Critical Reviews in Food Science and Nutrition

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Overview on the mechanisms of coffee

germination and fermentation and their
significance for coffee and coffee beverage quality

Deborah M. Waters, Elke K. Arendt & Alice V. Moroni

To cite this article: Deborah M. Waters, Elke K. Arendt & Alice V. Moroni (2017) Overview on
the mechanisms of coffee germination and fermentation and their significance for coffee and
coffee beverage quality, Critical Reviews in Food Science and Nutrition, 57:2, 259-274, DOI:
10.1080/10408398.2014.902804

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CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
2017, VOL. 57, NO. 2, 259–274
http://dx.doi.org/10.1080/10408398.2014.902804

Overview on the mechanisms of coffee germination and fermentation and their

significance for coffee and coffee beverage quality
Deborah M. Watersa, Elke K. Arendta, and Alice V. Moronib
a
School of Food and Nutritional Sciences, University College Cork, Western Road, Cork, Ireland; bDepartment of Food Science and Technology, Nestl
e
Research Centre, Vers-Chez-Les-Blanc, Lausanne, Switzerland

ABSTRACT KEYWORDS
Quality of coffee is a complex trait and is influenced by physical and sensory parameters. A complex Enzymes; fermentation;
succession of transformations during the processing of seeds to roasted coffee will inevitably influence germination; green coffee;
the in-cup attributes of coffee. Germination and fermentation of the beans are two bioprocesses that take microbiota; polysaccharides
place during post-harvest treatment, and may lead to significant modifications of coffee attributes. The
aim of this review is to address the current knowledge of dynamics of these two processes and their
significance for bean modifications and coffee quality. The first part of this review gives an overview of
coffee germination and its influence on coffee chemistry and quality. The germination process initiates
while these non-orthodox seeds are still inside the cherry. This process is asynchronous and the evolution
of germination depends on how the beans are processed. A range of metabolic reactions takes place
during germination and can influence the carbohydrate, protein, and lipid composition of the beans. The
second part of this review focuses on the microbiota associated with the beans during post-harvesting,
exploring its effects on coffee quality and safety. The microbiota associated with the coffee cherries and
beans comprise several bacterial, yeast, and fungal species and affects the processing from cherries to
coffee beans. Indigenous bacteria and yeasts play a role in the degradation of pulp/mucilage, and their
metabolism can affect the sensory attributes of coffee. On the other hand, the fungal population occurring
during post-harvest and storage negatively affects coffee quality, especially regarding spoilage, off-tastes,
and mycotoxin production.

Introduction
Coffee represents one of the most important crops in the world
and is the most widely traded tropical commodity (Coltro
et al., Ico Trade). The majority of coffee plants are from the
Rubiaceae family and genus coffea. There are around 100 spe-
cies, and the two most economic and of industrial importance
are cultivated for the production of coffee beverages. Coffea
arabica and Coffea canephora (robusta), respectively, represent
about 70% and 30% of the total coffee traded in the world, with
Brazil providing 30% of the total world market share (2006; de
Castro and Marraccini, 2006). C. arabica produces a better
quality coffee beverage than C. canephora, which is often used
as a blend with the former (de Castro and Marraccini, 2006). C.
arabica is an allotetraploid (four chromosomes) and mostly
autogamous (self-fertilizing) tree. In contrast, C. canephora is
diploid and may be a progenitor of C. arabica (Clarindo and
Carvalho, 2009). The life cycle of the coffee tree (Coffea sp.) has
been described using the extended Biologische Bundesantalt,
Bundessortenamt, and CHemische scale (BBCH Industrie,
Germany) (Arcila-Pulgar
ın et al., 2002). The BBCH scale does
not describe the true timing of coffee seed development in the
field; however, it does accurately illustrate its growth stages
(Arcila-Pulgar
ın et al., 2002). Mature coffee fruit is a red or yel-
low drupe with a pulpy mucilaginous flesh surrounding a
lignified endocarp (Figure 1), comprising two half-elliptical-
shaped seeds with a longitudinal furrow on their surface
(Dedecca, 1957). The endosperm is enveloped by the pericarp
and is made of three distinct layers: the outermost exocarp, the
middle mesocarp, and the innermost endocarp (parchment),
which is in contact with the silver skin containing the embryo
(Figure 1; Krug and Carvalho, 1939; Mendes, 1941). A number
of current reviews exist on the detailed physiology and cytology
of green coffee beans (de Castro and Marraccini, 2006; Eira
et al., 2006; DaMatta et al., 2007).
Apart from the variety and origins, coffee quality is influ-
enced by harvesting, post-harvest processing, transportation,
and storage. The mode of post-harvesting is believed to have
the biggest impact on the characteristics of the final product,
and efforts have been made to ameliorate/optimize the practi-
ces used for these processes, i.e. wet, dry, and semi-dry pro-
cesses (Schwan et al., 2012). It remains unclear how specific
processing steps affects coffee biochemistry and the final coffee
quality. Recent studies have shown that identical coffee samples
treated by wet and dry processes showed different qualities and
compositions (Selmar et al., 2012; Knopp et al., 2006). Thus, it
was suggested that changes in the composition of the beans due
to activation of their metabolism, i.e. germination, might have
caused such differences (Selmar et al., 2006). Coffee bean

CONTACT Elke K. Arendt e.arendt@ucc.ie School of Food and Nutritional Sciences, University College Cork, Western Road, Cork, Ireland.
Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/bfsn.
© 2017 Taylor & Francis Group, LLC
260 D. M. WATERS ET AL.
metabolism might not be the only one responsible for inherent
quality changes in the beans. In fact, the microbiota occurring
during fermentation in wet, dry, and semi-dry processes has
been suggested to have a significant impact on the properties of
the resulting coffee (Schwan et al., 2012; Evangelista et al., in
press). Germination can commence in the early stages of post-
harvesting and can induce a series of metabolic events that
influence the composition of coffee beans. At the same time, all
the post-harvest processes described in the literature are
accompanied by the evolution of a rather heterogeneous micro-
biota comprising bacteria, yeast, and filamentous fungi. The
interaction with the coffee substrate and the impact of bacteria
and yeast has not been clearly addressed, whereas moulds are
generally associated with spoilage and production of mycotox-
ins that are detrimental to coffee quality (Silva et al., 2000;
Food and Agriculture Organization (FAO), 2006; Taniwaki,
2006; Noonim et al., 2008; Velmourougane and Bhat, 2009).
The objectives of this paper are two-fold: (i) To review the
current knowledge on the biochemical modifications occurring
in the beans during germination and their influence on coffee
quality, and (ii) to address the incidence and role of coffee-
associated microbiota during fermentation and storage, and its
impact on quality of the final product.
Coffee seed: Dormancy and germination process
Coffee seeds are half-elliptical egg-shaped with longitudinal
furrow on the flat surface, covered by silver skin (Figures 1–3).
The endocarp surrounds the endosperm, which is a living tissue
with polyhedral cells, divided in hard external and soft internal
endosperm. The embryo comprises an axis and two cotyledons
and lies in the embryo cavity, enclosed by the endosperm cap
comprising compressed cells (da Silva et al., 2005, 2008). Rect-
angular cells are located in the close proximity of embryo and
the polygonal ones make up most of the rest of the endosperm.
The endosperm cell walls comprise cellulose and hemicellulose,
whereas galactomannas represent around 25% of the mass of
green mature grain, and also contain proteins, lipids, and min-
erals (da Silva et al., 2005; Pr
e et al., 2008; Rosa et al., 2010).
Coffee beans are neither orthodox, i.e. going through a des-
iccation period and metabolic dormancy before germination,
nor recalcitrant, i.e. having a high water content at the end of
maturity with germination commencing immediately. Instead,
they have been categorized into a new group between those of
orthodox and recalcitrant seeds. They are intermediate (non-
orthodox) seeds, and low-temperature damage to dry seeds is a
Figure 1. Two elliptical-shaped coffee beans present inside the mature coffee fruit
cherry (220–250 days after flowering). (de Castro and Marraccini, 2006).
notable feature (Roberts, 1973; Ellis et al., 1990). Another char-
acteristic of intermediate seed dormancy is defined as a block
in the progression of germination to a state of completion
under favorable conditions, which has evolved differently
across species to allow adjustment to the established environ-
ment (Hilhorst, 1995). As a member of the Rubiaceae family
and containing a spatulate axile embryo (Figure 2), coffee spe-
cies are regarded as being physiologically dormant (Baskin and
Baskin, 2004; Finch-Savage and Leubner-Metzger, 2006). This
state of arrest occurs within the fruit, likely by the joint effects
of abscisic acid (ABA) and the osmotic potential of fruit flesh
(Bytof et al., 2000). Release from dormancy leads to germina-
tion (Bytof et al., 2000; Finch-Savage and Leubner-Metzger,
2006). Eight embryo growth stages have been identified based
on the changes from imbibition to germination, and later from
germinated seed to seedling growth (da Rosa et al., 2010). The
first stage of germination commences with seed imbibition (up
to three days), followed by the appearance of a visible protuber-
ance in the endosperm (around day 5). Germination in sensu
strictu is completed with the protrusion of radicle through the
outer layer of endosperm (Figure 4; da Rosa et al., 2010). Before
radicle protrusion, changes in the metabolism occur in the
seeds. Such changes include protein hydration, structural
changes, respiration, and cell elongation (Selmar et al., 2006).
Two phases of endosperm weakening have been described (da
Silva et al., 2004). During the first phase, a proportion of water
from the thick cell walls of endosperm is gradually absorbed by
the embryo, which increases its pressure potential (da Silva
et al., 2004, 2008). Gradually, the endosperm structure is weak-
ened and cell walls of the endosperm are degraded. During the
second phase, the pressure potential from the embryo is
released and a protuberance occurs. The cell wall porosity in
the endosperm cap has increased, the compressed cells in the
endosperm cap have lost their integrity, and more water is
allowed to the embryo at this point. The observation of the
hydration status of the cells revealed that the thick wall cells
were less hydrated than the endosperm cap cells after five days
of imbibition, indicating a controlled water uptake mechanism
(da Silva et al., 2008). Alongside cell wall degradation, the cell
wall extensibility of the embryo increases, and an increase in
the length of cotyledons and embryonic axes was observed in
imbibed seeds (da Silva et al., 2004, 2008). Changes in plant
growth regulators and enzymatic activities are associated with
enzyme induction (Giorgini and Comoli, 1996; da Silva et al.,
2004, 2005). An increase in cellulase and mannanase activities
was shown to coincide with increased endosperm cell porosity
and decrease in puncture force during imbibition (da Silva
et al., 2004). Restricted enzymatic hydrolysis of the cells sur-
rounding the embryo creates space to increase the flexibility
and plasticity needed for embryo expansion and later for
reserve mobilization (da Silva et al., 2005, 2008). The accumula-
tion of solutes generated from cell wall hydrolysis could also
play a role in water uptake control (da Silva et al., 2008). The
hydrolytic enzyme activities plateau as the protuberance
enlarges, and increase again before the endosperm is punc-
tured, allowing the radicle to emerge through the endosperm
cap (Eira et al., 2006). Consequently, the degradation of lateral
endosperm commences, thereby mobilizing the seed reserves to
facilitate the growing embryo (da Silva, 2004). The germination

