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C H A P T E R

20
Melanoidins in Coffee
José A. Rufián-Henares, Silvia Pastoriza
Department of Nutrition and Food Sciences, School of Pharmacy, University of Granada, Campus Cartuja S/N,
Granada, Spain

List of Abbreviations because of both the large diversity of structures that couple
to build the different melanoidin fractions and the difficulty
MR  Maillard reaction
HMW  High-molecular weight in isolating and characterizing such fractions. A first step
LMW  Low-molecular weight in the elucidation of coffee melanoidin structure relies on
GMNs Galactomannans estimating their molecular weight. This has been done by
AGLs Arabinogalactans different separation techniques that isolate melanoidins by
AGPs  Arabinogalactan proteins
using their heavy molecular weight. Among these, ultra-
MRPs  Maillard reaction products
filtration, diafiltration, and dialysis are the most suitable
for isolating melanoidins, using membranes with different
molecular weight cut offs, ranging from 3 to 100 kDa.8–10
20.1 INTRODUCTION Although no general consensus regarding the real molecu-
lar weight of coffee melanoidins exists, it is more accepted
Coffee, one of the most consumed drinks in the world that they have a mass ranging from 3 to 22 kDa.8 In addi-
and with a distinct aroma and flavor, is prepared from the tion, these melanoidins could be linked in a noncovalent
roasted seeds of the Coffea plant.1 Different compounds manner to build a small fraction with a molecular mass
found in green coffee beans are degraded/transformed higher than 100 kDa. Other authors also state that in addi-
to some extent, giving rise to a decrease of the amount of tion to the HMW fraction, a low-molecular-weight (LMW)
carbohydrates and chlorogenic acids and the appearance fraction is also found.9 According to this article, 69% of
of melanoidins.2 Melanoidins are a heterogeneous group coffee melanoidins has a molecular weight higher than
of nitrogen-containing brown polymers produced during 12 kDa and 29% are LMW melanoidins. In addition, the
the last steps of the Maillard reaction (MR).3 Coffee mela- increasing formation of HMW melanoidins as a response
noidins are responsible for coffee’s characteristic brown to a severe roasting process has been demonstrated.11
color because they can absorb light at high wavelengths A second purification step relies on the different physi-
such as 420 nm.4 This absorbance is useful in evaluating cochemical properties of the HMW fraction previously
the melanoidins content in coffee brew,5 although other isolated. For example, depending on their hydrophobic-
methods, such as quantification by difference, also are ity, other LMW fractions were obtained using solid phase
possible.6 In the case of roasted coffee beans, the melanoi- extraction.9 A different approach uses the metal chelating
din content accounts for up to 25%,2 although this content ability of coffee melanoidins, separating the HMW com-
increases with stronger roasting processes.7 This chapter plex into fractions with different copper-chelating strengths
focuses on the structure and composition of melanoidins using copper affinity chromatography.12 Related to this,
and the mechanisms of their formation. because of the anionic behavior of coffee melanoidins, they
can also be isolated by anion exchange chromatography.7
In the case of melanoidin solubility, ethanol partitioning
20.2  CHEMICAL COMPOSITION can be used to obtain fractions with different polysaccha-
ride composition.13 At ethanol concentrations of 40–50%,
As stated above, coffee melanoidins are considered to galactomannans (GMNs) precipitate, whereas arabinoga-
be high-molecular-weight (HMW) brownish compounds. lactans (AGLs) precipitate at 75–80%; the remaining solu-
However, their chemical structure is not fully understood ble compounds have the lowest carbohydrate content.

Coffee in Health and Disease Prevention 183


http://dx.doi.org/10.1016/B978-0-12-409517-5.00020-6 © 2015 Elsevier Inc. All rights reserved.
184 20.  COFFEE MELANOIDINS

