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ABSTRACT The postharvest treatment and processing of fresh coffee cherries can
impact the quality of the unroasted green coffee beans. In the present case study, Received 17 August 2016 Accepted 21
October 2016
freshly harvested Arabica coffee cherries were processed through two different wet
Accepted manuscript posted online 28
and dry methods to monitor differences in the microbial community structure and October 2016
in substrate and metabolite profiles. The changes were followed throughout the Citation De Bruyn F, Zhang SJ, Pothakos V,
postharvest processing chain, from harvest to drying, by implementing up-to-date Torres J, Lambot C, Moroni AV, Callanan M,
techniques, encompassing multiple-step metagenomic DNA extraction, high- Sybesma W, Weckx S, De Vuyst L. 2017.
Exploring the impacts of postharvest
throughput sequencing, and multiphasic metabolite target analysis. During wet pro- processing on the microbiota and metabolite
cessing, a cohort of lactic acid bacteria (i.e., Leuconostoc, Lactococcus, and Lactobacil- profiles during green coffee bean production.
lus) was the most commonly identified microbial group, along with enterobacteria Appl Environ Microbiol 83:e02398-16. https://
doi.org/10.1128/AEM.02398-16.
and yeasts (Pichia and Starmerella). Several of the metabolites associated with lactic
Editor Johanna Björkroth, University of Helsinki
acid bacterial metabolism (e.g., lactic acid, acetic acid, and mannitol) produced in Copyright © 2016 American Society for
the mucilage were also found in the endosperm. During dry processing, acetic acid Microbiology. All Rights Reserved.
bacteria (i.e., Acetobacter and Gluconobacter) were most abundant, along with Pichia Address correspondence to Luc De Vuyst,
and non-Pichia (Candida, Starmerella, and Saccharomycopsis) yeasts. Accumulation of ldvuyst@vub.ac.be.
F.D.B., S.J.Z., and V.P. contributed equally to this
associated metabolites (e.g., gluconic acid and sugar alcohols) took place in the dry-
article.
ing outer layers of the coffee cherries. Consequently, both wet and dry processing
methods significantly influenced the microbial community structures and hence the
composition of the final green coffee beans. This systematic approach to dissecting
the coffee ecosystem contributes to a deeper understanding of coffee processing
and might constitute a state-of-the-art framework for the further analysis and subse-
quent control of this complex biotechnological process.
IMPORTANCE Coffee production is a long process, starting with the harvest of cof-
fee cherries and the on-farm drying of their beans. In a later stage, the dried green
coffee beans are roasted and ground in order to brew a cup of coffee. The on-farm,
postharvest processing method applied can impact the quality of the green coffee
beans. In the present case study, freshly harvested Arabica coffee cherries were pro-
cessed through wet and dry processing in four distinct variations. The microorgan-
isms present and the chemical profiles of the coffee beans were analyzed through-
out the postharvest processing chain. The up-to-date techniques implemented
facilitated the investigation of differences related to the method applied. For in-
stance, different microbial groups were associated with wet and dry processing
methods. Additionally, metabolites associated with the respective microorganisms
accumulated on the final green coffee beans.
KEYWORDS coffee bean fermentation, wet processing, dry processing, high-
throughput sequencing, metabolite target analysis, UPLC-MS/MS, green coffee beans
January 2017 Volume 83 Issue 1 e02398-16 Applied and Environmental Microbiology aem.asm.org 1
De Bruyn et al. Applied and Environmental Microbiology
P ostharvest processing of coffee cherries yields green coffee beans, which need to
be roasted and ground to obtain the desired characteristic coffee aroma and taste
(1). These processes are the main drivers of consumers’ perception of coffee beverage
quality. The cherries and beans are the fruits and seeds of the coffee plant (Coffea sp.,
family Rubiaceae), which is cultivated in plantations, mainly in the equatorial zone.
The on-farm postharvest coffee processing is essential for ensuring high coffee cup
quality (2) and constitutes a chain of interlinked phases mainly aimed to remove the
mucilage of the cherries and dry the beans to a low moisture content of 10 to 12%
(mass/mass). The final quality of the green coffee beans is thus dependent on the
agricultural and farm practices applied, which in turn depend on the coffee plant
cultivar, geography, weather conditions, and infrastructure available (3). Even when all
of these factors are fixed within one type of postharvest processing, a multitude of
variations exist, as a standardized pipeline for the production of green coffee beans is
RESULTS
Microbial community structures. The average numbers of raw V4 and internal
transcribed spacer 1 (ITS1) sequences per sample reached approximately 200,000 and
94,000 sequences, respectively. The estimated sequencing coverage ranged between
98.2 and 99.7%.