Figure 2. Images of fresh coffee bean: (A) husk, (B) de-hulled coffee bean with the silverskin intact, and (C) cross-cut coffee bean. (Unpublished pictures, University Col-
lege Cork).
process can be accelerated in vitro by removing the bean husk
(endocarp), which is severely inhibitory (Valio, 1976; Ellis
et al., 1990; Arcila-Pulgar
ın et al., 2002).
Light, temperature, and moisture influence the germination
capacity of the beans and the course of germination (Moraes,
1963; Valio, 1976; Resende et al., 2009; L€aderach et al., 2011).
Storage and moisture content greatly influence the germination
capacity of the beans. Different coffee species illustrate con-
trasting levels of desiccation sensitivity, which is related to their
ecological origins. This is shown by Arabica coffee, which has
intermediate seed storage behavior and is native to the dry and
cool regions of Ethiopia, while C. liberica (not of industrial
importance) shows more recalcitrant seed storage behavior and
is native to the hotter and more humid regions of Liberia
(Hong and Ellis, 1995). Optimum storage conditions of 10–
11% water content and approximately 10
C extend the viability
of green beans from Arabica coffee (Hong and Ellis, 1992). It
was also reported in the same study that C. arabica desiccation
sensitivity increases after imbibition for 3–10 days, which insin-
uates that high-moisture processing could play a negative role
in these beans viabilities (Hong and Ellis, 1995). In addition to
C. arabica and C. robusta (Ellis et al., 1990; Hong and Ellis,
1995), other seeds, such as Elaeis guineensis (oil palm seeds;
Ellis et al., 1991a), Carica papaya L. (papaya seeds; Ellis et al.,
1991b), and Zizania palustris (wild rice; Kovach and Bradford,
1992), also show intermediate seed storage behavior.
In addition to environmental conditions, hormones are
involved in the regulation of coffee germination. In general,
there are five main classes of hormones involved in the growth
of all plants: auxins, cytokinins, gibberellins (GAs), ABA, and
ethylene (Davies, 1995; Arteca, 1996). In general, auxins pro-
mote cell growth and division (Darwin and Darwin, 1881),
Figure 3. Scanning electron microscopy visualization of the longitudinal section of green coffee bean at different magnifications. (Unpublished pictures, University Col-
lege Cork).
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 261
cytokinins promote cell division (Haberlandt, 1913; Van Over-
beek et al., 1941), and GAs are involved in cell elongation regu-
lation (Mauseth, 1991; Salisbury and Ross, 1992; Davies, 1995;
Raven et al., 1998). ABA inhibits cell division (Salisbury and
Ross, 1992), and ethylene controls fruit ripening and can
inhibit the growth and development of plant cultures (Mauseth,
1991; Salisbury and Ross, 1992). Previous research into coffee
seed or embryo development has shown how hormones are
involved in the modulation of germination process. da Silva
and co-authors (2005) addressed the mechanisms of promo-
tion and inhibition of germination by GAs. Germination of
coffee seeds was shown to be dependent on the de novo
synthesis of the hormone, as for other species. The endoge-
nous GAs are responsible for both embryo cell elongation
and endosperm cap weakening (da Silva et al., 2005).
Supra-optimal concentrations of GAs can deregulate the
synchronization of germination process, and thus inhibit
germination (Valio, 1976; da Silva et al., 2005). ABA is syn-
thesized de novo during coffee seed imbibition and was
shown to have an important role in controlling seed germi-
nation (da Silva et al., 2004). ABA was suggested to inhibit
coffee embryo growth potential during the first stage of
endosperm weakening and to inhibit endo–mannanase
activities in the second stage (Silva et al., 2004). In a later
study by the same group, ABA was shown to inhibit the
accumulation of b-tubulin, cell growth division and growth,
nuclear replication of DNA, and organization of microtu-
bules during imbibition (da Silva et al., 2008). Kinetin has
been shown to reverse germination inhibition by both exog-
enous GA and ABA (Valio, 1976). Cytokinin-like hormones
were also shown to have a positive effect on C. arabica ger-
mination (Valio, 1976).
262 D. M. WATERS ET AL.

Figure 4. Germination of coffee beans over 45 days after imbibition by water at 30
C. (Unpublished pictures, University College Cork).

