CURRENT MICROBIOLOGY Vol. 42 (2001), pp.

252–256
DOI: 10.1007/s002840110213 Current
Microbiology
An International Journal
© Springer-Verlag New York Inc. 2001

Microbiological and Biochemical Study of Coffee Fermentation

Sylvie Avallone,1 Bernard Guyot,2 Jean-Marc Brillouet,3 Eugenia Olguin,4 Joseph-Pierre Guiraud1
1
USTL F-34095, GBSA-MBI CC 23 Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France
2
CIRAD-CP, Montpellier Cedex, France
3
CIRAD-FLHOR, Montpellier Cedex, France
4
Instituto de Ecologia, Xalapa, Veracruz, Mexico

Received: 21 August 2000 / Accepted: 25 September 2000

Abstract. The coffee fermentation microflora were rich and mainly constituted of aerobic Gram-negative
bacilli, with Erwinia and Klebsiella genuses at the highest frequencies. The best population increase was
observed with lactic acid bacteria and yeasts, whereas those microorganisms that counted on a pectin
medium remained constant during the fermentation step. Qualitatively, lactic acid bacteria belonged
mainly to Leuconostoc mesenteroides species but the others microflora were relatively heterogeneous.
The microorganisms isolated on pectin medium were Enterobacteriaceae, identified as Erwinia herbicola
and Klebsiella pneumoniae, not reported as strong pectolytic strains. Throughout coffee fermentation,
60% of the simple sugars were degraded by the total microflora and not specifically by pectolytic
microorganisms.