(i) Freshly harvested coffee cherries. The initial surface contamination of the
freshly harvested coffee cherries (CB sample) encompassed bacterial operational
taxonomic units (OTUs) belonging to the Enterobacteriaceae (especially Klebsiella
pneumoniae), AAB (Gluconobacter spp.), and soil-associated bacteria such as Dyella
kyungheensis (Fig. 2A and 3). Only a small portion of the reads was attributed to the
LAB species Leuconostoc mesenteroides/pseudomesenteroides. In regard to the fun-
gal diversity, Pichia kluyveri/fermentans was highly abundant (Fig. 2B and 3). Based
on the principal-coordinate analysis (PCoA) performed on all microbial community
structures, the CB sample was clearly differentiated from all other coffee-processing
samples (Fig. 4).
(ii) Wet coffee processing. Upon depulping (W1), the OTU Leuconostoc increased in
relative abundance, and it was the most prevalent bacterial taxon throughout the wet
processing, especially during fermentation (SW2 and EW2) (Fig. 2A and 3). The most
prevalent fungal taxon was Pichia, whereas the OTUs Starmerella and Candida increased
in relative abundances mainly during the fermentation and soaking stage (Fig. 2B and
3). Over the course of sun drying (SW4 and SW5), the arrangement of the OTUs stayed
nearly unvaried. Overall, all wet-processed samples grouped closer on the PCoA biplots,
depicting similarities in their microbial community structures that mainly encompassed
LAB and only limited fungal diversity (Fig. 4). However, certain differences in the
evolution of the microbial communities occurred. While Leuconostoc spp., Lactococcus,
and Weissella were predominant during fermentation in SW, a higher incidence of
lactobacilli was found from fermentation in EW. This indicated a shift toward more
acid-tolerant LAB communities, which corresponded to a decrease in the pH of the
fermenting mass from 4.5 (after 16 h of fermentation) to 4.0 (after 36 h of fermentation).
LAB-associated OTUs decreased during drying. Further, the abundance of enterobac-
terial taxa was lower in EW than in SW, especially after fermentation and soaking but
increased during drying. Also, a rise in the relative abundances of soil-associated OTUs
Acinetobacter, Janthinobacterium, and Cellulosimicrobium followed the decrease in
moisture content (Fig. 3; see also Fig. S1 in the supplemental material).
(iii) Dry coffee processing. During dry processing (both SD and HD), the LAB
species L. mesenteroides/pseudomesenteroides and AAB taxa (Acetobacter and Glucono-
bacter) were present in high relative abundances (Fig. 2A and 3). The Enterobacteriaceae
OTUs decreased compared to those for the CB and wet-processed samples. Other
bacterial OTUs appeared sporadically and in variable relative abundances over the
course of drying, for instance, reflecting environmental contamination (SD7). Further
examples are Lactobacillaceae, Enterobacteriaceae (mostly K. pneumoniae), Enterococ-
caceae, Brucellaceae (especially Ochrobactrum pseudogrignonense), Stenotrophomonas,
and Janthinobacterium lividum. The fungal diversity of the dry-processed samples was
greater than that of the wet-processed samples. Although Pichia was still the major
fungal taxon, the occurrences of Starmerella bacillaris and Candida spp. were higher
than in wet processing (Fig. 2). The PCoA underpinned the distinction between wet-
and dry-processing samples based on their microbial community structures (Fig. 4).
Significant differences between SD and HD were found. During the first 6 days of
HD, the heaped and nonstirred cherries remained moist, as liquid exuded from the
pulp, and no significant decrease in the moisture content was noted (Fig. S1). Also,
visible chalk-dust mycelia were formed on these cherries, and a strong odor developed,
indicating high microbial activity. Moreover, dominance of AAB and the presence of the
mold-like yeast Saccharomycopsis crataegensis were found during HD (Fig. 2 and 3). The
co-occurrence and coexclusion plot, based on a Spearman rank-order correlation matrix
(see Fig. S2 in the supplemental material) confirmed the positive relationship among
Gluconobacter spp., Acetobacter spp., and S. crataegensis as well as non-Pichia yeasts.
Metabolite target analysis. (i) Freshly harvested coffee cherries. The moisture
content of the mucilage (85%) and endosperm (51%) of the fresh coffee cherries as well
as the concentrations of all targeted metabolites differed substantially (Fig. 5A and B).
