World Journal of Microbiology and Biotechnology (2020) 36:186

https://doi.org/10.1007/s11274-020-02963-7

ORIGINAL PAPER

The use of mesophilic and lactic acid bacteria strains as starter

cultures for improvement of coffee beans wet fermentation
Luciana Silva Ribeiro1 · Maria Gabriela da Cruz Pedrozo Miguel1 · Silvia Juliana Martinez1 ·
Ana Paula Pereira Bressani2 · Suzana Reis Evangelista1 · Cristina Ferreira Silva e Batista1 · Rosane Freitas Schwan1 

Received: 27 April 2020 / Accepted: 12 November 2020

© Springer Nature B.V. 2020

Abstract 
The use of starter cultures during food fermentation aims to standardize the process and to obtain a higher quality product.
The objectives were to study mesophilic bacteria (MB) and lactic acid bacteria (LAB) isolated from wet coffee processing
and evaluate their performance in a pulped coffee medium. Eighty-six bacteria isolates (59 MB and 27 LAB) were assessed
for pectinolytic activity, metabolite production, and pH value decrease in coffee-based culture (CPM). Seven bacteria strains
(3 MB and 4 LAB) were selected and used as starter cultures in the wet fermentation of pulped coffee. The MB and LAB
populations varied from 4.48 to 8.43 log CFU g−1 for MB and 3.54 to 8.72 log CFU g−1 for LAB during fermentation.
Organic acid concentration (ranged from 0.01 to 0.53 for succinic acid; 0.71 to 8.14 for lactic acid and 0.06 to 0.29 for acetic
acid), and volatile compounds (44 compounds were detected in green beans and 98 in roasted beans) were evaluated during
fermentation. The most abundant compounds found in roasted beans belong to furans [15], ketones and esters [14], pyridines
[13], and pyrazines [12]). Leuconostoc mesenteroides CCMA 1105 and Lactobacillus plantarum CCMA 1065 presented
volatile compounds important for coffee aroma. Isovaleric acid; 2,3-butanediol; phenethyl alcohol; β-linalool; ethyl linoleate;
and ethyl 2-hydroxypropanoate could improve cupping qualities.

Electronic supplementary material  The online version of this

article (https​://doi.org/10.1007/s1127​4-020-02963​-7) contains
supplementary material, which is available to authorized users.

* Rosane Freitas Schwan

rschwan@ufla.br
Luciana Silva Ribeiro
luciana.ufla@gmail.com
Maria Gabriela da Cruz Pedrozo Miguel
mgcpmiguel@gmail.com
Silvia Juliana Martinez
silvia292004@gmail.com
Ana Paula Pereira Bressani
paulinhabressani@hotmail.com
Suzana Reis Evangelista
suzanareise@gmail.com
Cristina Ferreira Silva e Batista
cristinafsb@ufla.br
1
Department of Biology, Federal University of Lavras,
Campus Universitário, Lavras, MG 3037, Brazil
2
Food Science Department, Federal University of Lavras,
Lavras, MG, Brazil