Coffee preparation requires the elimination of the enve-
lopes from the beans and can be carried out by a dry or
wet process. In the wet method, coffee cherries are first
depulped in order to remove the skin (exocarp) and the
pulp (outer mesocarp). Beans are then put in a fermen-
tation tank with a water stream and allowed to ferment to
degrade a hygroscopic mucilaginous layer (inner meso-
carp) which constitutes an obstacle to the drying. The
quantity of water used in the wet process can be dimin-
ished by using dry pulping and water recycling [16, 26].
During the natural fermentation, the mucilaginous
substrate, rich in simple sugars and pectin, is liquefied [9,
11, 15, 17]. Coffee fermentation microflora are consti-
tuted by the classical microorganisms of vegetables,
fruits, and the hearth surrounding the trees [1, 14, 33].
The most frequent lactic acid bacteria isolated are Leu-
conostoc, Lactobacillus plantarum, and Lactobacillus
brevis [24, 32]. Yeasts have also been detected, but their
role in mucilage degradation is controversial [1, 14, 30].
The pectic substances would be degraded by the com-
bined action of microbial and/or endogenous coffee en-
zymes [2, 7, 13, 22, 32], but no biochemical proof was
ever done. Mucilage degradation is generally attributed
to pectolytic microorganisms, but further studies have
Correspondence to: J.P. Guiraud; email: guiraud@arpb.univ-montp2.fr.
demonstrated that the most frequent pectolytic strains,
Erwinia dissolvens, E. herbicola, and Klebsiella pneu-
moniae [4, 31], produce a pectatelyase unable to depo-
lymerize highly methylated pectins of coffee mucilage
[5, 10].
Until now, the evolution of each microflora (aerobic,
anaerobic, lactic, yeast, and pectolytic microflora) was
not studied as well as the biochemistry of the fermenta-
tion. The purpose of our work was to describe, quanti-
tatively and qualitatively, the microbial and biochemical
parameters of coffee fermentation in order to better un-
derstand the role of microorganisms in mucilage degra-
dation.
Materials and Methods
Plant material and processing. Coffee cherries (Coffea arabica L.
var. typica Cramer) were hand-picked at the mature stage in a planta-
tion from the Coatepec area (Xalapa, Veracruz, Mexico) during the
1998 year. External mesocarp (skin and pulp) was mechanically elim-
inated by wet or dry pulping (DV-225C PENAGOS depulper).
Depulped beans were then conveyed in a water stream to tanks and left
to ferment for 20 h. Every 5 h, samples were withdrawn from three
different locations at the middle depth of each tank and mixed.
pH, temperature and dry weight determinations. Fermented beans
(20 g) were gently swirled in distilled water, and the pH of the
supernatant was measured [20]. Temperature of the fermenting coffee
was determined with a portable electronic thermometer. Dry weight
S. Avallone et al.: Coffee Fermentation
was evaluated on samples left 3 h at 50°C and overnight at 60°C in a
vacuum oven.
Enumeration media. Fermented beans (20 g) were homogenized with
a Waring blender in bacto-NaCl solution (180 ml) during 1 min, and
decimal dilutions were then prepared in 8 g/l sterile sodium chloride.
Lactic microflora were counted on De Man, Rogosa and Sharpe agar
(MRS) [12], and yeast on Yeast Glucose Chloramphenicol agar (YGC)
containing 250 mg/l of antibiotic. Aerobic microorganisms were num-
bered on Plate Count Agar (PCA), and anaerobic bacteria on Meat-
Liver S.R. agar with incubation in anaerobic tanks. Microorganisms
able to grow with apple pectin (Sigma) as the sole source of carbon
were enumerated on Boccas medium, pH 6.8 [6]. After 2 days of
incubation at 30°C, colony-forming units (CFU) were counted in
duplicate, and data were expressed as the mean of the decimal loga-
rithm of the CFU/g of depulped bean dry weight. The most frequent
strains were isolated and stored at ⫺80°C in 30% glycerol. Strain
frequencies were calculated as the ratio of the respective population
density. All media used in our experiments were purchased from
Biokar Diagnostics company (France) except that of Boccas. Identifi-
cations were performed with API systems (BioMérieux, France), and
data were analyzed with an APILAB program calculating the most
probable bacteria.
Biochemical analysis. Simple sugars were extracted twice at room
temperature from whole pulped frozen beans with 80% ethanol for 1 h
under gentle stirring. Supernatants were pooled, concentrated, and the
volume was adjusted to 200 ml. The sugars were analyzed by high-
performance anion-exchange chromatography with a Dionex DX-300
system equipped with a PAD II detector and a Carbopac PA-1 column
(4 ⫻ 250 mm) with a Carbopac PA-1 guard column (4 ⫻ 50 mm). The
elution was performed at 1 ml.min⫺1 with 0.026 M NaOH and post-
column addition of 0.3 M NaOH. Organic acids were extracted at room
temperature from 5 g of pulped frozen beans with 5 mM H2SO4 (20 ml)
for 1 h. The solution was centrifuged and filtered on cellulose nitrate
(0.2 ␮m). Organic acids were then separated on an Applied Biosystems
Polypore H PPH-257 column (250 ⫻ 7 mm) eluted with 5 mM H2SO4
at 0.3 ml.min⫺1 [27]. Detection was achieved with a RID-6A Shimadzu
refractometer. Injected volume was 20 ␮l. Ethanol was measured by
gas-liquid chromatography on a packed Porapak Q column (1 m ⫻ 2.2
mm; 80 –100 mesh) at 170°C with nitrogen as carrier gas (20
ml.min⫺1). Injector and detector temperature was 200°C, and 2-propa-
nol was used as the internal standard.
253
Fig. 1. Physicochemical parameters of coffee fer-
mentation.
Results
Evolution of physical parameters. At the beginning of
the fermentation, the dry weight of depulped beans was
47%. The time necessary to decompose the mucilage
layer was approximately 20 h. During the first 10 h,
which corresponds to the night, the pH decreased slowly
because the outside temperature was rather cold (13°C)
(Fig. 1). Then acidification due to the microflora metab-
olism was observed. Coffee temperature variations were
moderate, but mesophilic microflora maintained a higher
temperature in tanks than outside during all the fermen-
tation step.
Quantitative study of coffee fermentation microflora.
The microbial counts are reported on a logarithmic scale
in Fig. 2. The initial wild microflora were rich and
remained constant during the first 10 h. This lag time was
due to the low temperature of the night and the time for
microorganisms to adapt their metabolisms. Between 10
and 15 h, the total microflora increased from 2.5 ⫻ 107
to 13.5 ⫻ 107 UFC/ml. The aerobic mesophilic micro-
flora were the quantitatively most important and dimin-
ished after 20 h because of low pH. Of the total aerobic
initial microflora, 0.1% grew on a medium with apple
pectin as the sole source of carbon, but no rise was
observed during the process.
Qualitative study of coffee fermentation microflora.
Aerobic bacteria. The evolution of the most numerous
strains is shown in Table 1. Most of the aerobic bacteria
were Gram-negative bacilli. The strains isolated with the
highest frequencies were coliforms commonly distrib-
uted in the environment and identified as Klebsiella
(73%) and Erwinia (28%). Bacteria having a respiratory
and fermentative metabolism with the production of a
low level of organic acids (Erwinia, Aeromonas) were
254 CURRENT MICROBIOLOGY Vol. 42 (2001)

