COFFEE BREW MELANOIDINS Structural and Functional Properties of Brown-Colored Coffee Compounds E. Koen Bekedam i Promotoren: Prof.dr. Gerrit Smit Hoogleraar Molecular Flavour Science Wageningen Universiteit Prof.dr.ir. Martinus A.J.S. van Boekel Hoogleraar Productontwerpen en kwaliteitskunde Wageningen Universiteit Co-promotor: Dr. Henk A. Schols Universitair hoofddocent bij de leerstoelgroep Levensmiddelenchemie Wageningen Universiteit Promotiecomissie: Prof.dr. Sacco C. de Vries Wageningen Universiteit Dr. Hans J. Bosma Douwe Egberts / Sara Lee Nederland, Utrecht Prof.dr. Lothar W. Kroh Technische Universität Berlin, Germany Prof.dr. Manuel A. Coimbra Universidade de Aveiro, Portugal Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG (Voeding, Levensmiddelentechnologie, Agrobiotechnologie en Gezondheid). ii COFFEE BREW MELANOIDINS Structural and Functional Properties of Brown-Colored Coffee Compounds E. Koen Bekedam Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof.dr. M.J. Kropff in het openbaar te verdedigen op vrijdag 5 september 2008 des namiddags te vier uur in de Aula. iii Bekedam, E. Koen COFFEE BREW MELANOIDINS Structural and Functional Properties of Brown-Colored Coffee Compounds Ph.D. thesis Wageningen University, The Netherlands, 2008 ISBN: 978–90–8504–951–7 iv Abstract The aim of the work presented in this thesis was the identification of structural and functional properties of coffee brew melanoidins, and their formation mechanisms, that are formed upon roasting of coffee beans. To this end, coffee brew was fractionated on the basis of e.g. molecular weight, charge, and hydrophobicity. The composition of the isolated coffee brew melanoidin populations was determined using a wide range of techniques leading to an improved insight in the structural and functional properties of coffee brew melanoidins. A new parameter, Kmix 405nm , was introduced that allowed the quantification of the melanoidin level in coffee brew fractions. The determined melanoidin levels correlated with both the protein contents and the nonprotein-nitrogen contents, from which it was concluded that proteins become part of melanoidin structures upon roasting. Additionally, it was found that intact chlorogenic acids are incorporated into melanoidin structures via the phenolic acid moiety through nonester-bonds. The extent of chlorogenic acid incorporation correlated with the melanoidin level, indicating that phenolic oxidation contributes to the brown color as well as Maillard reactions. Another finding was that coffee brew melanoidins were shown to expose negative charges at the pH of coffee. Furthermore, arabinogalactan proteins (AGPs) were found to participate in melanoidin formation upon roasting. A ‘pure’ AGP-melanoidin population could be isolated from coffee brew due to the high specificity of the reagent used for AGP precipitation. Characterization of low molecular weight melanoidins provided strong indications that sucrose is involved in the formation of melanoidins too. Electron spin resonance studies revealed that roasting leads to formation of antioxidative structures in coffee brew melanoidins. This should be due to the formation of novel roasting-induced antioxidative structures and due to the incorporation of chlorogenic acids in melanoidins. Investigation of the effect of the degree of roast on coffee brew melanoidins properties confirmed that proteins and chlorogenic acids are primarily involved in melanoidin formation. Furthermore, arabinogalactans seem to be more involved in melanoidin formation than galactomannans. Additionally, it was found that prolonged roasting especially led to accumulation of HMw coffee brew melanoidins. Finally, a scheme that describes melanoidin-related formation pathways for coffee beans compounds was introduced. The reaction pathways involved are explained in detail per coffee bean compound. KEYWORDS Coffee; brew; melanoidins; Maillard reaction; phenol oxidation; degree of roast; formation mechanisms; arabinogalactan proteins; chlorogenic acid incorporation; antioxidant; charge v vi Table of Contents Chapter 1 General Introduction 1 Chapter 2 High Molecular Weight Melanoidins from Coffee Brew 23 Chapter 3 Arabinogalactan Proteins Are Incorporated in Negatively 43 Charged Coffee Brew Melanoidins Chapter 4 Incorporation of Chlorogenic Acids in Coffee Brew 61 Melanoidins Chapter 5 Low Molecular Weight Melanoidins in Coffee Brew 79 Chapter 6 ESR-Studies on the Formation of Roasting-Induced 95 Antioxidative Structures in Coffee Brews at Different Degrees of Roast Chapter 7 Roasting Effects on Formation Mechanisms of Coffee Brew 113 Melanoidins Chapter 8 General Discussion 133 Summary 147 Samenvatting 151 Dankwoord 157 Curriculum Vitae 159 List of