The mucilage was rich in fructose (27% on dry mass), glucose (21%), sucrose (9%), and
organic acids (7.3%), among which malic acid, quinic acid, and gluconic acid were the
most abundant. In contrast, the endosperm had high levels of sucrose (8% on dry mass)
and low concentrations of monosaccharides, whereas the most prevalent organic acids
(2.4%) were citric acid, malic acid, and quinic acid. In addition, the trigonelline (1.0%)
and caffeine concentrations (0.9%) were higher in the endosperm than in the mucilage,
whereas the acetic acid concentration was lower.
(ii) Wet coffee processing. As the mucilage was completely removed from the
endosperm after fermentation, metabolite quantification of the mucilage was per-
formed up to the soaking stage. In the mucilage, sucrose was completely consumed by
the end of fermentation in both SW and EW. Fructose and glucose concentrations
decreased, and this drop was more intense during EW. A substantial accumulation of
metabolites associated with microbial activity occurred, including acetic acid, ethanol,
glycerol, lactic acid, and mannitol (Fig. 5A). These compounds started to accumulate
after depulping, and the concentrations increased proportionally to the time of fer-
mentation. The organic acid profile of the mucilage was also modified during fermen-
tation, as the concentrations of gluconic acid, malic acid, and quinic acid decreased.
Propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid,
and oxalic acid concentrations were below the quantification limits. The caffeine and
trigonelline concentrations decreased upon processing.
fructose concentrations decreased; however, this drop was more gradual in SD than in
HD. The glycerol and mannitol concentrations increased intensively and peaked in the
SD3 and HD5 samples. Arabitol, sorbitol, and xylitol concentrations also increased in the
outer layers during drying. Organic acid concentrations, encompassing lactic acid (1.0%
in SD5, 2.1% in HD3), gluconic acid (8.7% in SD5, 14.3% in HD3), and glucuronic acid
(1.7% in SD3, 3.1% in HD1) also increased during drying, with higher concentrations
generated in the HD samples than in the SD samples (Fig. 5C). A sharp increase in acetic
acid concentrations took place in SD1 and HD1, whereas they decreased afterwards.
Regarding the endosperm, the monosaccharide and sucrose concentrations de-
creased gradually during drying, whereas the glucose and fructose concentrations had
a secondary peak in the SD6 and HD3 samples (Fig. 4D). Small concentrations of
glycerol were found, reaching their highest values in the SD3 and HD3 samples. Acetic
acid, ethanol, and lactic acid reached their highest concentrations in the SD2 and HD3
DISCUSSION
The present case study on coffee processing represents a systematic approach for
the monitoring of both microbial community shifts and metabolite profiles associated
with coffee cherry substrates (i.e., pulp and mucilage) and coffee beans throughout the
postharvest processing chain. The molecular analysis conducted was based on an
enzymatic total DNA extraction method, targeting bacteria, yeasts, and filamentous
fungi, suitable for metagenomic purposes. The high-throughput sequencing (HTS) of
short-length amplicons of bacteria and fungi under the same barcode was an econom-
ical way to evaluate the total microbial diversity of this complex ecosystem. Challenges
were the equimolar pooling of the two DNA template libraries that resulted in a 2:1
ratio of generated reads (V4/ITS1) and adapter read-throughs in the ITS1 sequences
because of the various length of the ITS regions of Ascomycota and Basidiomycota (34).
The metabolite target analysis employed rapid sample preparation steps combined
with three different extraction methods and various chromatographic separation and
detection techniques, allowing high discriminatory power among structurally related
compounds in complex matrices.
The microbial community structure on the surface of the freshly harvested coffee
cherries was composed of Enterobacteriaceae, fungi, and soil microorganisms that
naturally occur in the phyllosphere (35, 36). As soon as the coffee cherries started
exuding, these prototrophic microorganisms were succeeded by fermentative LAB
species such as L. mesenteroides/pseudomesenteroides and the yeast species P. kluyveri/
the osmotic pressure facilitated the loss of monosaccharides and microbial metabolites
accumulated upon fermentation. Hence, the soaking step carried out on the fermented
coffee beans facilitated a significant washout of these compounds, which may impact
the quality of the brewed coffee because of a lower degree of acidity and will lead to
a milder flavor. It is well known that the loss of dry matter is associated with fermen-
tation and soaking due to endogenous metabolism and exosmosis, thereby influencing
coffee cup quality (4, 48). During drying of the coffee beans, the moisture content
decreased and shifted their microbial contamination to environmental taxa related to
soil (mainly Gram-negative bacteria) (49). Also, the drying step induced a drought stress
response and aerobic respiration in the endosperms (31), which slowed down the
glucose and fructose turnover rate (31). All of these processing steps contributed to
differences in the concentrations of coffee-specific compounds too. Consequently,
technological aspects (especially the duration of fermentation, soaking, and drying) can
entire chain of both wet and dry processing under favorable and less favorable
processing conditions. At the same time, specific endosperm metabolite changes were
followed. This was made possible by simultaneous HTS of metagenomic DNA and
metabolite target analysis of a broad range of chemical compounds. The results
showed that the different processing conditions influenced the composition and
activity of the microbial communities as well as metabolite accumulation in the
endosperms. In addition, the current findings corroborate the effect of microorganisms
on the chemical profiles of coffee beans and support the idea of using a starter culture
during coffee processing for improved process control and prevention of spoilage and
eventually steering the sensory differentiation of roasted coffee, as has been performed
for cocoa bean fermentation (62, 63). Further studies should ultimately allow strength-
ening of the understanding of the impact of the microbiota on coffee cup quality and
provide robust data for the development of commercial starter cultures.