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 186   Page 2 of 15
Graphic abstract
Keywords  Acid lactic bacteria · Arabica · Coffee aroma · Coffee fermentation · Mesophilic bacteria · Volatile metabolites
Introduction
The mucilage of coffee fruits is an insoluble hydrogel rich in
fermentable sugars and nitrogen that turn into a suitable sub-
strate for microbial growth (Silva 2015). Along with micro-
bial reproduction, those molecules are either transformed in
enzymes, acids, or volatile compounds. Previously described
enzymes include the polygalacturonase (PG), which cata-
lyzes the hydrolysis of 1,4-glycosidic bonds in polygalac-
turonic acid. The pectin lyase (PL) that catalyzes pectin
breakdown by releasing galacturonic unsaturated acids.
Lastly, the pectin methylesterase (PME) that is responsible
for de-esterification of the methoxy group of pectin-forming
pectic acid and methanol (Agate and Bhat 1966; Massawe
and Lifa 2010; Silva et al. 2013; Haile and Kang 2019).
Mutual interaction of extracellular enzymes and organic
acids leads to macromolecules’ hydrolysis (carbohydrates,
proteins, and polyphenols). It resulted in important aroma
precursors, such as reducing sugars, proteins, amino acids,
and phenolic compounds (Lee et al. 2015).
Changes of the volatiles profile and cupping qualities
in roasted coffee depend on the chemical composition of
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World Journal of Microbiology and Biotechnology (2020) 36:186
precursors present in green beans before roasting. Differ-
ent concentrations of flavor precursors in green beans can
provide different profiles of coffee beverages. For example,
furans are among the predominant coffee flavor groups and
produce caramel-like odors (Batista and Chalfoun 2015).
As furans, esters are associated with positive notes because
they positively influence beverage by producing fruity fla-
vors (Toledo et al. 2016). These chemical variations are also
influenced by the fermentation phase, just as reported in Lee
et al. (2015), Bressani et al. (2019), and Wang et al. (2019a),
which showed differences in the non-volatile profile of green
beans and the flavor profile.
Microorganisms have been used as starter cultures in dif-
ferent fermentative processes for thousands of years. The
evolution of taxonomy and identification methods has led
to various extensions of these microorganisms’ use in fer-
mented foods (Bourdichon et al. 2012; Elhalish and Zhao
2020). Yeasts’ use as starter cultures in natural and semi-
dry coffee has been documented (Evangelista et al. 2014;
Ribeiro et al. 2017; Martinez et al. 2017; Bressani et al.
2018); however, the use of bacteria in the wet process is still
unknown. There are numerous bacterial species possibilities
World Journal of Microbiology and Biotechnology (2020) 36:186 Page 3 of 15  186
as starter cultures that can and should be exploited, espe-
cially for wet processing.
Microbial starter cultures produce coffee with different
aromas and flavors, leading to new perspectives on cof-
fee quality. The relationship between coffee fermentation
and coffee aroma profile is very close (Jackels and Jackels
2005; Jackels et al. 2006; Gonzaléz-Rios et al. 2007; Lin
2010). With optimized parameters and selection of starter
cultures to remove mucilage, controlled fermentation might
confer specific desirable attributes to coffee beverages. The
targeted use of different bacteria species would lead to a
greater diversification of flavors, which is already evident in
the fermented food and wine industry (Marshall and Mejia
2011). Therefore, this work’s objective was to select bacteria
strains isolated from arabica coffee fruits capable of produc-
ing organic acids and volatile compounds during wet coffee
fermentation.
Material and methods
Bacteria isolates
The microorganisms were isolated during the wet process
from three different coffee varieties (Coffea arabica L.
var. Ouro Amarelo, Mundo Novo, and Catuaí Vermelho)
(Ribeiro et al. 2018). A total of 86 coffee isolates (including
59 Mesophilic Bacteria (MB) and 27 Lactic Acid Bacteria
(LAB)) (Table 1). The MB was reactivated on nutrient broth
NB (Himedia, Mumbai, India). The LAB was maintained on
MRS (Man Rugosa Sharp) broth (Merck, Darmstadt, Ger-
many) during the experiment conduct, and both scored in
an ultra-freezer at − 80 °C, containing 20% glycerol (w/w).
Experimental design
The work plan was conducted in two stages, as shown in
Fig. 1. First, 86 isolates were tested for their ability to pro-
duce pectinase and metabolites using a coffee pulp medium
(CPM). Secondly, the best-selected strains were inoculated
in wet coffee fermentation.
Characterization and selection of best bacteria
strains
Pectinase activity of microorganisms
The pectinolytic activity of the 86 strains was determined
as described in Schwan et  al. (1997) and Silva et  al.
(2013). The strains were grown on plates containing MP5
mineral medium (0.5% glucose, 0.5% polygalacturonic
acid, 0.6% ­KH2PO4, 0.1% yeast extract, 0.2% ­(NH4) ­SO4,
1.5% agar, and 0.1 mL of the solutions: F ­ eSO4 0.0001%,
­MgSO4 0.02%, ­CaCl2 0.0002%, ­H3BO3 0.0002%, 0.0002%,
­M nSO 4, 0.0014%, Z ­ nSO 47H 2O, 0.001%, C ­ uSO 45H 2O,
0.0002% ­MoO3) for determination of the activity of the PG
enzyme. The determination of the PL enzyme activity was
carried out using an MP7 medium; this had the same con-
stitution as described above, except for a substitution of
polygalacturonic acid for the citrus pectin. A CTBA solu-
tion 1% (polygalacturonic acid and pectin with cetylmethyl
ammonium bromide) was used as the revealed solution
for the enzymatic assay, being considered positive when
there was the formation of clear halo around the colony.
The yeast Kluyveromyces marxianus CCT 3172 (Schwan
et al. 1997) was used as a positive control.
Quantification of pectin lyase (PL) and pectin
methylesterase (PME) activity
The activity of PL and PME was quantified in culture
mediums with coffee, using only strains that showed posi-
tive qualitative pectinase results (test described above).
The PL activity was determined by increasing the absorb-
ance of the medium (Albersheim 1966), using the extinc-
tion coefficient of 5550 M−1 cm−1. A pectin solution (3 mL
citrus pectin 2.5% in 100 mM phosphate buffer, pH 6.8)
was mixed with 4.5 mL of culture supernatant. The mix-
ture was incubated at 40 °C, and 0.5 mL was collected at 0,
10, 15, 20, and 30 min; each time, the reaction stopped by
adding 4.5 mL of 0.01 M HCl. The samples were analyzed
at 235 nm. The equipment was calibrated with a pectin
solution and culture medium containing coffee without
inoculation.
The PL activity was expressed from the calculation of
the galacturonides concentration released by the PL action:
A = 𝜀 × l × C,
where A = the absorbance of the sample at 235 nm; ε = the
molar extinction coefficient ­(M−1 cm−1); 1 (one) = the opti-
cal path (1 cm); and C = analyte concentration, expressed
in M.
The PME activity quantification was determined by
titration according to the methodology proposed by Bara-
cat et al. (1989). The reaction was carried out with 3 mL of
the culture supernatant mixed with 20 mL of citrus pectin
1% (60% methoxylated or higher) in a solution of NaCl
0.1 M. The pH 7.5 was adjusted with NaOH 0.5 M. This
solution was incubated for 30 min at 30 °C, and the pH
was maintained at 7.5 by the addition of NaOH 0.02 M.
The PME activity was proportional to the NaOH volume
spent to keep the pH at 7.5. PME activity was expressed
as the micro equivalent of polygalacturonic acid ­mL−1 h−1.
The analyzes were carried out in triplicate.
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Table 1  Bacterial isolates used Species Number strain Species Number strain

in fermentative performance
in culture medium containing Bacillus subtilis group CCMA ­1228OA Rhizobium radiobacter CCMA ­1212MN
coffee peel and pulp CCMA ­1234 MN