Germination and green coffee biochemistry undergo modifications during roasting and impact the in-cup
quality of coffee beverage (Redgwell and Fischer, 2006). There-
Germination can start during post-harvesting and the meta-
fore, it is essential to understand their structural features and
bolic changes induced in the coffee beans can influence the final
the changes occurring during seed development and process-
quality of coffee (Eira et al., 2006; Selmar et al., 2006; Bytof
ing. The content and structural features of cell wall polysac-
et al., 2007). Germination-related reactions are activated during
charides have been addressed in various publications (Wolfrom
post-harvesting, and the extent and nature of these events
et al., 1961; Clifford, 1985; Bradbury and Halliday, 1990;
depend on the processing methods used. Recent studies (Sel-
Fischer et al., 2001; Nunes and Coimbra, 2001, 2002; Ooster-
mar et al., 2006; Bytof et al., 2007) demonstrated that coffee
veld et al., 2004). The total polysaccharide content and struc-
bean metabolism shows a different pattern in wet and dry proc-
ture of the Robusta and Arabica galactomannans are quite
essing by analyzing the expression of the specific germination
similar; however, their distribution and primary structure differ
enzyme, i.e. isocitrate lyase, and b-tubulin. In wet processing,
(Fischer et al., 2001; Nunes and Coimbra, 2001; Redgwell and
the onset of germination commences on the first day of fer-
Fischer, 2006; Arya et al., 2007). Characterization of these poly-
mentation, most likely due to the removal of the coffee fruit
saccharides is rendered difficult due to their insolubility and
flesh and consequent deactivation of suppression factors (Bytof
the compact structure of the cell walls where they are located
et al., 2007). On the contrary, the osmotic potential of the pulp
(Redgwell and Fischer, 2006). Overall, the methodology of
remains for longer during dry processing, as the fruit is initially
intact, and the germination starts only after few days of proc-
essing (Bytof et al., 2007). Table 1. Chemical composition of green coffee beans.
To date, only few investigations concerning the biochemical
C. arabica (% dry C. robusta (% dry
changes occurring during coffee seeds germination are avail- Compounds weight basis) weight basis)
able, and most of such studies are related to dried seeds that are
re-imbibed to induce germination (Selmar, 2006). The complex Caffeine 1.2 0.9 2.2 1.7
Trigonelline 1.0 1.0 0.7 0.9
events that take place during germination will be addressed in Ash 4.2 4.4
the following section with regard to the major coffee compo- Acid components
nents and influence on the quality of coffee (where possible). Chlorogenic 6.5 10.0 5.0–8.0x
acids
Aliphatic acids 1.0 1.0
Quinics 0.4 0.4
Carbohydrate composition and metabolism Sugar/carbohydrate/fiber components
The most abundant storage compounds in coffee are cell-wall Sucrose 8.0 8.4 4.0 6.4 7.0–11.0x
polysaccharides, which represent 48–60% of the bean dry Reducing sugars 0.1 0.4
Polysaccharides 44.0 48.0
weight (Bradbury, 2001). They primarily comprises galacto- Lignin 3.0 3.0
mannans (GM), arabinogalactans (AG), and cellulose (Wolf- Pectin 2.0 2.0
rom and Patin, 1965; Clifford, 1985; Redgwell and Fischer, Proteins 11.0 11.0 11.0–15.0x
Free amino 0.5 0.8
2006). Other major storage compounds in coffee include lipids acids
(10–16%), proteins (»11%), sucrose (4–8%), and chlorogenic Lipids 16.0 10.0 13.0–17.0x
acids (6.5%) (Jo€et et al., 2010; Table 1).
Adapted from Francis (2003).
Carbohydrates in coffee are important not only because they 
Results obtained using LCMS (Perrone et al., 2008).
x
are present in high concentrations but also because they Results adapted from Jo€et et al. (2010).