Regarding the physicochemical properties of coffee 80%.7,8 The proteins linked to melanoidins have a high
melanoidins, their solubility depends on the size of the concentration of aspartic and glutamic acids, as well as
polymer and its chemical nature. In coffee, melanoi- alanine and glycine, whereas the amounts of histidine,
dins are quite hydrophilic because of their intermedi- lysine, methionine, tyrosine, and arginine are lower than
ate molecular weight (3–33 kDa; see Table 20.1 for more those in green coffee proteins.16 This could be explained
information). However, in other foods with high protein by taking into account the protein degradation that
content, carbohydrates react through the MR to build a occurs as a consequence of the roasting process and the
cross-linked HMW network that is insoluble.3 In addi- extent of the MR. Then, some MR products derived from
tion, the LMW fraction of coffee melanoidins has a these amino acids, such a Nε-(fructosyl)lysine or Nε-
hydrophobic nature.9 Another interesting property of (carboxymethyl)lysine, also are included in the melanoi-
melanoidins is their polyanionic behavior.14 Coffee mel- din structure.12
anoidins are negatively charged, but the distribution of The amount of phenolic compounds in green coffee is
such charges is heterogeneous15 in that the HMW frac- high (up to 10%), so it is clear that some of them can
tion is more charged than the LMW one.9 According to be incorporated within the melanoidin structure. The
this, almost half of the anionic fractions have copper- main part of phenolic compounds in coffee is chlo-
chelating properties.12 In the case of coffee melanoidins, rogenic acids, which includes different acids such as
the source of these negative charges seems to rely on the caffeic, coumaric, or ferulic acid.19 Less than 1% of
presence of chlorogenic acids,16 although melanoidins the chlorogenic acids found in green coffee are incor-
from other foods without chlorogenic acids also show porated into melanoidins, although during roasting
such anionic behavior.14 Coffee melanoidins consists of the phenolics linked to melanoidins increase to up to
a mixture of carbohydrates, amino acids, and phenolic 29% of the overall phenolic compounds found in the
compounds that interact during coffee roasting. In the coffee brew.20 It is noteworthy that these chlorogenic
case of polysaccharides, the predominant species in green acids incorporated during the first stages of the pro-
coffee beans are GMNs and AGLs, which comprise up cess are later degraded. These acids have been demon-
to 50% of the bean’s dry weight.2 AGLs are covalently strated to be noncovalently linked to the melanoidin
linked to proteins in both green and roasted coffee17 in skeleton in soluble coffee21 through hydrogen, hydro-
the form of arabinogalactan-proteins (AGPs), and differ- phobic, or ionic bonding and can be released during
ent melanoidin fractions also contain 50% of brownish gastrointestinal digestion,22 which allows their possible
AGPs.15 Both GMNs and AGLs undergo different chemi- absorption at the intestinal level. In addition, the pres-
cal (caramelization, MR, oxidation, etc.) and structural ence of phenolic compounds covalently linked to the
modifications (e.g., AGL debranching and GMN polym- melanoidin core has been speculated using the Folin-
erization) because of the thermal treatment that occurs Ciocalteu reagent15 and the detection of phenols by
during the roasting process.13,18 These polysaccharides thermal degradation of coffee melanoidins.23,24 How-
are covalently linked to the different melanoidin popu- ever, the presence of chlorogenic acids in the melanoi-
lations,7,8,12 although the exact types of linkages is yet din structure was confirmed by alkaline fusion, which
unknown (for a more detailed explanation see Ref. 2). released different chemical species derived from caffeic
Proteins are especially rich in the melanoidin fraction and ferulic acids.12 The linking site of such phenols is
with a higher solubility at an ethanol concentration of still being studied, although it has been proposed that

TABLE 20.1  Physicochemical Properties of Melanoidins


Foods Containing Melanoidins Color Intensity Appearance Water Solubility Hygroscopicity