CCMA ­1235CV Rhizobium pusense CCMA ­1200CV

CV
CCMA ­1236
CCMA ­1237CV Rhodococcus pyridinivorans CCMA ­1214CV
CV
CCMA ­1238 CCMA ­1282CV
CV
CCMA ­1239
CCMA ­1240CV Pantoea vagans CCMA ­1208OA
CV
CCMA ­1241 CCMA ­1209OA
CV
CCMA ­1242 CCMA ­1210OA
CV
CCMA ­1244
Bacillus pumilus group CCMA ­1248CV Pantoea agglomerans CCMA ­1211CV
CV
CCMA 1249 CCMA ­1277CV
CCMA ­1250MN Microbacterium paraoxydans CCMA ­1152CV
CCMA ­1251CV CCMA ­1153CFW
CCMA1252CV Microbacterium testaceum CCMA ­1147CFW
CCMA ­1272CV CCMA ­1148CFW
CCMA 1273 CV CCMA ­1150CFW
CCMA 1274 CV CCMA ­1151MN
Bacillus safensis CCMA ­1232OA CCMA ­1082OA
CCMA ­1083OA
OA
Bacillus megaterium CCMA ­1245 CCMA ­1084OA
VC
CCMA ­1246 CCMA ­1085OA
CCMA ­1087MN
OA
Bacillus clausii CCMA ­1229 Leuconostoc mesenteroides CCMA ­1089MN
OA
CCMA ­1230 CCMA ­1090MN
OA
CCMA ­1231 CCMA ­1105CV
OA
CCMA ­1283 CCMA ­1106CV
OA
Bacillus licheniformis CCMA ­1233 CCMA ­1108CV
CCMA ­1109CV
OA
Bacillus asahii CCMA ­1247 CCMA ­1110CV
CCMA ­1111CV
OA
Bacillus horneckiae CCMA ­1265 CCMA ­1112CV
CV
CCMA ­1266 CCMA ­1115CV
CCMA ­1117CV
MN
Bacillus humi CCMA ­1217 CCMA ­1120CV
MN
Bacillus simplex CCMA ­1216 Lactobacillus plantarum CCMA ­1058OA
CCMA ­1059OA
OA
Lysinibacillus fusiformis CCMA ­1263 CCMA ­1064OA
MN
CCMA ­1264 CCMA ­1065OA
CCMA ­1067MN
MN
Lysinibacillus macroides CCMA ­1280 CCMA ­1071CV
MN
CCMA ­1281 CCMA ­1072CV
CCMA ­1076CV
MN
Brevibacillus parabrevis CCMA ­1218 CCMA ­1078CV
CCMA ­1080CV
CV
Psychrobacillus psychrotolerans CCMA ­1215 Paenibacillus cookii CCMA ­1267OA
CFW
Paenibacillus illinoisensis CCMA ­1269 Paenibacillus lactis CCMA ­1268MN
OA
Paenibacillus konsidensis CCMA ­1253 Arthrobacter koreensis CCMA ­1157CV
CCMA ­1279CV
OA MN CV CFW
ISOLATED FROM: Coffea arabica var. Ouro Amarelo; Mundo Novo; Catuaí Vermelho; Cof-
fee fermentation water

