETC., 3

ROBUSTA COFFEE GROWING AREAS MAJOR PRODUCERS: VIETNAM, BRAZIL, INDONESIA, INDIA, AFRICAN CONTINENT, ETC., SOURCE : SCAA Raw coffee composition: an overview Pericarp or Exocarp: the outer and colored layer (skin) Mesocarp: the jelly -like layer (pulp) Endocarp: covers the bean (parchment) Epidermis: silver skin (chaff) Endosperm: seed (bean) 4

Harvesting: the cherry ripeness Color change: chlorophyll is degraded>> production of pigments Pulp softening (soluble compounds): pectin is converted from a water insoluble form to a soluble one by degrading enzymes Maximum sugar and dry material contents: acids >> sugars Aromatic compound presence: volatile compounds (esters) creation COFFEE MOISTURE % Green cherry 60-70 Ripe Cherry 60-65 Over-ripe Cherry 30-40 Post-harvesting Methods Natural Coffee: dry process Pulped Coffee: wet or washed and fermented process Natural Pulped Coffee or Honey: semi-wet process 5

Comparison between methods Differences in chemical composition affect the sensory properties: Natural: higher body, higher sugars Wet: higher acidity, more aromatic Semi wet: higher sweetness Composition of Green Coffee CONSTITUENT ARABICA* ROBUSTA* COMPONENTS Soluble carbohydrates 9-12.5 6-11.5 Monosaccharides 0.2-0.5 0.2-0.5 Fructose, Glucose, Galactose, Arabinose (traces) Oligosaccharides 6-9 3-7 Sucrose (>90%), Raffinose (0-0.9%), Stachyose (0-0.13%) Polysaccharides 3-4 3-4 Polymers of Galactose (55-65%), Arabinose (20-35%), Mannose (10-20%), Glucose (0-2%) Insoluble polysaccharides 46-53 34-44 Hemicelluloses 5-10 3-4 Polymers of Galactose (65-75%), Arabinose (25-30%), Mannose (0-10%) Cellulose, β(1-4)mannan 41-43 32-40 H.-D. Belitz, W. Grosch & P. Schieberle, Food Chemistry, 2004, p. 942 *% dry matter 6

Composition of Green Coffee CONSTITUENT ARABICA* ROBUSTA* COMPONENTS Acids and Phenols Volatile Acids 0.1 0.1 Nonvolatile Aliphatic Acids 2-2.9 1.3-2.2 Citric, Malic, Quinic Chlorogenic Acids 6.7-9.2 7.1-12.1 Mono-, Dicaffeoyl-, and Feruloylquinic Lignin 1-3 1-3 Lipids 15-18 8-12 Wax 0.2-0.3 0.2-0.3 Oil 7.7-17.7 7.7-11.7 Main Fatty Acids: 16:0 and 18:2 (9,12) H.-D. Belitz, W. Grosch & P. Schieberle, Food Chemistry, 2004, p. 942 *% dry matter Composition of Green Coffee CONSTITUENT ARABICA* ROBUSTA* COMPONENTS N Compounds 11-15 11-15 Free Amino Acids 0.2-0.8 0.2-0.8 Main Amino Acids: Glu, Asp, Asn Proteins 8.5-12 8.5-12 Caffeine 0.8-1.4 1.7-4.0 Traces of Theobromine and Theophylline Trigonelline 0.6-1.2 0.3-0.9 Minerals 3-5.4 3-5.4 H.-D. Belitz, W. Grosch & P. Schieberle, Food Chemistry, 2004, p. 942 *% dry matter 7

Insoluble Polysaccharides in Green Coffee Insoluble Polysaccharides in Green Coffee 8