Fig. 2. Coffee fermentation microflora.

Table 1. Main species of aerobic mesophilic microflora during coffee Table 3. Main species of yeast microflora during coffee fermentation
fermentation
Time(h) Strains Frequencya (%)
Time (h) Strains Frequencya (%)
0 Cryptococcus laurentii 40
0 K. ozaenae 27 15 Kloeckera apis apicuata 61
15 K. oxytoca 18 Cryptocccus albidus 33
E. herbicola 28 20 Candida guilliermondii 26
20 K. ozaenae 73 Kloeckera apis apiculata 26

a a
Frequency related to the total aerobic mesophilic population. Frequency related to the total yeast population.

Table 2. Main species of lactic acid microflora during coffee

fermentation
Time (h) Strains
Frequency (%)
10 Ln. mesenteroides dextranicum
15 Ln. mesenteroides dextranicum
20 Lb. brevis
a
Frequency related to the total lactic acid population.
detected. Aerobic microflora seemed to be more hetero-
geneous when water was extensively used in the process.
Pseudomonas cepaciae and Chrysomonas luteola were
then detected (data not shown). The water used for
pulping was usually highly contaminated by aerobic me-
sophilic microflora (5.2 ⫻ 106 CFU/ml), mainly com-
posed of enterobacteria (2.3 ⫻ 104 CFU/ml) (data not
shown). This water must contaminate the endogenous
coffee microflora.
Lactic acid bacteria. Identified species (Table 2) (Ln.
mesenteroides dextranicum, Lb. brevis) were heterofer-
mentative with production of acetic and lactic acids. At
the final stage, a change of lactic acid population was
observed with the Lb. brevis appearance. These lactic
acid bacteria are usually present in vegetable fermenta-
tions: Lb. brevis would consume galacturonic acid as the
a
sole source of carbon, and Ln. mesenteroides produces a
pectinase activity [19, 29].
67
10 Yeast. Yeasts isolated were varied and consisted of
14 classical strains found in plants like Kloeckera, Candida,
and Cryptococcus genuses. These microorganisms have
a good fermentative capacity with ethanol production
(Table 3). Apiculated strains, like Kloeckera apis apicu-
lata, are generally found in vegetable fermentations,
when the alcohol level is low, as observed in coffee
fermentation. The identified strains were not reported as
pectolytic microorganisms [21, 25].
Pectolytic microflora. Bacteria isolated on pectin me-
dium were Gram-negative bacilli (71%), and the most
frequent strains were K. pneumoniae and E. herbicola
with a relative frequency from 31% to 46% (Table 4).
These microorganisms were not reported as strong pec-
tolytic strains [25]. No yeasts were identified on this
medium.
Biochemical results. Mucilages from fermented and
unfermented beans were analyzed for sugars and organic
acids contents (Fig. 3). A significant decrease (⬃60%) of
total simple sugars (glucose, fructose, and sucrose) was
S. Avallone et al.: Coffee Fermentation 255