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World Journal of Microbiology and Biotechnology (2020) 36:186 Page 5 of 15  186
Fig. 1  Flowchart of the bacteria
selection process
Growth assessment in coffee‑based medium
The bacteria fermentation were carried out in a culture
medium containing coffee pulp (CPM) as described in
Silva et al. (2013):100 g of coffee cherries were homog-
enized in a ­Stomacher ® with 1 L of distilled water for
10  min; it was then filtered, and 0.5% of glucose was
added. The pH value was adjusted to 5.5 with HCl (0.1 M)
solution, and the media were sterilized in flowing steam
for 20 min.
The bacteria were grown up to a concentration of
­10  CFU mL−1 in NB for MB and MRS for LAB. Subse-
7
quently, the cells were centrifuged and resuspended in ster-
ile peptone water (0.1%). The inoculum ­(107 CFU mL−1)
was transferred separately to 500 mL flasks containing
CPM medium (100 mL) and kept at 30 °C for 48 h. Sam-
ples were collected at intervals (12, 24, and 48 h of fer-
mentation and final drying) for chemical and microbio-
logical analysis. The fermentations were carried out in
triplicate.
Microbiological analysis
Serial decimal dilutions were performed with sterile pep-
tone water (0.1% bacteriological peptone) to assay the MB
and LAB population. The MB population was quantified on
nutrient agar, and LAB was quantified on MRS agar. The
plates were incubated at 30 °C and 35 °C, respectively, for
48 h.
Organic acids
Malic, lactic, acetic, butyric, propionic, citric, oxalic, suc-
cinic, and tartaric acid were analyzed by high-performance
liquid chromatography (HPLC). Samples of fermented
coffee medium were centrifuged (10.000 rpm at 4 °C for
10 min), and the supernatant was filtered using a 0.22 μm
cellulose acetate filter. The extracts were analyzed using an
HPLC system (Shimadzu, Japan). A Shimpack SCR-101H
(7.9 mm × 30 cm) column was used with a 100 mM solu-
tion of perchloric acid with a flow rate of 0.6 mL/minute as
the mobile phase. The oven temperature was kept at 50 °C,
detected with a 210 nm UV detector. The compound quan-
tification was performed using calibration curves, differ-
ent standard concentrations were analyzed using the same
conditions used for the samples, and the analyses were per-
formed in triplicate.
Second experimental stage
Coffee fermentation inoculated with bacteria
Selected bacteria strains were used to carry out fermenta-
tion on a larger scale and the control fermentation, which
is without inoculation. Coffee cherries of the Catuaí Ver-
melho variety were mechanically harvested on a farm
located in Lavras, Minas Gerais State, Brazil, at 900 m
above sea level. Three kilograms of pulped coffee were
fermented in plastic buckets with 2 L of sterile distilled
water. A population of ­107 ­CFUg−1 of coffee of the cho-
sen strains was inoculated separately in each vessel. The
fermentation time was 48 h. After fermentation, the water
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 186   Page 6 of 15
was discarded, and coffee was transferred to suspended
terraces for sun drying until 11–12% moisture [Gehaka
model G650J]. Samples were collected for microbiologi-
cal and chemical analysis. The coffee fermentation was
performed in triplicate.
Parameters evaluated in inoculated coffee
The bacterial population and organic acid concentration
in the inoculated coffee were assessed according to the
above items.
Analyses of volatile compounds were done on green
and roasted coffee by gas chromatography/mass spectrom-
eter (GC/MS). Volatile compounds were extracted using a
manual headspace-solid-phase microextraction procedure
(HS-SPME), according to Evangelista et al. (2014). The
compounds were analyzed using a Shimadzu QP2010 GC
model equipped with mass spectrometry (MS) and a silica
capillary Carbo-Wax 20 M (30 m × 0.25 mm × 0.25 mm)
column. Operating conditions were performed as described
by Ribeiro et  al. (2017). The volatile compounds were
identified by comparing the mass spectra to the NIST11
library. Besides, an alkane series (C10–C40) was used to
calculate the retention index (RI) for each compound, which
was compared with RI values found in the literature data
(Czerny and Grosch 2000; Eom and Jung 2013; Piccino
et al. 2014).
Statistical analysis
The organic acid data were evaluated by variance analy-
sis (ANOVA), and the Scott–Knott test was used for com-
parison between means. An 8 × 4 factorial arrangement
of treatment was used to analyze the results of the organic
acid: ten treatments (control, CCMA 1234, CCMA 1251,
CCMA 1151, CCMA 1082, CCMA 1105, CCMA 1065,
CCMA 1072) and five sample collection times (T12, T24,
T48, dried coffee). The data were analyzed under the fol-
lowing model:
X = 𝜇 + Treatj + Timek + Treat × Timejk + ejk.
where µ  =  global mean; Treatj  =  treatment effect
(j = control, CCMA 1234, CCMA 1251, CCMA 1151,
CCMA 1082, CCMA 1105, CCMA 1065, CCMA 1072);
Timek = time collected effect (k = T12, T24, T48, dried
coffee); Treat × Timejk = effect of interaction between
treatment and time collection time; and ejk = experimen-
tal error. The tests were performed using Sisvar 5.6 soft-
ware (Ferreira 2014). The significance level was defined
at p < 0.05 level.
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World Journal of Microbiology and Biotechnology (2020) 36:186
Results
Evaluation of the fermentative performance
of bacteria
The first criterion was the ability to decrease the pH value,
the initial pH was 5.5, and a variation between the strains
tested was observed (Fig. 2), where some strains promoted
increased, others decreased the pH value. Microbacterium
testaceum CCMA 1147 and 1148 the strains that presented
the most considerable pH reduction, significantly different
from the others (3.73 and 3.55, respectively). In compari-
son, the LAB strains lowered the pH value to around 3.7
(Fig. 3). Leuconostoc mesenteroides CCMA 1082, 1105,
and 1117 were the strains that showed the most significant
pH reduction, significantly different from the others (3.68,
3.40, and 3.67, respectively). The decrease in pH values
was from 26 to 38% in fermentation using LAB as a starter
culture, whereas fermentation inoculated with MB reduced
the pH values by up to 32%.
The results population counting criterion showed that
the initial population inoculated was 7 log CFU g−1(Fig. 2),
strains that increase their population, or remain in the ini-
tial community are of interest. Arthrobacter koreensis
CCMA 1157 (8.66 CFU g−1), Microbacterium testaceum
CCMA 1151 (8.84 CFU g−1), Leu. mesenteroides CCMA
1082, 1090, 1105 (8.05; 8.08; 8.04 CFU g−1, respectively)
and Lactobacillus plantarum CCMA 1065 and 1072 (7.95
and 7.93 CFU g−1, respectively) were the strains that pre-
sented the largest final population, significantly different
from the others (Figs. 2, 3).
Eighty-six bacterial isolates tested on plates, 22 strains
showed a positive result for PL, and 4 showed a positive
outcome for PME, and the results were based on growth or
non-growth (data not shown). Only MB showed positive
results for pectinolytic activity, Bacillus subtilis CCMA
1234, 1236, and 1237, and Paenibacillus konsidensis
CCMA 1253 showed higher results for PL activity (0.073;
0.064; 0.057 and 0.073 U/mL, respectively). B. subtilis
CCMA 1238 and 1239 showed the highest results for PME
activity of 0.483 and 0.450 micro equivalent m ­ L−1 h−1,
respectively (Table 2).
Metabolites results presented below are associated
with fermentation in coffee-based medium (Supplemen-
tary Material). The citric acid production was mainly
characterized when inoculated with Bacillus pumilus
CCMA 1248; B. pumilus CCMA 1251; and B. horneckiae
CCMA 1265, and Leuconostoc mesenteroides CCMA
1105. Malic and tartaric acid production were associated
with Pantoea agglomerans CCMA 1277 and between the
LAB, Leu. mesenteroides CCMA 1082 and Leu. mesenter-
oides CCMA 1106 was related to malic acid production.
World Journal of Microbiology and Biotechnology (2020) 36:186 Page 7 of 15  186
Fig. 2  Mesophilic bacterial population ­(Log10 CFU g-1 ) and pH
( ) of culture medium containing coffee pulp at final fermenta-
tion. (1–11) B. subtilis; (12–19) B. pumilus; (20) B. safensis; (21–22)
B. megaterium; (23–26) B. clausii; (27) B. licheniformis; (28) B. asa-
hii; (29–30) B. horneckiae; (31) B. humi; (32) B. simplex; (33–34) L.
fusiformys; (35–36) L. macrolides; (37) B. parabrevis; (38) P. psy-
chrotolerans; (39) P. illinoisensis; (40) P. konsidensis; (41) P. cookie;
Fig. 3  Lactic acid bacterial population ­(Log10 CFU g-1 ) and pH
( ) of culture medium containing coffee pulp at final fermenta-
tion. (1–17) Leu. mesenteroides; (18–27) Lac. plantarum. The stand-
Arthrobacter koreensis CCMA 1157 and P. dispersa
CCMA 1278 were mainly characterized by propionic acid
production.
Succinic and acetic acids were primarily associated with
Microbacterium paraoxydans CCMA 1152, Rhizobium
radiobacter CCMA 1212, and R. pusense CCMA 1200, and
(42) P. lactis; (43–44) A. koreensis; (45) R. radiobacter; (46) R. puse-
nse; (47–48) M. paraoxydans; (49–52) M. testaceum; (53–54) R. pyri-
dinivorans; (55–57) P. vagans; (58–59) P. agglomerans. The stand-
ard deviation of the mean pH was less than 0.05. Error bars represent
standard deviations of means of population. There was a statistical
difference at p < 0.05 by the Scott–Knott test
ard deviation of the mean pH was less than 0.05. There was a statisti-
cal difference at p < 0.05 by the Scott–Knott test
almost all Leu. mesenteroides (CCMA 1083, CCMA 1085,
CCMA 1087, CCMA 1090, CCMA 1109, and CCMA 1112)
tested were characterized by acetic acid production.
Microbancterium testaceum CCMA 1147 and 1148,
Lactobacillus plantarum CCMA 1065, and CCMA 1072
were characterized by lactic acid production. Strains
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Table 2  Pectin lyase (PL) No. Strain Species PLa (U/mL) PMEb ­(mL−1 h−1)
and pectin methyl esterase
(PME) activities quantified in 1 CCMA 1234 Bacillus subtilis 0.073 ± 0.01c 0.100 ± 0.01a
CPM medium fermented with
2 CCMA 1228 0.013 ± 0.03a 0.250 ± 0.00b
mesophilic and lactic acid
bacteria 3 CCMA 1236 0.064 ± 0.00c 0.070 ± 0.00a
4 CCMA 1237 0.057 ± 0.02c 0.100 ± 0.06a
5 CCMA 1238 0.033 ± 0.01b 0.483 ± 0.03d
6 CCMA 1239 0.021 ± 0.00b 0.450 ± 0.00d
7 CCMA 1240 0.024 ± 0.03b nd
8 CCMA 1241 0.005 ± 0.02a 0.250 ± 0.00b
9 CCMA 1242 0.029 ± 0.00b 0.383 ± 0.03c
10 CCMA 1244 0.001 ± 0.02a 0.250 ± 0.00b
11 CCMA 1248 Bacillus pumilus 0.016 ± 0.00a nd
12 CCMA 1251 0.025 ± 0.01b nd
13 CCMA 1273 0.010 ± 0.02a 0.250 ± 0.00b
14 CCMA 1253 Paenibacillus konsidensis 0.073 ± 0.01c 0.100 ± 0.06a
15 CCMA 1211 Pantoea agglomerans 0.033 ± 0.01b 0.070 ± 0.04a
16 CCMA 1278 Pantoea dispersa 0.055 ± 0.05b 0.633 ± 0.03e
17 CCMA 1279 Arthrobacter korensis 0.001 ± 0.00a nd