extraction used to characterize cell wall polysacharrides in cof-
fee can have an effect on the final assessment of their primary
structure and distribution. Frequently, only a part of arabinoga-
lactans and galactomannans were extracted and analyzed in the
published studies. Galactomannans are the most resistant poly-
mers to solubilization, and thus influence the rate of extraction
during production of soluble coffee (Clifford, 1985). They com-
prise 1,4-linked b-mannan chains and are present as an heter-
ogenous mixture of unsubstituted and substituted polymers at
the O-6 position with residues of galactose. The frequency of
this substitution and the physical environment of the polysac-
charides (in mature or young beans) strongly impact the solu-
bility of galactomannans, where greater Gal/Man ratios
enhance solubility due to the disruption of inter-chain hydro-
gen bonds (Redgwell et al., 2003). In mature coffee beans, levels
of substitution for Gal/Man ratio have been reported as varying
from 1:130 (Bradbury and Halliday, 1990) to 1:30 (Fischer
et al., 2001), 3:1, and 9:1 (Oosterveld et al., 2004) and between
1:7 and 1:40 (Redgwell et al., 2003). In the latter study, up to
90% of the cell wall galactomannans could be solubilized prior
to analysis and their solubility was found to be positively corre-
lated with their degree of substitution (Redgwell et al., 2003).
Galactomannans were also reported to be acetylated (Nunes
and Coimbra, 2002; Oosterveld et al., 2004), and the presence
of acetyl groups could influence their solubility. Nunes et al.
(2005) also provided evidence that arabinosyl residues were
present in galactomannans at the O-6 mannose residues.
Higher amount of soluble galactomannans were isolated from
Robusta coffee variety ROM than in Arabica, and the branch-
ing degree in the former was higher (Fischer et al., 2001). Type
II arabinogalactans mainly comprises b-(1!3)-linked galacto-
syl residues, substituted at the O-6 position with arabinosyl and
galactosyl residues (Bradbury et al., 2001). Redgwell and co-
authors (2002) demonstrated that all or some of the arabinoga-
lactans occur in the beans as arabinogalactans–protein com-
plexes in both Arabica and Robusta varieties, and that the
polysaccharides have a negative charge due to the presence of
10% glucuronosyl residues at the non-reducing end of galacto-
syl side chains. Coffee arabinogalactans are widely heteroge-
neous, mainly due to the degree of branching and to the
variable monosaccharide composition of side chains (Redgwell
et al., 2002). Fischer et al. (2001) reported that the Robusta cof-
fee variety ROM contains more soluble arabinogalactans, which
were more branched and possessed longer side chains than
Arabica coffee.
During seed growth and development, the content and
structural features of the polysaccharides progressively change.
To date, changes in the carbohydrate composition and features
during post-harvesting stages have not been fully addressed
(Redgwell and Fischer, 2006). At an early stage of development,
the concentration of free sugars in endosperm and perisperm
increases, whereas little changes are observed in sugar alcohols,
and oligosaccharides do not undergo extensive modifications
(Rogers et al., 1999). At the beginning of seed growth, cellulose
and arabinogalactans are the main polysaccharides, whereas
mannans synthesis progressively increases as the beans
approach maturity (Redgwell and Fischer, 2006). It was shown
that galactomannans in the young beans account only for 10%
of the polysaccharides and they are highly substituted
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 263
(Redgwell et al., 2003). During the development of the beans,
the content of galactomannans progressively increases up to
50% of the total polysaccharides in the mature beans. In paral-
lel, the degree of substitution of galactomannans is decreased,
mainly due to the concomitant increase in a-galactosidase
activity, which liberates galactose in the beans (Redgwell et al.,
2003). Coffee-derived a-D-galactosidase (E.C. 3.2.1.22)
(a-Gal), similar to other galactohydrolases, is able to cleave
a¡1,6 links between galactose units from the N-terminal end
of galactomannans seed storage tissue (Buckeridge and Die-
trich, 1996). In a comprehensive study on the characterization
of a-Gal in coffee beans (Marracini et al., 2005), the authors
showed that its activity is below the limits of detection at an
early stage of bean development. As the growth progressed,
a-Gal was accumulated and its activity increased to reach a
peak in 30 weeks after flowering in the pericarp and especially
in the endosperm (Marraccini et al., 2005). The activation of
the enzyme also coincided to the hardening of endosperm, as
previously suggested by Redgwell and co-authors (2003). Mar-
raccini an co-authors (2005) also showed that a-Gal in coffee
beans is extracellular, which ensures that the high Gal/Man
ratios in newly synthesized galactomannans are maintained
and their solubility is retained. At maturation, the accumulated
a-Gal can remove a-galactosyl residues from mannan chains
and thus contribute to the deposition of insoluble polysacchar-
ides in the cell wall, ensuring endosperm hardening (Marracini
et al., 2005). The Arabica and Robusta varieties analyzed in this
study, Arabica var. Caturra and Robusta var. ROM, showed
a-Gal activities, which were inversely correlated with the degree
of branching of galactomannans, as previously analyzed by
Fischer et al. (2001). Marracini et al. (2005) showed that the
a-Gal activity was very low in imbibed seeds, but increases in
germinating coffee beans, reaching a peak after 40 days of ger-
mination. a-Gal was purified and characterized in germinating
coffee beans by Shen and co-authors (2009). An early study
showed that the content of oligosaccharides, sucrose, raffinose,
and stachyose decreased during soaking and short germination
of Arabica and Robusta beans, together with an increase in
a-galactosidase activity (Shadaksharaswamy and Rachaman-
dra, 1968). However, the results of this study may not be con-
clusive, as the germination process could not be carried out for
longer than 72 h after soaking due to fungal spoilage of the
beans.
Endo-b-mannanases (man) are essential for the metabolism
of cell wall polysaccharides (Marracini et al., 2001). Mannanase
activity was first discovered in coffee beans by Takaki et al.,
(1979). Marraccini and co-authors (2001) cloned and
sequenced two man cDNAs, i.e. manA and manB, from Coffea
Arabica. The two enzymes shared 56% sequence identity, and
the conserved regions were shown to be homologous to other
plants endo-b-mannanases (Marracini et al., 2001). The puri-
fied manB enzyme was active on mananans comprising five or
more mannose units (Marracini et al., 2001). Marracini et al.
(2001) also followed the expression of man-encoding genes
during the germination of coffee beans. A peak of transcripts
was detected in the beans at around 20 days after imbibition,
slightly earlier than the measured peak of enzymatic activity (at
28 days after imbibition). Protrusion of radicle, however, was
observed before the occurrence of these two peaks. This
264 D. M. WATERS ET AL.
suggests that a very low man activity was enough to weaken the
endosperm, and the initial man localization was most likely to
be in the tissue around the embryo (Marraccini et al., 2001). In
a previous work, Giorgini and Comoli (1996) followed the man
activity in germinating beans by indirect measurements, i.e. by
analyzing the capability of the homogenized endosperm tissue
to decrease the viscosity of guar galactomannans solution. In
contrast to the findings of Marracini et al. (2001), the authors
could not identify any man activity in the beans’ endosperm
prior to radicle protrusion (Giorgini and Comoli, 1996). These
results led the authors to conclude that the man activity
becomes significant only after the seeds have completed germi-
nation. However, the methodology used to detect man activity
in Giorgini and Comoli (1996) might have not been adequately
sensitive to detect low man activities. The role of GA3 in the
development of coffee beans also seems contradictory. In an
earlier report, GA3 was shown to stimulate man activity,
whereas the opposite effect was described in a more recent pub-
lication (Giorgini and Comoli, 1996; Marracini et al., 2001).
Mannose has been shown to inhibit ATP synthesis and hexose
metabolism (Herold and Lewis, 1977). However, there are con-
flicting reports about the effect of mannose on germination
inhibition, with studies saying that both may be unrelated (da
Silva et al., 2005) or inter-dependent (Valio, 1976; Takaki et al.,
1979). Cellulases are detected before coffee bean germination
commences, which may indicate a role in endosperm cap soft-
ening before radical protrusion (Takaki et al., 1979). The con-
centration of this enzyme was enhanced upon addition of GA3
during initial coffee bean imbibition (Takaki et al., 1979).
Storage proteins, nitrogenous compounds, and metabolism
Nitrogenous compounds, free amino acids and peptides, are
significant precursors of coffee volatile compounds and influ-
ence coffee aroma. Changes in these compounds during post-
harvesting process can influence the final in-cup quality of cof-
fee. Proteins represent around 10% of the coffee endosperm,
followed by amino acids, caffeine, and trigonelline (Shimizu
and Mazzafera, 2000; Table 1). Albumins and globulins
(according to Osborne classification) have been isolated in cof-
fee, with the latter storage proteins being the most abundant
one. Two homologous (>98% sequence homology) legumin-
like seed storage proteins (11S storage globulin) were first iso-
lated as the main storage proteins from C. arabica endosperm
using acidic aqueous buffers (Acu~na et al., 1999). Immunocyto-
chemistry investigation revealed that the legumin storage pro-
tein is stored in cytoplasm-located vacuoles and accounts for
most of the cell volume (Acu~ na et al., 1999). Under reducing
conditions, the mature 55-kDa precursor form generates two
polypeptides of molecular size of around 24 and 33 kDa. These
subunits can be identified by two-dimensional (2D) profiles of
green coffee proteins (Rogers et al., 1999). Ludwig and co-
workers (2000) analyzed the peptide and relative amino acid
composition of Robusta and Arabica commercial and freshly
harvested coffee samples. The authors observed no significant
differences between the peptide contents of freshly harvested
and commercial beans, but suggested that the composition of
peptides varied between the two sets of samples. Both varieties
harbored peptides in the molecular size range of 8 to 10 kDa,
and few differences in the specific molecular size distribution
could be observed between Arabica and Robusta beans. The
most abundant amino acids were glutamine/glutamic acid, fol-
lowed by glycine, serine, and asparagine (Ludwig et al., 2000).
Murkovic and Derler (2006) reported that the most abundant
amino acids in Arabica and Robusta coffee beans were alanine
and asparagine. The qualitative determination of free amino
acids in Arabica green beans revealed that glutamine/glutamic
acid, and asparagine and aspartic acid were among the most
abundant ones in the matrix (Shimizu and Mazzafera, 2000).
Upon germination, organized enzymatic hydrolysis of these
reserves commences and peptides/amino acids are translocated
for use by the growing embryo (Callis, 1995). The type and
amount of proteins and amino acids present in the beans will
later influence the coffee roasting chemistry. Shimizu and Maz-
zafera (2000) published a comprehensive study addressing pro-
tein changes in germinating coffee beans over a six-week
period. Analysis of the free amino acids in coffee seeds revealed
that the most abundant ones corresponded to glutamine/gluta-
mic acid and glycine followed by asparagine, aspartic acid, ala-
nine, lysine, and serine. Over the germination time, the
qualitative analysis of protein changes by SDS-page revealed
that the staining intensity of the two major bands with the
molecular size of 23.9 and 35.7 kDa, corresponding to the legu-
min-like protein subunits, decreased progressively. On the
other hand, quantitative analysis confirmed an overall decrease
in free amino acids by over 50% during germination, but no
significant changes in the total protein content of the seeds was
detected. In contrast, tyrosine was the only amino acid detected
in increasing levels as germination progressed (Shimizu and
Mazzafera, 2000).
Very limited studies have been published on the characteri-
zation of green coffee proteases/peptidases so far. One research
group isolated a number of endo-peptidases from C. arabica
and C. robusta freshly harvested and commercial green beans
(Ludwig et al., 2000). Five to seven active enzymes were found
in the green beans and their activity did not change from fresh
to harvested beans. This class of enzymes was found to be resis-
tant to serine and pepsin inhibitors but susceptible to inactiva-
tion by heavy metals and iodacetamide (Ludwig et al., 2000).
However, no activity profile or data regarding the roles of
enzymes during germination were hypothesized or proved.
More recently, Lepelley and co-workers (2012) investigated the
presence and expression of cysteine proteases (CP) and cysteine
proteinases inhibitor (CPI) genes in Robusta coffee seeds dur-
ing maturation and germination. Two CP genes, CcCP1 and
CcCP4, were found to be exclusively expressed at these stages.
The former was suggested to be involved in the modifica-
tion of proteins during the late stage of maturation and ger-
mination, whereas CcCP4 was indicated as playing a role in
protein and/or cell remodelling during late germination as
well as in the programmed cell death during post-germina-
tion (Lepelley et al., 2012). Four CPI genes were found to
be expressed in coffee, two of which having specific func-
tions, i.e. modulating CP activity (CcCPI-1 and CcCPI-4)
and protection of coffee seeds from insects and pathogens
(CcCPI-4). This work highlighted the need for further
investigations on possible links between variations in genes
encoding for CP and CPI, and the flavor/aroma associated
with coffee varieties (Lepelley et al., 2012).