digestion was achieved by incubation of these suspensions with 0.5 mg of proteinase K (Merck) at 56°C
for 1 h. Thereupon, 100 l of a 5 M NaCl solution was added to the suspensions and incubated at 65°C
for 2 min, after which 80 l of a 10% (mass/mass) cetyltrimethyl ammonium bromide solution was added,
and the mixtures were incubated at 65°C for 10 min. Following this, 600 l of chloroform-phenol-isoamyl
alcohol solution (49.5:49.5:1.0) was added, and the lysates were shaken vigorously for 5 min. Finally, the
solutions were centrifuged at 13,000 ⫻ g for 5 min in 2-ml vials (Phase Lock Gel Heavy; 5 Prime, Hilden,
Germany). The DNA contained in the supernatants was purified by binding on and elution from a
cellulose acetate membrane, using the DNeasy blood and tissue kit (Qiagen, Venlo, The Netherlands)
according to the manufacturer’s instructions. DNA concentrations were measured with a NanoDrop
ND-2000 (Thermo Scientific, Wilmington, DE).
(ii) Amplification of group-specific loci for HTS. Group-specific loci of both bacterial and fungal
DNA were amplified through PCR. The V4 hypervariable region of the bacterial 16S rRNA gene was
amplified using the primers F515 (5=-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGTGCCAGCMGCC
GCGGTAA-3=) and R806 (5=-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAA
T-3=) with an Illumina platform-specific 5= tag (underlined) (64). The PCR assay conditions consisted of an
initial step at 94°C for 3 min, followed by 35 cycles at 94°C for 45 s, 50°C for 60 s, and 72°C for 90 s. A
was first separated from the beans, then the mucilage was scraped off, and finally the parchment
was detached manually. In the case of dry-processed samples, all dried outer layers were removed
manually. In all cases, representative samples (20 g) of cherry layers or endosperm were first frozen
in liquid nitrogen (Air Liquide, Louvain-la-Neuve, Belgium) and ground into fine powders with a malt
miller (Corona Mill, Bogotá, Colombia).
Three types of extraction were used to analyze the metabolites targeted in the final samples. Water
extracts were prepared by submerging 0.1 to 0.5 g of sample in 5 g of ultrapure water (Milli-Q; Merck
Millipore, Billerica, MA) at room temperature for 30 min. Methanol extracts were prepared by treating 0.1
to 0.5 g of sample in 5 g of 40% (vol/vol) methanol (Sigma-Aldrich) at 40°C for 20 min. Acidic extracts
were prepared by adding 0.1 to 0.5 g of sample to 5 g of 0.01 N HCl and ultrasonicating (Ultrason 2510;
Branson, Danbury, CT) at room temperature for 20 min. All samples were microcentrifuged (14,000 rpm,
10 min), deproteinized with acetonitrile (Sigma-Aldrich) at a 1:3 ratio, and filtered through 0.2-m
pore-size filters (Whatman filters; GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) prior
to injection. All samples were both prepared and analyzed in triplicate.
(ii) Determination of free carbohydrates and sugar alcohols. The concentrations of free carbo-
hydrates (fructose, galactose, glucose, and sucrose) were determined by high-performance anion-
SUPPLEMENTAL MATERIAL
ACKNOWLEDGMENTS
We acknowledge financial support from the Research Council of the Vrije Univer-
siteit Brussel (SRP7 and IOF342 projects), the Hercules Foundation (grant UABR09004),
and Nestec S.A., a subsidiary of Nestlé S.A.
Cyril Moccand and Jay Siddharth are acknowledged for critical reading of the
manuscript and Arne Glabasnia and Frédéric Mestdagh for their advice on the analytical
methods.
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