nd not detected. a–e mean values with different letters are significant at p < 0.05 for each treatment based
on the Scott–Knott test
a
 Expressed as the amount (in μmol) of unsaturated galacturonides/min per ml of culture supernatant U/mL)
b
 Expressed as micro equivalents of pectic acid released per ­ml−1 h−1)

Fig. 4  Population count (­log10 ­CFUg-1) of mesophilic bacteria (a) (----); Lac. plantarum CCMA 1065 (.....); Microbacterium testaceum
and lactic acid bacteria (b) during fermentation and drying of wet- CCMA 1151 (♦); Bacillus pumilus CMMA 1251 (▲) and Bacillus
processed coffee. Control (•); Leu. mesenteroide CCMA 1105 (*); subtilis CCMA 1234 (■)
Leu. mesenteroide CMMA 1082 (×); Lac. plantarum CCMA 1072

belonging to the Lac. plantarum species increased the
lactic acid concentration by more than 50%. Based
on these criteria, the selected strains were B. subtilis
CCMA 1234, B. pumilus CCMA 1251, Microbacterium
testaceum CCMA 1151, Leu. mesenteroides CCMA
1082 and CCMA 1105, Lac. plantarum CCMA 1065
and CCMA 1072.
Use of starter selected bacteria in wet coffee
fermentation
Three MB and four LAB strains were inoculated separately
in wet coffee fermentation. Among the MB count tested,
there was a significant difference (p < 0.05) in the treatments
and fermentation time (Fig. 4a). During fermentation (T12
to T48), there was a significant decrease (p < 0.05) in MB in