Publications 161 Overview of Completed PhD Training Activities 163 vii viii CHAPTER 1 General Introduction ABSTRACT In this chapter, an introduction into the topic of coffee brew melanoidins is given, starting with brief information on coffee growing and processing. The composition of green beans, and the compositional changes that occur during roasting are discussed. The characteristics of coffee bean carbohydrates, proteins, and chlorogenic acids are discussed in further detail. Subsequently, the Maillard reaction is reviewed with special attention for the non-volatile, macromolecular, nitrogenous, and brown-colored compounds that are formed during roasting, the melanoidins. Studies dealing with melanoidins are reviewed while making a distinction between melanoidins from model systems and real food systems. Knowledge reported in the literature on coffee brew melanoidins is summarized with special attention for the chemical and functional characteristics. The last part of this chapter describes why research on coffee brew melanoidins is needed and why research on coffee melanoidins is expected to provide valuable information for both the scientific world as well as for coffee producing industries. Finally, the aim and outline of this thesis are defined. 1 Chapter 1 COFFEE These days, coffee is reckoned to be the most widely traded commodity in the world after oil. In 2005, the worldwide coffee bean production was more than seven million tons (1). The cultivation of coffee beans is gradually spread across the world, with Brazil as the world’s largest producer (1). The 2 species that are of commercial importance are Coffea arabica and Coffea canephora, which are known under their trade names Arabica and Robusta coffee beans, respectively. Coffee beans as we know them are actually the seeds of bright red cherries of the coffee plant (Figure 1A). After harvesting of the cherries, the coffee cherries are ‘wet’ or ‘dry’ processed resulting in cherries that have a moisture content of about 12%. Subsequently, the outer casing is removed, yielding the green beans (Figure 1B). After shipment to coffee processing industries, green beans are roasted till the desired degree of roast (Figure 1C). After grinding, the last step is the infusion of the ground and roasted coffee beans with water yielding the well known cup of coffee (Figure 1D). Detailed information on growing of coffee and coffee processing can be found in the literature (2, 3). Due to the enormous size of the coffee market, much research has been devoted to this product. Much research focused on growing, roasting, infusion techniques, and consumer perception (3). Additionally, the chemical composition and functional properties of coffee beans and brew were investigated thoroughly. The chemical composition of green and roasted coffee beans and changes induced by roasting will be discussed below and references to detailed reviews on each subtopic will be given. A B C D Figure 1. Pictures of (A) coffee cherries, (B) green beans, (C) roasted beans, and (D) a cup of coffee brew. GREEN BEANS The most abundant components in green coffee beans are carbohydrates, proteins, lipids, and chlorogenic acids as can be seen from the compositional data for green Arabica coffee beans (Table 1). This compilation provides a resume of the data available and, although these were produced in 1975, only relatively few additional data have been produced since (2, 3). These major green coffee bean components will be discussed in detail below except for 2 General Introduction Table 1. Compositional Data (% of Dry Matter) for Green and Roasted Arabica Coffee Beansa Green Beans Roasted Beans Minerals 3.0 – 4.2 3.5– 4.5 Caffeine 0.9 – 1.2 ~ 1.0 Trigonelline 1.0 – 1.2 0.5 – 1.0 Lipids 12.0 – 18.0 14.5 – 20.0 Chlorogenic acids 5.5 – 8.0 1.2 – 2.3 Aliphatic acids 1.5 – 2.0 1.0 – 1.5 Oligosaccharides 6.0 – 8.0 0.0 – 3.5 Polysaccharides 50.0 – 55.0 24.0 – 39.0 Amino acids 2.0 – Proteins 11.0 – 13.0 13.0 – 15.0 Melanoidins – 16.0 – 17.0 a Data were extracted from Clarke and Macrea (2) and were originally produced by Clifford (4). lipids since I) no papers were found that reported on the involvement of lipids on coffee brew melanoidins formation, and II) lipids are not extracted into coffee brew due to their hydrophobic properties. We refer to the literature for elaborate information on coffee lipids (3) and lipid involvement in food browning (5). Chlorogenic Acids in Green Coffee Beans Chlorogenic acids (CGAs) are the main phenolic compounds in coffee beans and the CGA content might be as high as 8% in green beans (Table 1). Several beneficial health effects have been attributed to CGAs which may be largely explained by their potent antioxidant activities (6).