Lipids and germination
Lipid metabolism during post-harvesting can affect the quality
features of coffee. The lipid fraction in Arabica and Robusta
green coffee represents between 10% and 17% (dry weight) of
coffee bean solids (Table 1), and are located in the endosperm.
A comprehensive overview on the composition of lipid fraction
in green coffee has been published by Speer and co-workers
(2004). The lipid fraction mainly comprises triacylglycerols
(TGAs), which give a major contribution to aroma in roasted
coffee, esters of diterpene alcohols, and fatty acids. The most
abundant fatty acids in TGAs present as free fatty acids (FFAs)
in coffee beans are C18:2, C16:0, and C18:1. Arabica and Robusta
varieties show similar content of fatty acids, but vary in the rel-
ative stearic and oleic acid proportions (Speer et al., 2004,
1993). Diterpenes in coffee are mainly pentacyclic diterpene
alcohols based on the kauran skeleton. Arabica coffees contain
cafestol and kahweol, and Robusta coffees contain cafestol,
small amounts of kahweol, and 16-O-methylkahweol, which
can be found only in this variety. Tocopherols are also found in
coffee beans, and the predominance a-tocopherol is a key fea-
ture in coffee beans in comparison to its content in fruits and
vegetables.
The content of FFAs and free diterpenes can change during
post-harvesting. For example, during storage, moisture and
temperature regulate the liberation of FFAs (Speer et al., 2004).
Speer and co-authors (2004) postulated the presence of a lipase
activated during storage. A recent paper by Patui and co-work-
ers (2014) sheds a first light on the characterization of lipase
activity in green coffee and its role in lipid degradation during
germination. Coffee seeds, with and without parchment, were
germinated and the lipase activity was detected during germi-
nation using a colorometric method. The authors identified a
protein of 60 kDa, which cross reacted with an anti-lipase anti-
body, and the enzyme was inactivated by the polyclonal anti-
body and tetrahydrolipstatin. A biphasic behavior was
observed in the enzyme’s activity during the first stage of ger-
mination, similar in seeds with or without parchment. How-
ever, at the intermediate stage the lipase activity continued to
increase gradually until the end of the process in seeds with
parchment, whereas in the “naked” seeds there was a sharp
peak of activity, followed by a decrease and delay in germina-
tion. Accordingly, the FFAs accumulated steadily during germi-
nation of coffee seeds with parchment, unlike those without
parchment, where the peak of lipase activity was followed by a
decrease in FFAs. Thus, the authors suggested that the parch-
ment regulates the lipase activity in germinating beans by the
regulation of oxygen permeation and by the inhibitory effect
exerted by chlorogenic acid (Patui et al., 2014).
Overall, the degree of coffee metabolism and the activation
of endogenous enzymes during post-harvesting can be expected
to influence the content and structure of coffee polysaccharides,
proteins, and lipids, and thus influence the final sensory charac-
teristics of green and roasted coffee. However, to date, there are
only a limited number of studies that clearly link specific bio-
chemical changes in the developing bean with specific altera-
tions in the sensory attributes of coffees. Thus, more detailed
work coupling biochemical/chemical analysis of coffees treated
by different post-harvest methods with sensory analysis is
warranted.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 265
Post-harvest processing and fermentation
During post-harvesting, a heterogeneous microbiota compris-
ing bacteria, yeast, and filamentous fungi develops on coffee
cherries and beans. Few scientific reports have addressed the
occurrence of these microbial communities, and although their
relevance for the final coffee quality is uncontested, little is
known on the functionality and importance of microbial asso-
ciations and metabolites produced on the quality of coffee. A
recent communication has highlighted the importance of
addressing in more detail this aspect of post-harvesting as a
way to potentially influence taste of coffee, and offers new cof-
fee experiences (Folmer, 2014).
Post-harvest processing
To prepare green coffee beans, which are suitable for storage,
transport, and roasting, the outer layers of the coffee fruit,
including skin, mucilage, and pulp, must be removed from the
seeds (Jones and Jones, 1984). There are two primary methods,
dry (natural) and wet processing, and the third procedure is
called semi-dry processing Figure 5. A comprehensive overview
on these processes is given by Schwan et al. (2012). Dry process-
ing is generally used for Arabica and Robusta coffees from Brazil,
Ethiopia, Haiti, Indonesia, and Paraguay. This process produces
the so-called “natural” or “non-washed” coffee. Coffee cherries
are usually handpicked and this results in the inclusion of vary-
ing proportions of green and over-ripe cherries as well as raisins
and dry shriveled fruits. Following harvesting, dry processing
involves the fermentation of sun-dried coffee on platforms (typi-
cally made from cement, concrete, tarmac, or earth), which are
heaped at night and spread each morning for 10–25 days until
they reach humidity levels around 11–12%. Wet processing is
applied for Arabica coffee and is employed in Colombia, Kenya,
countries of Central America, and Hawaii. This process generates
the so-called “washed” coffees, which generate softer, less bodied,
and more acidic beverages than natural coffees. Coffee fruits are
selectively harvested, mechanically de-pulped (demucilation) and
fermented for 24–48 h in water tanks, and subsequently dried
(da Silva et al., 2012). Semi-dry processing is a variation of wet
process and produces “pulped natural” coffee. Coffee fruits are
pulped and fermented/dried as in the dry process. Fermentation
by yeasts, bacteria, and moulds can occur in both wet and dry
processing. The occurrence of certain microbes, their growth,
metabolism, and interaction with the cherries/beans can influ-
ence the final in-cup quality of coffee (Vilela et al., 2010). The
ecology and dynamics of the coffee-associated microbiota depend
on many factors such as the coffee variety, type of processing
methods, and environmental factors.
The nature of wet and dry processing results in different
conditions of fermentation. During wet processing, the muci-
laginous layer adheres to the beans and this is the substrate
used by endogenous microbes for fermentation. In this process-
ing, coffee beans are exposed to contamination coming from
the machinery and handling, and the wet stage offers high
moisture and anaerobic conditions for microbial growth (Jones
and Jones, 1984). The duration of fermentation is considered
short, as the wet stage lasts for around 48 h, and the pH
decreases fast (Schwan et al., 2012). Bacteria and yeasts are
266 D. M. WATERS ET AL.
Figure 5. Coffee processing by wet and dry methods, with an outlook on fungal contamination and potential use of bacteria and/or yeasts as starters.
commonly associated with wet-processing, whereas the occur-
rence of filamentous fungi in this process is less frequent
(Schwan et al., 2012). In dry processing, the pectins in the pulp
represent the main carbon source for the fermenting microbes.
Due to the nature of this process, its length (up to 20 days), and
low moisture conditions, the growth of yeasts and filamentous
fungi is facilitated versus that of bacteria, which can dominate
at the early stage due to the presence of free sugars and higher
moisture in the pulp (Schwan et al., 2012). The practices of re-
spreading and heaping of coffee bean piles can also influence
the course and microbial diversity of fermentation. In addition,
the effect of rainfall and the high ground temperatures in dry
processing lead to differences in predominant microbiota (Silva
et al., 2000).
To date, more emphasis has been put on the development of
moulds during post-harvesting due to the production of toxins
and off-flavors, whereas less attention has been given to the
bacterial and yeast communities occurring during post-harvest
processing. The aim of the following sections is to address the
existing knowledge on the ecology and dynamics of fermenta-
tion, which occur during wet and dry processes, as well as its
influence on the quality of coffee.
Bacteria and yeasts
Table 2 gives a comprehensive overview of bacterial and yeast
species associated with dry, semi-dry, and wet processed coffee
beans. From the first analysis of published literature, one can
conclude that the identification techniques used for coffee-asso-
ciated bacteria and yeasts are often based on phenotypic char-
acterization, and thus are not up-to date. The reason behind
this scenario is most likely the little importance that has been
attributed to the role of yeasts and, in particular, bacteria in the
fermentation process, and in turn the quality of coffee. Overall,
a rather heterogenous microbiota has been identified during
the processing of coffee. Independent of processing, strains of
Bacillus, Acinetobacter, Enterobacter, and Candida, and Saccha-
romyces and Pichia were the most frequently isolated strains
among bacteria and yeasts, respectively.
Wet fermentation occurs in an anaerobic environment, at
low water temperature, and with inner mucilage as main sub-
strate for microbial growth (Schwan et al., 2012). A limited
number of studies are available on the microbiota of wet proc-
essing, and these investigations show that mainly gram-nega-
tive strains belonging to Kleibsella, Flavobacterium, and
Erwinia spp. dominated the fermentation as well as the lactic
acid bacteria (LAB) Lactobacillus brevis and Leuconostoc mes-
enteroides. Yeasts of the genera Saccharomyces, Pichia, and
Candida were found to dominate during different stages of wet
process (Agate and Bhat, 1966; Avallone et al., 2001; Masoud
et al., 2004). During fermentation, the microorganisms produce
organic acids, which can migrate from the pulp and influence
the quality of coffee. The production of excessive amounts of
butyric, propionic, or acetic acids can give rise to “alcoholic” or
“stinker” tastes in coffee (da Silva et al., 2008). On the other
hand, the presence of malic and citric acids in given concentra-
tions can confer desirable acidity to the product (Avallone
et al., 2002; Schwan et al., 2012; Evangelista et al., 2014).
The main aim of fermentation during wet processing is the
efficient removal of the pectinaceous layer adhering to the
beans for a rapid and efficient drying. The exact role played by
endogenous microorganisms in degrading the mucilaginous
layer on the beans has yet to be fully understood. It is often
assumed that the microorganisms occurring during wet proc-
essing are responsible for the degradation of the mucilage pectic
substances that surround coffee beans (Frank et al., 1964, 1965;
Agate and Bhat, 1966; Masoud and Jespersen, 2006). Agate and
Bhat (1966) demonstrated that the yeasts associated with the
wet-processed Robusta coffee harbored pectinolytic Saccharo-
myces species, and that yeasts, rather than bacteria, had a vital
role in the degradation of mucilage. On the other hand, the
studies from Frank et al. (1964, 1965) showed that in Kona cof-
fee microbial growth was necessary to induce hydrolysis of
mucilage, and that gram-negative bacteria were responsible for
the pectinolytic activities during fermentation. The contrasting
findings from these studies might be related to differences in
the origins of the beans, different environmental conditions
occurring during processing, and the microbiota associated
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 267

Table 2. Bacteria and yeast species identified in wet, dry, and semi-dry processing.