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World Journal of Microbiology and Biotechnology (2020) 36:186 Page 9 of 15  186
Fig. 5  Organic acid concentration (mg  g−1) of culture medium con-
taining coffee pulp fermented with Mesophilic Bacteria at final fer-
mentation. a Succinic acid; b lactic acid; c acetic acid. Control (•);
Leu. mesenteroide CCMA 1105 (*); Leu. mesenteroide CMMA 1082
almost all the fermentations. However, in treatments inocu-
lated with B. subtilis CCMA 1234 and B. pumilus CCMA
1251, there was an increase in population (from 4.91 to 5.34
and 5.34 to 6.04 log CFU g−1, respectively). After T48 of
fermentation, the MB decreased its population. There was
a significant increase (p < 0.05) in the LAB population in
fermentations inoculated with MB. The fermentation inocu-
lated with Lac. plantarum CCMA 1065, CCMA 1072, and
Leu. mesenteroide CCMA 1105 showed a decrease of the
LAB population (reaching the end of fermentation [T48]
with an average population of 7,24; 6,85 and 6.65 log
CFU g−1, respectively).
Succinic, lactic, and acetic acids were detected, and
there was a significant difference (p < 0.05) among treat-
ments (Fig. 5), and these acids, the lactic acid was pro-
duced in greater concentration in all fermentations. There
was a decrease in the succinic acid concentration (Fig. 5a)
inoculated with MB and Leu. mesenteroides CCMA1082.
Inoculated fermentation with LAB (Leu. mesenteroides
CCMA 1105, Lac. plantarum CCMA 1065, and CCMA
1072) showed an increase in the succinic acid concentra-
tion (Fig. 5a). At the end of drying, there was an increase
(×); Lac. plantarum CCMA 1072 (----); Lac. plantarum CCMA 1065
(.....); Microbacterium testaceum CCMA 1151 (♦); Bacillus pumilus
CMMA 1251 (▲) and Bacillus subtilis CCMA 1234 (■)
in succinic acid concentration in all treatments. The fer-
mentation with Leu. mesenteroides CCMA 1105 presented
the highest concentration of succinic acid (0.54 mg g−1),
and Leu. mesenteroides CCMA 1082 presented the lowest
concentration (0.22 mg g−1).
There was an increase (p < 0.05) in lactic acid con-
centration, with the highest increase in final drying time
(dried coffee) (Fig. 5b). The fermentation inoculated with
LAB showed a higher lactic acid concentration, which was
already expected. The fermentation with MB showed 5.0
and 5.9 mg g −1 of lactic acid, while fermentation with
LAB showed 7.6 and 8.1 mg g −1. The highest concen-
tration of acetic acid was found in fermentation without
inoculation (control—0.18 mg g−1) in 48 h of fermenta-
tion (Fig. 5c). All treatments showed an increase in ace-
tic acid concentration at the end of fermentation (T48)
(Fig. 5c). Acetic acid was not detected in the fermentation
inoculated with Leu. mesenteroides CCMA 1105 at any
sampling time, and in fermentations with Lac. plantarum
CCMA 1072 and 1065 presented low concentrations of
this acid, which seems to be a positive point for the use of
these starter cultures.
13
 186   Page 10 of 15
Forty-four volatile compounds were detected by HS-
SPME GC–MS analysis in the green coffee fermented with
bacteria inoculation and spontaneous fermentation (Table 3).
Some compounds were detected in specific samples, such as
1-phenoxypropan-2-ol (B. subtilis CCMA 1234), phytol, and
ethyl linoleate (C. cellulans CCMA 1186) and octadecane
(Leu. mesenteroides CCMA 1105). The fermentation with
M. testaceum CCMA 1151 and Lac. plantarum CCMA 1065
showed a higher percentage of aldehydes and ketones, pre-
dominately found after 48 h. Isovaleric acid was detected in
spontaneous fermentation, Leu. mesenteroides CCMA 1105,
and d Lac. plantarum CCMA1065. While 2,3-butanediol
in coffee beans fermented by C. cellulans CCMA1186).
Phenethyl alcohol was detected in samples from coffee beans
without inoculation, Leu. mesenteroides CCMA 1105, and
Lac. plantarum CCMA1065. β-linalool (Leu. mesenteroides
CCMA 1105); 2-hexenal (without inoculation and Leu. mes-
enteroides CCMA 1105); ethyl 2-hydroxypropanoate (with-
out inoculation and Leu. mesenteroides CCMA 1105); and
tetracosane (Leu. mesenteroides CCMA 1105) influenced
coffee quality.
A total of 98 volatile compounds was detected in the
roasted coffee (Table 4). Among these compounds, acids
(6), aldehydes and ketones (14), esters (14), furans (15), lac-
tones (4), phenols (4), pyrans (3), pyrazines (12), pyridines
(13), pirroles (8) and other compounds (alkaloids, alcohol,
sulfur compounds and amines) were classified. Some com-
pounds were detected in all samples, which means that they
are compounds inherent in the coffee variety studied and
compounds naturally present in roasted coffee. Fermenta-
tion with Leu. mesenteroides CCMA1105 and B. pumilus
CCMA1251 showed the highest percentages of aldehydes
and ketones. M. testaceum CCMA1151 had the highest rate
of esters. Leu. mesenteroides CCMA1082, Lac. plantarum
CCMA1072 and spontaneous fermentation (control) had a
higher percentage of furans. Fermentations inoculated with
Leu. mesenteroides CCMA1105 presented a higher rate of
lactones, phenols, and pyrans, respectively.
Pyrazines and pyridines were shown in higher percent-
ages in the fermentations with M. testaceum CCMA1151
and Lac. plantarum CCMA1065. The Leu. mesenteroides
CCMA1082 had a higher percentage of pyrrole. Some com-
pounds were detected in a few samples, such as 2,3-octane-
dione (B. subtilis CCMA1234); 2,4-pentanedione, 3-methyl-
(Leu. mesenteroides CCMA1105); phthalic acid, hept-3-yl
isobutyl ester (without inoculation); phthalic acid, hex-3-yl
isobutyl ester; phenol, 2-methoxy- (B. pumilus CCMA1251);
vinyl crotonate; dibutyl phthalate; pyrazine, 2-ethyl-methyl-;
ethanone, 1-(2-hydroxy-5-methylphenyl); 3-hydroxypyridine
monoacetate (M. testaceum CCMA1151); undecanoic acid
isopropyl ester; acetamide, N-(3-methyl-2-oxobutyl)- (Lac.
plantarum CCMA1072); 1H-indole, 3-methyl- (Leu. mes-
enteroides CCMA1082).
13
World Journal of Microbiology and Biotechnology (2020) 36:186
Discussion
We used four criteria for selecting bacteria to be used as
starter cultures in wet coffee fermentation. And these were
(i) pH decrease, (ii) increase in bacteria population, (iii)
pectinases, and (iv) organic acids (lactic, malic, succinic,
citric, and propionic acids) production in the coffee pulp
medium. pH values decreased over the fermentation time
when evaluated in a coffee-based (CP) medium (Figs. 2,
3). During coffee fermentation, the pH value decreased
due to acid production by microorganisms; thus, the ability
to survive in a coffee fermentation medium is an essential
feature for the starter culture’s viability maintenance. The
pH value decreased due to acid production by microorgan-
isms (Elhalish and Zhao 2020); thus, the ability to survive
in a coffee fermentation medium is essential for the starter
culture’s viability maintenance. It is worth remembering
that until now, Leu. mesenteroides CCMA 1105, in addi-
tion to increasing its population during fermentation in a
coffee-based medium, also decreased the pH value in the
first experimental stage performed in the present study.
Species of the lactic acid bacteria group are increasingly
being used in fermentation processes. These have shown
positive results as presented by Wang et al. (2019a), who
used LAB to ferment green coffee beans and evaluate
changes in final roasted coffee flavors.
In the present study, only mesophilic bacteria showed
pectinolytic activities (Table 2). The low pectinase produc-
tion occurred due to several factors: (i) the bacteria were
isolated from pulped coffee where there was not a large
amount of pectic substrate to be degraded, (ii) pectinolytic
enzymes could be suppressed by excess or lack of mono-
saccharides (Schwan et al. 1997; Henick-Kling et al. 1998;
Masoud and Jespersen 2006), (iii) lower availability of the
nitrogen source (Silva et al. 2013), (iv) rapid lowering of
pH value (Figs. 2, 3), which made the enzymatic activity
difficult (pH 3.5–5.5) (Silva 2015).
Like the MB, the LAB population showed a significant
difference (p < 0.05) (Fig.  5). The population of LAB
varies from ­104 to 1­ 08 log CFU g−1 (Avallone et al. 2002;
Pereira et al. 2020; Evangelista et al. 2015). Haile and
Kang (2019) reported that it would be better to use lac-
tic acid bacteria to remain as close as possible to natural
fermentation.
Production of metabolites was another important fea-
ture in selecting starter cultures for coffee fermentation of
the present study. Acetic, succinic, malic, tartaric, citric,
propionic, and lactic were detected during fermentation
using a coffee-based medium (supplementary material).
The synthesis of some acids, such as acetic and propionic,
occurs due to bacteria present on the fruit’s surface, which
can migrate to the mucilage and pulp (Silva et al. 2013).
World Journal of Microbiology and Biotechnology (2020) 36:186 Page 11 of 15  186