Insoluble Polysaccharides in Green Coffee Galactomannan Found also in Locust Bean Gum (Carob Gum) (D-Mannose (1-4) with Galactose (1-6) every 4th Mannose) Molecular weight 330,000 ±30,000 Neutral - relatively unaffected by ions, ph Not soluble in cold water Fully hydrated if heated 10 minutes at 80 C Solutions are cloudy, off-white Pseudoplastic - shear thinning, zero yield value Acids and Phenols Aliphatic Acids O H Formic Acid C H 3 O Acetic Acid C H 3 O Lactic Acid O HO O Tartaric Acid C H 3 O O Pyruvic Acid HO O O O HO O Citric Acid (0.6-1.4%) Malic Acid (0.2-0.7%) Quinic Acid (0.3-0.6%) O HO O They contribute to the pleasantly sour taste of coffee 9

Acids and Phenols Chlorogenic Acids CQA = Caffeoylquinic Acid FQA = Feruloylquinic Acid pcoqa = p-coumaroylquinic Acid In green coffee beans (C. arabica) Chlorogenic acid % 5-caffeoylquinic acid 4.79 4-caffeoylquinic acid 0.71 3-caffeoylquinic acid 0.51 3,5-dicaffeoylquinic acid 0.43 4,5-dicaffeoylquinic acid 0.29 5-feruloylquinic acid 0.28 3,4-dicaffeoylquinic acid 0.20 3,4-diferuloylquinic acid < 0.1 3,4-dicoumaroylquinic acid < 0.1 Very high alkyl peroxyl radical scavenger activity Acids and Phenols Lignin is a class of complex, high molecular weight polymers whose exact structure varies. It is an amorphous, i.e., not crystalline, polymer that acts as a binding agent to hold cells together. Lignin also occurs within cell walls to impart rigidity. Like cellulose and hemicellulose, lignin is made from carbon, oxygen, and hydrogen. However, these elements are arranged differently so that they are not classified as carbohydrates. They are instead classified as phenolics, and the polymer is based on the phenylpropane unit. original source: Adler, E. 1977. Lignin chemistry-past, present and future. Wood Sci. Technol. 11, 169-218. 10

Acids and Phenols Isoflavonoids & Lignans (phyto-oestrogens) Mazur et al., Br. J. Nutr., 1998, 79, 37-45. Isoflavonoids & Lignans possess several biological activities, such as antioxidant and (anti)oestrogenic properties, and thus may reduce the risk of certain cancers as well as cardiovascular diseases (Milder et al, 2005; Osoki & Kennelly 2003) Coffee Oil in Green Beans CONSTITUENT %TOTAL LIPIDS Triacylglycerols 70-80 Free fatty acids 0.5-2.7 Diterpene esters 15-18.5 Free diterpenes 0.1-1.2 Triterpenes, sterols, and sterol esters 5-Hydroxytryptamides and derivatives 1.4-3.2 0.3-1.0 Tocopherols 0.3-0.7 Phosphatides 0.3 A. Illy & R. Viani, Espresso Coffee, 1995, p. 33 Water distribution Oil distribution R. Toffanin et al., Proceedings of the 19th ASIC Colloquium, Paris, 2001. 11

Coffee Oil in Green Beans Triacylglycerols: 70-80% Karl Speer and Isabelle Kölling-Speer Braz. J. Plant Physiol., 18(1):201-216, 2006 Free fatty acids: 0.5-2.7% Coffee Oil in Green Beans DF1 = -8.87 C16:0-5.40 C18:0-3.59 C18:1-9.26 C18:2-1.29 C18:3-1.46 C22:0 Green Arabica Green Robusta Roasted Arabica Roasted Robusta Martín et al., Talanta 54 (2001) 291 297 Total fatty acids 12

Diterpenes in Coffee Oil OCH 3 Cafestol Kahweol 16-OMethyl-Cafestol ARABICA* 0.55-0.95 0.31 absent ROBUSTA* 0.19-0.23 0-traces 0.07-0.15 *% dry matter Diterpene esters* 15-18.5 Free diterpenes* 0.1-1.2 A. Illy & R. Viani, Espresso Coffee, 1995, p. 35 *% total lipids Coffee Oil in Green Beans Karl Speer and Isabelle Kölling-Speer Braz. J. Plant Physiol., 18(1):201-216, 2006 Diterpene esters: 15-18.5% 13