References Location Type of processing Bacteria Yeasts Identification techniques

Silva et al., 2008 Brazil Dry B.cereus C.saitoana Bacteria: bac-tray, API
Acinetobacter sp. C.fermentati
B.polymixa Stephanoascus smithiae Yeasts: morphology, spores,
Arthrobacter sp. D.polymorphus Assimilation; fermentation
B.subtilis P.guillermondii
B.macerans C.membranifaciens
E.agglomerans D.hansenii
Yersinia sp. P.burtonii
B.megaterium P.anomalus
Arxula adeninivorans
P.sydowiorum
P.subpelliculosa
S.cerevisiae
Dry green coffee B.subtilis P.anomala Bacteria: bac-tray, API
Stored in jute or B.cereus D.hansenii
polystyrene bags
B.megaterium Yeasts: morphology, spores
B.macerans Assimilation; fermentation
Kurthia sp.
Tatumella ptyseos
S.rubidea
S.plymutica
Acinetobacter sp.
P.ohmeri
Vilela et al., 2010 Brazil Semi-dry Acinetobacter sp. Arxula sp. ARDRA, DGGE,
Bacillus sp. C.ernobii 16S, 23S-sequencing
B.subtilis C.fukuyamaensis
E.agglomerans C.membranifaciens
E.herbicola C.carpophila
E.coli H.uvarum
K.pneumoniae Kloeckera sp.
L.brevis Kluyveromyces sp.
L.plantarum P.anomala
L.lactis P.caribbica
L.mesenteroides R.mucilaginosa
Srratia sp. S.bayanus
Saccharomyces sp.
T.delbrueckii
Silva et al., 2000 Brazil Dry Aeromonas sp. Pichia sp. Bacteria: bac-tray, phenotypic
Enterobacter sp. Candida sp.
Pseudomonas sp. Arxula sp.
Serratia sp. Saccharomycopsis sp. Yeasts: morphology, biochemical
Hafnia sp. S.cerevisiae
Tatumella ptyseos S.pombe
Flavobacterium odoratum S.fibuligera
Klebsiella sp. Sporopachydermia cereana
Salmonella choleraesuis Trichosporonoides oedocephales
Chromobacter violaceum Willopsis saturnus
Pasteurella haemolytica
Bacillus sp.
Cellulomonas sp.
Arthrobacter sp.
Microbacterium sp.
Dermabacter sp.
Lactobacillus sp.
Avallone et al., 2001 Mexico Wet Klebsiella ozanae Cryptococcus laurentii API (bacteria and yeast)
Klebsiella oxytoca Kloeckera apis apicuata
Erwinia herbicola Cryptococcus albidus
Ln mesenteroides Candida guillermondii
Lactobacillus brevis
K.pneumoniae
Agate and Bhat, 1966 Wet (70 h) Flavobacterium sp. K.marxianus Morphology, fermentation
S.bayanus Assimilation
S.cerevisiae
S.pombe
Masoud et al., 2004 Tanzania Wet P.kluyveri
C.pseudointermedia
Only yeasts P.anomala
K.marxianus
P.kudriavzevii
T.delbrueckii
H.uvarum
268 D. M. WATERS ET AL.
with local processing. Masoud and Jespersen (2006) recently
demonstrated that strains of Pichia anomala and Pichia kluy-
veri, previously isolated from wet-processed coffee, were able to
produce extracellular polygalaturonase (PG) in in-vitro assays.
Since this enzyme was shown to be active at pH range of 4.5–
5.5, which is within the range of the fermentation step (6.0 to
3.5), the authors assumed that the yeasts could have an active
role in mucilage degradation (Masoud and Jespersen, 2006).
The authors also observed that the free monosaccharides in cof-
fee as substrate for yeast fermentation might be a prerequisite
for polygalaturonase secretion. Studies from Avallone and co-
workers (2001a, 2001b, 2002) have challenged the assumption
that microorganisms are primarily involved in the degradation
of mucilage pectins. The authors analyzed the composition of
the mucilaginous layer before and after fermentation (20 h)
and found that the composition and content of alcohol insolu-
ble residues (AIRs), hot water soluble crude pectic substances
(PECTs), and hot water insoluble residues (RESs) did not vary
before and after fermentation. However, depolymerization of
extracted PECTs and de-esterification of RESs were observed in
the fermented beans. Strains belonging to Erwinia herbicola
and Kleibsella pneumoniae were most dominant during fer-
mentation, but their number did not increase through the pro-
cess. The authors showed that these strains could not efficiently
produce pectolytic enzymes active in the pH range of fermenta-
tion (Avallone et al., 2002). Therefore, it was suggested that the
microbial population is involved in the liquefaction of pectins
because of the production of organic acids (Avallone et al.,
2002). Microorganisms can be isolated from coffee cherries in
the field prior to any contamination related to processing
(Sakiyama et al., 2001; Iamanaka et al., 2014). Sakiyama and
co-workers (2001) have isolated and characterized endophytic
bacteria from ripe cherries after the sterilization of surface.
They were the first to isolate a strain of Paenobacyllus amyloly-
ticus, which produced extracellular pectin-lyase. This type of
enzyme is normally associated with fungal strains, and thus
this finding suggests that bacteria could potentially be involved
in the degradation of pulp.
More systematic investigations are needed to address the
various theories regarding the mechanism of degradation of
mucilage during wet processing. Are the fermenting microor-
ganisms utilizing complex pectinaceous substrate or free sugars
from the pulp? Is the degradation of mucilage due to enzymes
and/or organic acids secreted by the yeasts or bacteria, or is it
due to the (combined) interaction with endogenous enzymes?
Future investigation should address the ecology and dynamics
of fermentation using comprehensive and modern biomolecu-
lar identification techniques, combined with the analysis of the
chemistry of mucilage. The data collected could be integrated
into a database to address the ecology of this complex system
and to understand which criteria apply for the dominancy of
certain strains of bacteria and yeasts during fermentation. Ulti-
mately, such data collection could represent an opportunity for
standardizing/controlling the fermentating microbiota and the
quality of coffee.
The conditions for fermentation during dry processing differ
from that used during wet processing. The pulp is not removed
from the beans, the cherries are in contact with the soil/ground,
the conditions are aerobic, and the fermentation occurs at
ambient temperature and lasts for up to 20 days (Schwan et al.,
2012). Under these conditions, a wider variety of bacteria and
yeasts species have been identified. Silva et al. (2000) character-
ized the microbiota associated with dry-processed C. Arabica
during two consecutive years in Brazilian farms. The authors
observed that the total microbial counts and composition of
microbiota varied widely among the farms at different stages of
processing. The bacterial species most frequently isolated
belonged to gram-negative Aeromonas, Pasedomonas, Entero-
bacter, and Serratia genera, and species of Bacillus, Cellulomo-
nas, Arthobacter, Microbacterium, Brochothrix, Dermabacter,
and Lactobacillus were identified among gram-positive bacteria
(Silva et al., 2000). The most frequently yeasts isolated were fer-
mentative species belonging to the genera Pichia, Candida,
Arxula, and Saccharomycopsis. Unfortunately, the authors used
phenotypic characterization techniques and did not provide
any details on the dynamics of microbial population (only sam-
ples at mid-fermentation were collected). Indeed, it was pointed
out that the sampling frequency and size might not have been
sufficient to address variability in the microbial population and
the association of microbial data to the conditions at farms
(Silva et al., 2000). Nonetheless, this study has been the first
attempt to address the occurrence and incidence of specific
microbes dominating during the dry processing of coffee,
which is a prerequisite to enable better process control and con-
stant coffee quality. In a more recent study, the authors have
investigated the dynamics of the microbiota in dry-processed
coffee from Brazil (Silva et al., 2008). The results indicate that
the occurrence of the microbes was influenced by the process-
ing stage and moisture content in the cherries and beans. Bacte-
ria dominated the fermentation during the first days and
progressively decreased in number toward the eighth day,
when yeasts outgrew them. Fungi represented the highest per-
centage of isolates, followed by yeasts and bacteria, during the
last days of drying. Fungal species persisted in significant num-
bers during storage of dry beans. The storage method influ-
enced the occurrence of microbial species. In fact, bacteria
occurred among the dominant microbes only in the beans kept
in polystyrene bags and not in those stored in jute bags (Silva
et al., 2008). This difference might be due to anaerobic condi-
tions favored in the former storage conditions. As previously
observed in Brazilian dry-processed coffee, Bacillus species
were predominant throughout the fermentation and drying
(Silva et al., 2000, 2008). The dominancy of species belonging
to Bacillus genus might be correlated to its ubiquity in soil envi-
ronments and to its cellulitic activity, which might represent a
competitive advantage for substrate utilization. Gram-negative
bacteria represented around 20% of bacterial isolates and
belonged mainly to the genera Enterobacter and Serratia. Enter-
obacter aerogenes, E. cloacae, and Klebsiella isolates were also
isolated, and T. ptyseos, P. putrefaciens, E. aerogenes, Acineto-
bacter sp., and P. mirabilis were found to produce pectin lyase,
indicating a likely role in the degradation of highly methylated
coffee pulp pectin, thus accelerating the fermentation process
(Silva et al., 2008). Among yeasts, the most frequently isolated
species belonged to Debaromyces, Pichia, and Candida, as
observed in the previous study (Silva et al., 2000). The compet-
iveness of these genera might be due to the production of anti-
fungal substances, especially in the case of isolates belonging to