Table 3  Volatile compounds identified in green coffee at the end of spontaneous and inoculated wet fermentation via headspace-solid phase
microextraction/gas chromatography-mass spectrometry (HS-SPME/GC–MS)
Compounds Treatments*
A A1 B B1 C C1 D D1 E E1 F F1 G G1 H H1 I I1 J J1
Acids
Acetic acid
Isolvaleric acid
Hexadecanoic acid
Tetradecanoic acid
Hexanoic acid
Octanoic acid
Alcohols A A1 B B1 C C1 D D1 E E1 F F1 G G1 H H1 I I1 J J1
1-Hexadecanol
2-Heptanol
2,3-Butanediol
5-methyl-2-hexanol
Benzyl alcohol
1-phenoxypropan-2-ol
Phenethyl alcohol
Pentadecanol
Tetradecanol
Tridecanol
Dodecanol
β-linalool
Phytol
Aldehydes and Ketones A A1 B B1 C C1 D D1 E E1 F F1 G G1 H H1 I I1 J J1
Acetoin
Benzaldehyde
Pentadecanal
2-Hexenal
9,17-Octadecadienal, (Z)-
2-Pentadecanone, 6,10,14-
trimethyl-
(E)-Geranyl acetone
2,4-Hexadienal, (E,E)-
Esters A A1 B B1 C C1 D D1 E E1 F F1 G G1 H H1 I I1 J J1
Methyl hexadecanoate
Ethyl hexadecanoate
Diisobutyl phthalate
Ethyl 2-hydroxypropanoate
Ethyl linoleate
Ethyl benzeneacetate
Hydrocarbons A A1 B B1 C C1 D D1 E E1 F F1 G G1 H H1 I I1 J J1
Heptadecane
Hexadecane
Hexacosane
Tetracosane
Octadecane
Others** A A1 B B1 C C1 D D1 E E1 F F1 G G1 H H1 I I1 J J1
Methyl salicylate
Vanillin
Heneicosane
Caffeine
*Treatments: Spontaneous fermentation (A); B. subtilis CCMA1234 (B); B. pumilus CCMA1251 (C); C. cellulans
CCMA1186 (D); P. dispersa CCMA1203 (E); M. testaceum CCMA1151 (F); Leu. mesenteroides CCMA1082 (G); Leu.
mesenteroides CCMA1105 (H); Lac. plantarum CCMA1065 (I); Lac. plantarum CCMA1072 (J). Capital letters without
number: the beginning of fermentation; Capital letters with subscript number: end of fermentation. **Included: Alkaloids.
*Treatments: spontaneous fermentation (A); B. subtilis CCMA1234 (B); B. pumilus CCMA1251 (C); C. cellulans CCMA1186 (D); P. dispersa
CCMA1203 (E); M. testaceum CCMA1151 (F); Leu. mesenteroides CCMA1082 (G); Leu. mesenteroides CCMA1105 (H); Lac. plantarum
CCMA1065 (I); Lac. plantarum CCMA1072 (J). Capital letters without number: the beginning of fermentation; Capital letters with subscript
number: end of fermentation
**Included: alkaloids

13
 186   Page 12 of 15 World Journal of Microbiology and Biotechnology (2020) 36:186

Table 4  Volatile compounds Compounds Treatments*

identified in roasted coffee A B C D E F G H I J
processed by spontaneous Acids
and inoculated wet Acetic acid
fermentation via headspace- Propanoic acid
solid phase microextraction/ 3-methyl-2-butenoic acid
gas chromatography-mass Isovaleric acid
Tetradecanoic acid
spectrometry (HS-SPME/
Hexadecanoic acid
GC–MS) Aldehydes and Ketones A B C D E F G H I J
1,2-Cyclopentanedione, 3-methyl-
2-Cyclopenten-1-one, 3-ethyl-2-hydroxy-
2-Propanone, 1-hydroxy-
2,3-Hexanedione
2,5-Hexanedione
2,3- Octanedione
2,5-Octanedione
Ethanone, 1-(2-hydroxy-5-methylphenyl)-
5,9-Dodecadien-2-one, 6,10-dimethyl-, (E,E)-
4-Methyl- 1,5-Heptanediene
1-Penten-3-one, 2,4-dimethyl-
2-Propanone
2,4-Pentanedione, 3-methyl-
Cyclobutane, 1,2-bis(methylene)-
Esters A B C D E F G H I J
Acetic acid, methyl ester
2-Butanone, 1-(acetyloxy)-
Acetic acid, (acetyloxy)-
Phthalic acid, hept-3-yl isobutyl ester
2-Propanone, 1-(acetyloxy)-
1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester
1,2-Ethanediol, diacetate
Acetic acid ethenyl ester
1,2-Ethanediol, monoacetate
Phthalic acid, hex-3-yl isobutyl ester
Propanoic acid, 2-oxo-, ethyl ester
Vinyl crotonate
Dibutyl phthalate
Undecanoic acid isopropyl ester
Furans A B C D E F G H I J
Ethanone, 1-(2-furanyl)-
2-Furanmethanol, acetate
2-Furancarboxaldehyde, 5-methyl-
2-Furanone, 2,5-dihydro-3,5-dimethyl
2-Furanmethanol
trans-Furfurylideneacetone
Furan, 2,2'-[oxybis(methylene)]bis-
2,5-Dimethyl-4-hydroxy-3(2H)-furanone
Benzofuran, 2,3-dihydro-
5-Hydroxymethylfurfural
Bis(2-furfuryl)disulfide
Furan, 2-[(methyldithio)methyl]-
2-Furfurylthiol
3(2H)-Furanone, dihydro-2-methyl-
2-Furylmethyl formate
Lactones A B C D E F G H I J
Butyrolactone
2(5H)-Furanone
2(3H)-Furanone, dihydro-4-hydroxy-
2-hydroxy-gamma-butyrolactone
Phenols A B C D E F G H I J
Methyl salicylate
4-Hydroxy-2-methylacetophenone
Phenol, 2-methoxy-
4-Hydroxy-3-methylacetophenone
Pyrans A B C D E F G H I J