Table 4. Main species isolated on pectin medium during coffee Discussion

fermentation
The initial coffee fermentation microflora were rich and
Time (h) Strains Frequencya (%) significantly acidified the coffee mass after a lag phase.
Aerobic microflora were predominant and more hetero-
10 K. pneumoniae 31
E. herbicola 21
geneous when water was extensively used in the process.
20 K. pneumoniae 46 During fermentation, lactic acid bacteria and yeasts
E. herbicola 29 grew, whereas pectolytic microflora remained stable. At
the end of the fermentation, yeasts were quantitatively
a
Frequency related to the total population on pectin medium. important when the pH was low because of their higher
resistance to acid conditions. These microorganisms
could be responsible for the alcoholic taste of the coffee
beverage after overfermentation [18, 20]. Qualitatively,
the yeast population was heterogenous, and aerobic bac-
teria were mainly Gram-negative bacilli. Lactic acid
bacteria belonged to Ln. mesenteroides specie in agree-
ment with others observations [24, 32]. This strain could
initiate the acidification as described for sauerkraut [23],
and lactobacilli would then acidify the fermentation me-
dium with the highest organic acid production [33].
Furthermore, Ln. mesenteroides would play a role in the
solubilization of pectic substances [19].
Contrary to the hypothesis of several authors [1, 2],
no rise of the pectolytic microorganisms was observed
during the fermentation step. These microflora were not
Fig. 3. Simple sugar content of the mucilage during coffee fermenta- more adapted to the substrate than the others. Further-
tion. more, the most frequent pectolytic strains, identified as
K. pneumoniae, E. dissolvens, and E. herbicola, were not
reported as strongly producing enzyme bacteria [4, 25].
Contrary to previous assertions and cocoa fermentation
[1, 28], no yeasts were involved in the eventual pectoly-
sis of mucilage.
Sugar consumption by the microflora was an impor-
tant metabolism. The lactic acid bacteria identified were
heterofermentative, but other metabolisms could take
place as mixed acid fermentation by Enterobacteriaceae.
In general, microorganisms first consume the substrate
that is easily metabolized, such as a monosaccharide,
before hydrolyzing a polysaccharide. Viewing the high
sugar content of the mucilage, bacteria must consume
these sugars preferably before using pectin breakdown
products. It is probably the reason that no rise of pecto-
Fig. 4. Organic acids and ethanol content of the mucilage during coffee lytic microflora number was observed throughout the
fermentation. fermentation.
These results are in opposition to the hypothesis of
mucilage degradation by pectolytic microorganisms [2,
observed throughout the fermentation time. Concomi- 8, 13, 14] but confirm the microscopical observations
tantly, microflora produced lactic and acetic acids, in- that prove that mucilage was an organized tissue after
ducing acidification from pH 6.3 to 4.1 (Fig. 4). At the fermentation and that the pectic substances of the cell
end of the fermentation, the acetic acid content (7.5%/ walls were still present [3]. Furthermore, they confirm
MS) was higher than lactic acid (4.5%/MS). The micro- previous works which demonstrated that the major cof-
flora greatly consumed simple sugars, but only produced fee-fermenting microorganisms (E. dissolvens, E. herbi-
a low level of organic acids and ethanol. cola and K. pneumoniae) produced only pectatelyase
256
activity unable to depolymerize highly methylated pec-
tins of coffee mucilage [4, 5, 10].
These microbiological and biochemical results show
that the mucilage, rich in monosaccharide, allows the
growth of the total mucilage microflora and not specifi-
cally pectolytic microorganisms. If pectin degradation
occurred during coffee fermentation, it must be very
limited. Polysaccharide analysis would be necessary to
confirm that pectolytic microflora play a minor role in
mucilage liquefaction.
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