Debaromyces, and to the potential of synthesizing pectin lyases.
Overall, this is the first study to address the succession and inci-
dence of the microbial population during dry processing and
storage, and to relate the observed trends back to the environ-
mental conditions and substrate composition. Furthermore, the
two studies published by Silva et al. (2000, 2008) confirm cer-
tain consistency in the microbial species identified as dominant
in dry-processed coffee.
The species identified in dry-processed coffee have also been
found in coffee produced by semi-dry processing. Vileila et al.
(2010) applied for the first time a polyphasic approach to study
microbial diversity and dynamics in semi-dry-processed beans.
The bacteria and yeast population increased during the first
days of processing, and remained stable until the last day of
drying, with counts around 105–107 CFU/g. In contrast, the fil-
amentous fungi decreased in number and failed to grow toward
the end of the process. The bacterial diversity increased during
the fermentation step. In particular, Enterobacter agglomerans,
Escherichia coli, Bacillus cereus, Bacillus megaterium, Bacillus
macerans, and Lactobacillus plantarum were the predominant
bacteria detected during this processing step. Remarkably, a
Bacillus isolate was present at a significant level (103 CFU/g) in
the cherries before processing commenced. The yeasts diversity
also increased during fermentation. Among yeasts, Pichia
anomala, Torulaspora delbrueckii, Saccharomyces spp., and
Rhodotorula mucilaginosa were the dominant species.
Overall, information on the microbial ecology and dynamics
for wet- and dry-processed coffee is limited and very often
gathered via obsolete identification techniques, mainly based
on phenotypic profiling. Nonetheless, these studies indicate
that understanding the mechanisms behind the diversity and
incidence of specific microbial population during post-harvest-
ing could enable a more efficient control of fermentation, and
eventually would allow driving the quality of coffee beans.
Therefore, scientists have the opportunity of creating a database
interlinking the substrate composition, fermentation condi-
tions, occurrence, and dominancy of specific microbes during
processing and storage of coffee.
This may enable improved process control, and eventually
may encourage the use of selected bacteria and yeasts to drive
fermentation and obtain coffee with consistent safety, and con-
stant and eventually differentiated organoleptic quality.
An evidence of this innovative approach in coffee processing
has been recently provided by Evangelista et al. (2014). The
authors evaluated the potential of using yeast starters to drive
the dry fermentation of washed and non-washed coffee using
four yeast strains previously isolated from dry- and semi-dry-
processed coffee. The starter strains Saccharomyces cerevisiae
UFLA YCN727, S. cerevisiae UFLA YCN724, Candida parapsi-
losis, UFLA YCN448, and Pichia guilliermondii UFLA YCN731
were chosen based on their ability to utilize the pectinaceous
substrate by producing pectinolytic enzymes and by the prod-
ucts of their fermentation, i.e. organic acids and volatiles, as
tested in vitro. The selected candidates were applied on coffee
cherries, their dominancy over the fermentation was monitored
via PRC-DGGE, and the qualitative and quantitative profiles of
their metabolites were evaluated. All strains persisted through-
out the fermentation and some outgrew the indigenous strains,
indicating their robustness in the process. Interestingly, strains
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 269
of Saccharmoyces, Pichia, Torulaspora, and Debaryomyces were
also detected in some fermentations or associated with the
fruits before processing, as observed before (Silva et al., 2008;
Vileila et al., 2010). Depending on the starter strains used, dif-
ferent chemical profiles were found in the samples in terms
organic acids and volatiles. In none of the samples, butyric or
propionic acids could be detected. On the other hand, alcohols
and aldehydes were the most abundant compounds in green
grains, and the volatile profile was greatly influenced by the
washing step applied before fermentation. The washing might
lead to the migration of solutes from the bean to wash water.
The sensory analysis of the final coffee indicated that the strain
and process used (washed, non-washed coffee cherries) influ-
enced the flavor notes of the final product. In particular, a cof-
fee with caramel, fruity, and herb notes could be produced by
using the starters S. cerevisiae, UFLA YCN727 and C. parapsilo-
sis, UFLA YCN448.
Thus, the authors showed how the coffee produced using
coffee beans from the same variety, the same origins and proc-
essed under the same conditions could be differentiated by
selected fermentation. The outcomes of this research prove that
by using starters to drive the fermentation, it is possible to
modulate the final in-cup attributes and to differentiate the sen-
sorial properties of roasted coffee.
Overall, a careful selection of strains from the pool of the
“autochthonous” microbial population, based on metabolic
traits and interaction with the substrate, may lead to the devel-
opment of robust starters, which can be applied to drive fer-
mentation and influence the quality of the beans. This type of
approach has indeed been applied for other complex systems,
such as cocoa and sourdough (Lefeber et al., 2011; G€anzle et al.,
2014). The potential of using starters could have a great impact
in the reduction and/or control of fungal spoilage. In fact,
yeasts seem also to play an important role in inhibiting the
growth/germination of toxin-producing fungi or at least sup-
pressing their production of mycotoxins (Masoud and Kaltoft,
2006).
Fungal microbiota and mycotoxins
Numerous studies have addressed the occurrence and incidence
of filamentous fungi during post-harvesting. Fungal growth is
associated with spoilage and concurrent production of toxins
and off-flavors, which can increase the amount of defective
beans (Iamanaka et al., 2014). Interestingly, the genus Clado-
sporium has been associated with good quality coffee beverage
(for review refer to Chalfoun, 2010). It is assumed that filamen-
tous fungi do not play a primary role in the actual fermentation
process. In fact, as shown by Silva et al. (2000, 2008), fungal
growth commences during the last days of fermentation or
only during storage for dry processed beans, and fungi are
rarely regarded as significant contaminants during wet process-
ing. The dynamics and the extent of fungal contamination
depend on environmental factors, such as humidity and tem-
perature, farming practices, and storage. A comprehensive
overview on the fungal species occurring during dry and semi-
dry processes is reported by Schwan et al. (2012). Research has
shown that the main fungi present associated with the coffee
cherries, which are subject to dry and semi-dry processing, are
270 D. M. WATERS ET AL.
species of Aspergillus, Penicillium, Cladosporium, Fusarium,
Pestalotia, Paecelomyces, and to a lesser extent Cylindrocarpon,
Eurotium, Fusariella, Geotrichum, Mucor, Phoma, and Ulocla-
dium (Silva et al., 2008; Vilela et al., 2010). The predominant
toxigenic genera are represented by Aspergillus, Penicillium,
and Fusarium (Silva et al., 2000; Batista et al., 2003; Taniwaki
2006; Batista et al., 2009).
The fungal toxins that are potentially produced by toxigenic
fungi associated with coffee are ochratoxin A (OTA), aflatoxins
and fumosins (Batista et al., 2003; Taniwaki 2006; Noonim
et al., 2008; Bokhari and Aly, 2009b). OTA is mainly produced
by Aspergillus and Penicillium species (Bucheli et al., 2000; Pitt,
2000; Taniwaki et al., 2008; Schwan et al., 2012). It was first
reported in green coffee by Levi et al. (1974) and in roasted cof-
fee by Tsubouchi et al. (1987). The level of OTA present in
green and roasted coffee varies considerably and there is little
known about which conditions induce isolates to produce OTA
(Batista et al. 2003). However, it is know that Aspergillus ochra-
ceus and other Aspergillus spp. are the most frequently isolated
OTA producers in coffee (Schwan et al., 2012). Various con-
tamination levels with OAT in green and roasted coffee from
different origins were reported (Levi et al., 1974; Micco et al.,
1989; Romani et al., 2000; Bucheli and Taniwaki, 2002; Leong
et al., 2007; Tozlovanu et al., 2010). In a study from Batista
et al. (2003), strains belonging to Aspergillus ochraceus, A. sul-
phreus, A. elegans, A. sclerotiorum, A. auricomus, A. insulicola,
A. petrakii, and A. sulphureus were isolated in coffee fruits. In
addition, the Penicillium species P. verrucosum and P. nordi-
cum were described as producers of OTA (Larsen et al., 2011).
Aflatoxins could also be produced by coffee-associated fungi.
Among others, aflatoxin FB1 has been given status of a class 1
human carcinogen (Cancer 1993). Magnoli and co-authors
(2008) identified A. niger, A. flavus, and A. parasiticus as the
main contaminants of green and roasted Colombian coffee.
The latter two were identified as potential aflatoxin FB1 pro-
ducers, but levels of aflatoxins in green and roasted beans were
below the detection limit (Magnoli et al., 2008). Similarly, A.
flavus isolates from commercial Arabic green coffee were iden-
tified as producers of aflatoxin B1; none of the isolates of A.
parasiticus could produce the toxin (Bokhari, 2007). In commer-
cial samples, the levels of aflatoxin B1 were on average seven-
fold lower than the maximum level produced in vitro by A. fla-
vus, and contamination with OTA was more significant
(Bokhari, 2007). Partial degradation of aflatoxins could be
achieved via roasting, depending on the conditions used (Bokhari
and Ali, 2009a). Fumonisin B2 (FB2) is a carcinogenic mycotoxin
normally associated with Fusarium, but also Aspergillii species,
and has been detected in low concentration in coffee beans
(Noonim et al., 2009). A. niger species isolated as a predominant
fungi in C. arabica and C. robusta beans in Thailand were found
to be capable of producing FB2 and FB4 (Bokhari and Aly,
2009b). However, Aspergillus spp. need high concentrations of
carbohydrates to produce significant amount of fumosin FB2,
and thus only a limited risk of fumonisin contamination can be
associated with coffee beans (Bokhari and Aly, 2009).
Overall, the presence of toxigenic fungi in coffee beans is not
necessarily correlated with the production of mycotoxins (in
significant amounts); however, preventing their growth can
reduce the risk of contamination. The origins and storage
conditions used can dramatically influence the types of con-
taminant fungi, and amount and types of the toxic compounds.
Novel control strategies to control fungal spoilage, such as the
application of protective bacteria and yeasts during post-har-
vesting and storage, should be explored.
Apart from the toxigenic potential of fungi, these microbes
can also dramatically influence the quality of coffee beverage.
Iamanaka et al. (2014) have recently investigated the relation-
ship between fungal contamination and sensory attributes of
coffee beverage. A wide diversity in fungal species was identi-
fied in the samples collected at different stages of processing,
i.e. cherries and stored coffee beans. Overripe cherries from the
trees and cherries from the ground produced beverages with
negative sensorial notes, such as fermented, “strinker,” and
mouldy. The occurrence of Aspergillus section Nigri and A.
westerdijikiae in these samples was suggested as being partly
responsible for off-flavors in the beverage (Iamanaka et al.,
2014). Rio off-flavor in Brazilian coffee has also been associated
with the infestation of beans by fungi (and bacteria), and conse-
quent degradation of the coffee cell structure (Spadone et al.,
1990). To this regard, the deterioration of barley kernel ultra-
structure following infection by Fusarium spp. was recently
addressed by Oliveira and co-workers (2012). Interestingly, the
sensory attribute “fermented” is detrimental for coffee bever-
age, and it seems to be associated with the growth of fungi,
rather than the actual fermentation by yeasts and bacteria.
Due to the negative impact of filamentous fungi on the over-
all quality of coffee and coffee beverage, it is desirable to pre-
vent and/or control their growth on the beans during the last
steps of post-harvesting and storage (Figure 5). Fungal contam-
ination is currently preventable through good agricultural prac-
tices, careful post-harvest processing (Bucheli et al., 2000),
appropriate storage (Bucheli et al., 1998) of separated/graded
green coffee beans, and discarding defective beans (Federation,
2005). The use of bacterial and/or yeast starters could represent
a new avenue to avoid fungal infection.
A recent study has evaluated the potential of L. plantarum
strains isolated from the indigenous population of coffee pulp
to antagonize the growth of A. carbonarius strains, previously
isolated from coffee beans. The in-vitro tests revealed that cer-
tain L. plantarum strains exhibit anti-fungal activities toward
the Aspergillus isolates (Djossou et al., 2011). No further details
on the mechanisms of such inhibition were given in this study.
Yeasts also seem to play an important role in inhibiting the
growth/germination of toxin producing fungi or at least sup-
pressing the production of mycotoxins (Masoud and Kaltoft,
2006). The authors have investigated the antifungal potential of
yeast strains isolated during the wet processing of coffee, i.e. P.
anomala, P. kluyveri, and H. uvarum against A. ochraceus iso-
lates from coffee. Results from in-vitro tests indicated that the
three yeast strains could inhibit the growth and germination of
spores of Aspergillus when co-cultured with the fungus, and the
strains of P. anomala and P. kluyveri had a stronger antagonis-
tic effect (Masoud and Kaltoft, 2006). In particular, the inhibi-
tory effects of the yeasts strains were attributed to the
competition with fungi for nutrients and to the production of
antifungal metabolites, such as volatiles and toxins. The yeasts
investigated in this study inhibited the production of OTA by
A. ochraceus, which was most likely a result of production of