Coffee ++++ Large, iridescent, flat flakes ++++ ++

Chocolate +++ Hardened agglomerate ++ ++++

Balsamic vinegar +++ Short, flat flakes +++ ++

Sweet wine ++++ Short, flat flakes ++ +++

Dark beer +++ Powder +++ ++

Pilsner-style beer ++ Powder +++ ++

Bread crust + Spongy mass + ++

Breakfast cereals + Spongy mass + ++

Biscuits + Spongy mass + +

I.   INTRODUCTORY AND GENERAL TEXT


20.3  Mechanistic Formation and Structure of Coffee Melanoidins 185
chlorogenic acids could be linked to the melanoidin rise to many different biological effects such as antioxi-
skeleton through proteins25 or through the arabinose dant,29 antimicrobial,30 or antihypertensive activities31
residues of GMNs and AGLs.16 The most recent results that depend strongly on fraction’s chemical composition
in the field26 indicate that phenolic compounds interact (Table 20.2), as reviewed elsewhere.2–4
with coffee storage proteins (legumin-like seed stor-
age proteins of the 11S class) thanks to polyphenol oxi-
dase activity. These chlorogenic acids react covalently 20.3  MECHANISTIC FORMATION AND
with lysine at the α chain and undergo oxidation by STRUCTURE OF COFFEE MELANOIDINS
polyphenol oxidase, giving rise to colored compounds
ranging from light yellow to dark green. The develop- The formation of melanoidins is quite complicated
ment of dark pigments because of the presence of poly- in real foods and has been studied using model sys-
phenols also has been seen in melanoidins from other tems. In these systems, reaction time and temperature
foods such as sweet wine or grape syrup.4 dictate that the molecular weight of coffee melanoi-
Other LMW compounds have been found in cof- dins, since pH influences the development of chromo-
fee melanoidins and melanoidins from different foods phores.4 In general, there are three theories about how
and model systems. Some of the LMW compounds HMW melanoidins are formed. The first theory states
attached to melanoidins are odor-active thiols, such that the HMW complexes are formed from LMW inter-
as 2-furfurylthiol, 3-methyl-2-butenthiol, 3-mercapto- mediates coming from the MR (pyrroles, furans, etc.)
3-methylbutyl formate, 2-methyl-3-furanthiol, and that polymerize during the later stages of the reaction.28
methanethiol, that influence the flavor of the coffee As stated in the previous section, the LMW chromo-
brew.27 Others are furans accompanied by pirazines, phores are reaction intermediates, which means that
pyrroles, pyridines, and carbonyl compounds.4 Most of their polymerizing activity depends strongly on the
these compounds are chromogenic, with different colors heating time. A second theory32 proposes that the HMW
such as blue (pyrrolopyrrole dimer), red (pyrrolopyrrole melanoidin complexes are formed by the cross-linking
azepine), and yellow (acetyl pyrimidine and acetyl activity of chromophoric LMW compounds and the
azepinone). These colorants have an extraordinary reactive side chains of several amino acids (lysine,
polymerizing activity, which suggests that they are arginine, cysteine). Different chromophores have been
reaction intermediates for melanoidin formation.28 The isolated through the reaction of α-dicarbonyl com-
involvement of such LMW colored compounds in the pounds, furans, or their nitrogen-containing deriva-
formation of melanoidins is discussed in the next section. tives with arginine, lysine, or casein.4 The nitrogen
All the fractions mentioned above—LMW and HMW atoms of such colored compounds can then be included
proteins, polysaccharides, and phenolic compounds—give within proteins via the furan-derived structures pro-
duced upon the thermal degradation of carbohydrates
TABLE 20.2  Biological Activities of Coffee Melanoidins
or the amino and guanidine groups of amino acids.
The third theory postulates that the melanoidin core is
Biological Activity Active Structure made of sugar derivatives (degradation products such
Prebiotic AGLs and GMNs as α-dicarbonyl compounds, oligosaccharides, and/
or polysaccharides) branched with amino acids.33 The
Anticariogenic Polymeric backbone
main structure of such saccharides remains uncharged
Food preservation Polymeric backbone in low-moisture media, reacting with amino acids at
Fiber-like antioxidant activity AGLs and GMNs their reducing end. In addition, a small part of the
glycosidic bonds is broken, allowing transglycosyl-
Antioxidant activity in coffee HMW and LMW fractions
ation reactions during the first stages of the MR that
brew
increase the branching state of the melanoidin skel-
Plasma antioxidant capacity N-methylpyridinium eton. This is the situation in real foods such as coffee
Toxic compound binding HMW fraction or cocoa, where the MR takes place at low moisture
content and high temperature (>150 °C) in short time
Aroma compound binding AGLs, GMNs, or chlorogenic acids
periods (ranging from minutes to a few hours). On the
Cation binding HMW fraction other hand, α-dicarbonyl compounds react with pro-
Chemopreventive HMW fraction teins via aldolic condensation in high-moisture media
to increase the complexity of the melanoidin back-
Anticarcinogenic HMW fraction
bone.4 This is how melanoidins form in high-moisture
Antihypertensive HMW and LMW fractions foods such as beer or sweet wine, where the MR
AGL, arabinogalactan; GMN, galactomannan; HMW, high-molecular weight; takes place for longer periods (fermentation or stor-
LMW, low-molecular weight. age for days or months) at low temperatures (<50 °C).