Sterols in Coffee Oil Structures of some common phytosterols and phytostanols Karl Speer and Isabelle Kölling-Speer Braz. J. Plant Physiol., 18(1):201-216, 2006 Tocopherols (Vitamin E) in Coffee Oil α = 349 825 mg/kg oil β = 455 749 mg/kg oil γ = 31.9 113 mg/kg oil Jham et al.,jafc (2007) 55 5995 α = 1.37 ± 0.47 µg β = 2.09 ± 0.67 µg Alves et al.,food Chem (2009) 115, 1549 Very small contribution to the recommended daily Vitamin E intake (15 mg of α-tocopherol) Complement to the overall antioxidant effect provided by the brew 14

Amino Acids in green coffee Caffeine and Other Purine Alkaloids Caffeine Theobromine Theophylline 15

Caffeine and Other Purine Alkaloids Tea Cocoa 25,000-55,000 2,000 700-1,700 12,000 20-130 (mg/kg, dwb) Chlorogenic Acid Caffeine Complex low binding energy 20 25% bound caffeine Horman & Viani, 1971;1972; D Amelio et al., 2009 16

Trigonelline and its Derivatives second most abundant alkaloid in green coffee C. arabica: 7.9 10.6 g/kg C. canephora: 6.6 6.8 g/kg undergoes significant degradation during roasting: O O -CO 2 N + -CH 3 NH 2 CH 3 N N N-methylpyridinium Nicotinic acid Nicotinamide (Niacin) Minerals in Green Coffee Potassium: relevant source (Gillies & Birkbeck, 1983). 40% of total mineral content. Magnesium: substantial source. A mean of 63.7 µg/cup (100 ml) has been reported (Astier-Dumas & Gounelle de Pontanel, 1974) Manganese: significant contribution (Gillies & Birkbeck, 1983) Chromium: 8% daily intake (Santos et al., 2004) Rubidium: up to 180 mg/kg in green beans 17

Changes During Storage of Green Coffee Many things depend on water activity: 1. Lipid oxidation 2. Nonenzymatic browning 3. Hydrolytic reactions 4. Ascorbic acid loss 5. Enzymatic activity 6. Growth of moulds 7. Growth of yeasts 8. Growth of bacteria Moisture: ~5% ~12.5% The roasting process Temperature ( C) 250 200 150 100 50 Roaster temperature Coffee temperature 0 0 2 4 6 8 10 Roasting time (min) 51 18

Browning development Porosity increase Green coffee beans Roasted coffee beans 15 µm 45 45 µm mm 45 µm Adapted from Schenker et al., J. Food Sci., 65, 452-457, 2000 55 19

Chemical Changes During Roasting of Green Coffee Polysaccharides Sucrose Proteins Amino acids Chlorogenic acids Trigonellin Water Caffeine Minerals Aliphatic acids Quinic acid Volatile aroma compounds Melanoidins CO 2 Adapted from: A. Illy & R. Viani, Espresso Coffee, 1995, pp. 98-99 Chemical Changes During Roasting of Green Coffee CONSTITUENT ARABICA* ROBUSTA* Carbohydrates GREEN ROASTED GREEN ROASTED Sucrose 8.0 0 4.0 0 Reducing sugars 0.1 0.3 0.4 0.3 Polysaccharides 49.8 38.0 54.4 42.0 Proteins and amino acids Proteins 9.8 7.5 9.5 7.5 Amino acids 0.5 0 0.8 0 Lipids 16.2 17.0 10.0 11.0 Caramelization and condensation products (by difference) 25.4 25.9 Adapted from: A. Illy & R. Viani, Espresso Coffee, 1995, pp. 98-99 *% dry matter 20