inhibitory compounds by the yeasts (Masoud and Kaltoft,
2006). The first study into the on-farm biocontrol of A. ochra-
ceus has been published recently (Velmourougane et al., 2011).
The affordable and readily available Saccharomyces cerevisiae
commercial bakers’ yeast was used as a fungal antagonist, and
resulted a reduced incidence of ochratoxigenic moulds and
OTA contamination during green coffee processing (Velmour-
ougane et al., 2011).
The potential of coffee-associated bacteria, especially LAB,
and yeasts to prevent and/or retard fungal growth should be
further explored. LAB and yeasts with antifungal activity repre-
sent a promising alternative to chemical preservation and have
been employed in many foods. The antagonist effect of LAB
and yeast on fungal growth is a complex mechanism and has
been associated with the competition for nutrients and space,
and production of organic acids and secondary metabolites,
such as toxins, volatiles, bacteriocins, and peptides. (Lavermi-
cocca et al., 2003; Lowe and Arendt, 2004; Lind et al., 2005;
Schnurer and Magnusson, 2005; Strom et al., 2005; Rouse and
van Sinderen 2008; Trias et al., 2009; Ryan et al., 2011; Crowley
et al., 2013). Yeast and LAB starters have been successfully
applied during storage of grains to reduce the counts of natu-
rally occurring moulds (Olstorpe et al., 2010).
Therefore, yeasts and bacteria starter cultures could poten-
tially be employed to control and/or prevent the growth of fila-
mentous fungi during dry processing and storage of coffee
(Figure 5). The choice of starter strains should rely primarily
on their capability to dominate the fermentation step and pro-
duce active anti-fungal compounds. This selection should be
based on comprehensive data on the microbial diversity and
dynamics occurring during post-harvesting. The effects on the
overall quality of final coffee beverage by application of these
starters should be then addressed.
Conclusions and future prospects
Germination and fermentation are two post-harvest-associated
events that can influence the overall quality of coffee and the
final coffee beverage. The metabolic status of beans during
post-harvesting influences the composition and relative
amount of major storage components (carbohydrates, protein,
and lipids), which affects the chemistry and processing quality
of beans. The course and impact of coffee fermentation on and
during wet and dry processes were also discussed. There is
some knowledge of microbial diversity associated with these
fermentations, but the role and impact of coffee-associated
microorganisms in the transformation of cherries to dry beans
remains to be fully elucidated. Novel aspects of this processing
step, such as the use of starter strains to control the process,
modulate coffee quality, and prevent spoilage, may have an
important role in the future of coffee production.
Overall, the mechanisms, significance, and correlation of
germination and fermentation during post-harvesting to the
quality changes of coffee should be investigated in more detail.
Acknowledgments
The authors would like to thank Michael Callanan, James Mc Carthy, and
Wilbert Sybesma for comments and critical reading of the manuscript.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 271
Funding
Funding was provided by Nestl
e, Lausanne Switzerland.
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