I.   INTRODUCTORY AND GENERAL TEXT


186 20.  COFFEE MELANOIDINS

Then, because of the extensive polymerization, food by the authors is that the formation of the melanoidin
melanoidins (especially those coming from liquid itself and the brown compounds during coffee roast-
foods) are richer in HMW compounds than in LMW ing have distinct origins. On one hand, the presence
ones. of soluble sugar in coffee is one of the main factors
The different chemical composition of coffee mela- for the development of LMW brownish compounds
noidins suggests that they can be formed through dif- linked to proteins and polysaccharides. On the other
ferent pathways. In this sense, previously published
articles seem to indicate that the above three theo-
ries may occur during coffee roasting (Figure 20.1).
The presence of volatile compounds, such as furans
and carbonyl compounds, in thermally degraded
coffee melanoidins,23 as well as the formation of the
polymerizing CROSSPY radical during coffee roast-
ŽīĞĞ
ing, reinforce the plausibility of the first theory. In ŵĞůĂŶŽŝĚŝŶƐ
the case of the second and third theories, other stud-
ies have pointed out the presence of different popula-
tions of modified and nonmodified proteins12,16,26 and
polysaccharides7,8,12,15 on the melanoidin backbone
(Figures 20.2 and 20.3). Nunes and coworkers34 FIGURE 20.1  The mechanisms of melanoidin formation in
recently studied these several formation pathways coffee. Adapted with permission from Nunes and Coimbra.25 Copyright©
through a novel “in bean” model. The first idea stated 2010, Springer.

FIGURE 20.2  Relevance of proteins and phenolic compounds in the formation of coffee melanoidins. Adapted with permission from Nunes
and Coimbra.25 Copyright© 2010, Springer.

FIGURE 20.3  Relevance of arabinogalactan proteins and galactomannans in the formation of coffee melanoidins. Adapted with permission
from Nunes and Coimbra.25 Copyright© 2010, Springer.

I.   INTRODUCTORY AND GENERAL TEXT


20.5 Summary Points 187

ŵĂƚĞƌŝĂů

ŽīĞĞ
ŵĞůĂŶŽŝĚŝŶƐ

FIGURE 20.4  Main chemical species of coffee melanoidins. Adapted with permission from Nunes and Coimbra.25 Copyright© 2010, Springer.

hand, sugars such as sucrose do not increase the final Therefore, the knowledge about the amount and forma-
amount of HMW melanoidins. In the same way, AGPs tion of melanoidins in different foodstuffs will allow the
were the other source (in addition to chlorogenic acid) formulation of new foods with improved sensorial or
of brown compounds, in accordance with other arti- technical and biological activities.
cles.15 This could be related to the reaction of the arab-
inose side chains with reducing sugars upon roasting.
Finally, Nunes and coworkers also found that the cell 20.5  SUMMARY POINTS
wall material is responsible for generating a large
amount of melanoidins. Then, the roasting-induced • C offee melanoidins are brown nitrogen-containing
pyrolysis of cell wall polysaccharides such as GMNs polymers produced during coffee roasting.
accounts for 47% of the melanoidin formed. All the • The amount of coffee melanoidins accounts for up
chemical species responsible for the formation of cof- to 25% of the roasted coffee and increases with the
fee melanoidins are depicted in Figure 20.4. severity of roasting.
• Coffee melanoidins are composed of different
chemical species such as GMNs, AGLs, proteins,
20.4  FINAL REMARKS chlorogenic acids, and MR products.
• The formation of melanoidins is explained by three
In the past decade the elucidation of melanoidins’ different theories involving the polymerization of
structure, composition, and formation pathways cen- LMW MR products, cross-linking activity of LMW
tered the interest of many research groups worldwide. compounds over proteins, and/or the reaction of
The European COST Action 919 “Melanoidins in food polysaccharides with proteins.
and health” was the driving force behind melanoidin • Coffee melanoidins are generated by these three
research in Europe. In the past 10 years research activity mechanisms, including the additional reaction of
moved toward the biological activity of these polymers. phenolic compounds.

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188 20.  COFFEE MELANOIDINS

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I.   INTRODUCTORY AND GENERAL TEXT