Chemical Changes During Roasting of Green Coffee CONSTITUENT ARABICA* ROBUSTA* GREEN ROASTED GREEN ROASTED Caffeine 1.2 1.3 2.2 2.4 Trigonelline (and roasted by-products) 1.0 1.0 0.7 0.7 Organic acids Aliphatic 1.1 1.6 1.2 1.6 Quinic 0.4 0.8 0.4 1.0 Chlorogenic 6.5 2.5 10.0 3.8 Volatile aroma compounds traces 0.1 traces 0.1 Minerals (as oxide ash) 4.2 4.5 4.4 4.7 Water 8-12 0.5 8-12 0.5 Adapted from: A. Illy & R. Viani, Espresso Coffee, 1995, pp. 98-99 *% dry matter The Maillard reaction Modified from Hodge, J. Agric. Food Chem., 1, 928-943, 1953 59 21

The Maillard reaction Heat intensity MILK, UHT MILK, CANNED LIGHT BEER PASTA First stages of the Maillard reaction Intermediate stages of the Maillard reaction Amadori product Dycarbonyl Dicarbonyl compounds Carboxy reductones HMF Other volatile compounds BAKERY PRODUCTS DARK BEER COCOA ROASTED MEAT COFFEE Advanced stages of the Maillard reaction Strecker aldheydes Pirazine Melanoidins Heterocyclic amines Melanoidins 60 Melanoidins Proteins Macromolecular, nitrogenous, brown-colored Maillard reaction end-products formed during home and industrial heat processing of foods, widely distributed in our diet (coffee, cocoa, bread, malt, honey...) Phenolics nonenzymatic browning Proteins Carbohydrates Phenolics Carbohydrates 22

Melanoidins: Challenging Chemistry Nunes & Coimbra Phytochem Rev (2009) 9(1), 171-185 Coffee Aroma About 300 compounds in green coffee aroma 1000 compounds in roasted coffee aroma 23

Why is Aroma so important in coffee? At least 70% of the characteristic impression of the cup Aroma Influence global perception Negative notes positive notes - - - +++ Positive notes in coffee aroma Caramel Bread Nutty Fruity Flowery Chocolate Spicy Browning reaction of sugars Smell of bread, bakery Nuts, almonds, peanuts, hazelnut Citrus, lemon, melon, apple, pear, fruit salad Rose, jasmine, orange blossom Cocoa smell, or chocolate note Cinnamon, cloves, or others 24

Negative notes in coffee aroma Earthy Peasy Woody Rancid Stinker Juta Rio Burnt Mouldy, earthy Bacterial contamination of cherries Smell of dry wood Due to lipid oxidation, old crop Raw beans are stinker after scraping Typical smell of humid juta bags Medicinal, iodine-like, corky, phenolic Overroasted, burnt What s in the aroma? Smelly compounds Volatile compounds Small molecules Low polarity, lipophilicity 25

Aroma Compounds Several concomitant reactions lead to a very high number of volatile compounds Aroma Compounds in Roasted Coffee Family Number of Number of Family compounds compounds Aliphatic hydrocarbons 45 Pyrazines 89 Aromatic hydrocarbons 35 Quinoxalines 11 Alcohols 25 Furanones 128 Aldehydes 37 Oxazols 35 Ketones 85 Thiols 7 Acids 28 Sulphides 13 Esters 33 Disulphides 10 Amines 13 Thiophenes 28 Pyrrols 72 Thiazoles 27 Pyridines 20 Phenols 49 26

Aroma Compounds J. Chrom. A, 1186 (2008) 123 143 Aroma Compounds Dominant Robusta Robusta Robusta H.-D. Belitz, W. Grosch & P. Schieberle, Food Chemistry, 2004 27

Aroma Analysis Aroma Extract Dilution Analysis (AEDA) Through dilution of the extract (dilution factor: 2, 4, 8, 16 2 n ) Odor threshold Minimum concentration for DETECTION (I smell something) RECOGNITION (I smell lemon) Aroma Compound Analysis 1. EXTRACTION 2. SEPARATION into single components (by gas-chromatography) 3. IDENTIFICATION chemical structure (by mass spectrometry) smell (by GC-sniffing) 4. QUANTIFICATION 28

Extraction of Aroma Compounds Handle 10 ml Sample Vial Syringe Needle Fiber green coffee SPME (Solid Phase Micro Extraction) Liquid liquid techniques Solid phase techniques GC-MS Olfactometry 29

GC-MS Olfactometry Separation & Identification 30

Aroma Defects in Coffee methyl 2-methylbutanoate and methyl 3-methylbutanoate 3-isopropyl-2- methoxypyrazine (E)-β-damascenone 2-methoxy-5-vinylphenol 2-methoxy-4-vinylphenol Scheidig et al., J. Agric. Food Chem. 2007, 55, 5768-5775 Immature green beans compared to normal Light Immature Normal Beans Dark Immature Isovaleraldehyde Terpinen-4-ol 120 Phenylethanol 2-heptanol 100 t-2-nonenal Linalool β-damascenone 2-ethyl-3,5-dimethylpyrazine 2-ethyl-3,6-dimethylpyrazine 80 60 40 20 0-20 Nonanal t-2-hexenal Ethyl-2- methylbutanoate Ethylisovalerate Trimethylypyrazine Ethyltiglate 2,4-decadienal Ethylhexanoate E,E-2,4-nonadienal Phenylacetaldehyde 1-octen-3-ol Hexanal 1-octen-3-one Methional 31

Off-Flavor Compounds in Coffee Stinker Beans Rioy Beans S S CH H 3 C CH3 3 H 3 C S Cl Cl beans Cl OCH 3 Cl Dimethyl sulfide Dimethyl disulfide 2,4,6-Trichloroanisole Cl CH 3 Cl Mouldy Beans H 3 C CH 3 Geosmin CH3 Earthy Beans N CH 3 C H 3 CH 3 2-Methylisoborneol Peasy Beans N CH 3 OCH3 2-Isopropyl-3-methoxypyrazine A. Illy & R. Viani, Espresso Coffee, 1995 Chemical Toxins: Acrylamide O NH 2 J. Agric. Food Chem. 2008, 56, 6081 6086 32

Chemical Toxins: Acrylamide O NH 2 Top Eight Foods by Acrylamide Per Portion Food AA concentration (μg/kg) Portion size (g) AA per portion (µg) French fries (OB) 698 70 48.8 Prune juice 214 140 30.0 French fries (RF) 404 70 28.3 Postum 93 240 22.3 Potato chips 598 30 17.9 Breakfast cereal 119 55 6.6 Canned black olives 243 15 3.6 Brewed coffee 7.8 240 1.9 "no observed adverse effect level" (NOAEL) for acrylamide neuropathy: 0.5 mg/kg body weight/day FAO, 2002 Font: FDA, 2006 Chemical Toxins: Furan O 2-7 mg/kg 5 µg/cup a furan dose of 30 µg/kg bw was proposed as the no-observed adverse effect level for hepatic toxicity Toxicol Pathol 2010, 38:619-630 33

Chemical Toxins: PAH Polycyclic Aromatic Hydrocarbons potentially carcinogenic, B[a]P eq, in coffee brews (moka) range from 0.008 to 0.060 µg/l A maximum level of 5 µg/kg in smoked fish and meat (Commission Regulation 2005/208/EC) coffee contributes with very insignificant quantities to the daily human intake Food and Chemical Toxicology 47 (2009) 819 826 The perfect espresso Dose 7.0 ± 0.5 g Water temperature 90 ± 2 C Extraction pressure 9.0 ± 0.5 atm Percolation time 30 ± 5 s Volume 25 ± 5 ml No other drink manages to convey such a pleasant, unforgettable taste and flavour at the moment of drinking, and for 30 minutes thereafter, as a good espresso does Ernesto Illy (1925 2008) 34