13
World Journal of Microbiology and Biotechnology (2020) 36:186 Page 13 of 15  186

Table 4  (continued) Maltol
4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
Ethanone, 1-(2-hydroxy-5-methylphenyl)
Pyrazines A B C D E F G H I J
Pyrazine, methyl-
Pyrazine, 2,6-dimethyl-
Pyrazine, 2-ethyl-6-methyl-
Pyrazine, trimethyl-
5H-5-Methyl-6,7-dihydrocyclopentapyrazine
1-(6-Methyl-2-pyrazinyl)-1-ethanone
Pyrazine, 2-ethyl-5-methyl-
Pyrazine, 2,5-dimethyl-
Pyrazine, 3-ethyl-2,5-dimethyl-
2-Butyl-3-methylpyrazine
2-Isoamyl-6-methylpyrazine
Pyrazine, 2-ethyl-methyl-
Pyridine A B C D E F G H I J
Pyridine
2-Pyridinecarboxylic acid
2-Pyridinecarboxaldehyde
Picolinamide
Fampridine
3-Pyridinol
4-Pyridinamine, N,N-dimethyl-
4(H)-Pyridine, N-acetyl-
1,2-Ethanediol, 1,2-di-pyridinyl-
2-Aminopyridine
3-Aminopyridine
3-Hydroxypyridine monoacetate
2-Pyridylhydroxymethanesulfonic acid
Pyrroles A B C D E F G H I J
1H-Pyrrole-2-carboxaldehyde, 1-methyl-
Ethanone, 1-(1H-pyrrol-2-yl)
1H-Pyrrole-2-carboxaldehyde
Indole
1H-Pyrrole, 1-(2-furanylmethyl)-
2-Pyrrolidinone, 1-butyl-
Pyrrole
1H-Indole, 3-methyl-
Others** A B C D E F G H I J
2-Thiophenemethanol
Diacetylsulphide
Caffeine
Cyclobutanol
Acetamide, N-(3-methyl-2-oxobutyl)-
*Treatments: Spontaneous fermentation (A); B. subtilis CCMA1234 (B); B. pumilus CCMA1251 (C); C. cellulans
CCMA1186 (D); P. dispersa CCMA1203 (E); M. testaceum CCMA1151 (F); Leu. mesenteroides CCMA1082 (G); Leu.
mesenteroides CCMA1105 (H); Lac plantarum CCMA1065 (I); Lac. plantarum CCMA1072 (J). Capital letters without
number: the beginning of fermentation; Capital letters with subscript number: end of fermentation **Included: Alkaloids,
sulfur compounds, alcohols, and amines.
*
 Treatments: Spontaneous fermentation (A); B. subtilis CCMA1234 (B); B. pumilus CCMA1251 (C); C.
cellulans CCMA1186 (D); P. dispersa CCMA1203 (E); M. testaceum CCMA1151 (F); Leu. mesenter-
oides CCMA1082 (G); Leu. mesenteroides CCMA1105 (H); Lac plantarum CCMA1065 (I); Lac. plan-
tarum CCMA1072 (J). Capital letters without number: the beginning of fermentation; Capital letters with
subscript number: end of fermentation **Included: Alkaloids, sulfur compounds, alcohols, and amines

These acids, such as acetic and succinic in high concentra-
tions (more significant than one mg mL−1), create negative
deterioration factors in coffee (onion flavor) and interfere
in a deprecatory way in the final quality of the beverage
(Lopez et al. 1989). However, these acids were described
in the present study; they were detected in concentrations
below mg g −1. The variation of acids during fermenta-
tion due to microbial metabolism changed the pH value
and influenced the microbiota present, and the diffusion
of these acids into the bean could affect the final bever-
age flavor and quality (Silva et al. 2013; Evangelista et al.
2014; Silva 2015).
The succinic acid was detected in some fermentation
carried out in this study using the coffee-based medium.
Succinic acid is obtained naturally during coffee process-
ing and contributes to the final product (Bressani et  al.
2019; Wang et al. 2019b; Avallone et al. 2001). This acid
may be produced by Bacillus spp. (Silva et al. 2013), by
heterofermentative LAB (Swiegers et al. 2005), and yeast
(Martinez et al. 2017; Bressani et al. 2019). Stains of Bacil-
lus subtilis and Pantoea dispersa were used as bacteria start-
ers in Martinez et al. (2019), showing that they produced
succinic acid during wet fermentation. Wang et al. (2019b)
reported that this acid could remain higher even during cof-
fee roasting.
Lactic acid was the acid detected in the highest concentra-
tion in coffee-based fermentation (Supplementary Material)
and bucket fermentation (Fig. 4) with the selected bacte-
ria. Evangelista et al. (2015) also found that lactic acid was
the primary acid detected in wet coffee fermentations. The
high concentration of lactic acid produced by the LAB and
the consequent lowering of pH would cause the dissocia-
tion of the bean mucilage. The high production of citric,
succinic, and lactic acids, and the low production of acetic
and propionic acids in a coffee pulp medium were used as
selection factors to determine the strains most suitable for

13
 186   Page 14 of 15
conducting the wet coffee fermentation. According to Mar-
tinez et al. (2019), lactic, acetic citric, and succinic acids
were produced when they inoculated bacteria of the genus
Bacillus and Pantoea during wet fermentation. According
to Pothakos et al. (2020), LAB showed to be dominant and
very important during coffee processing, from green coffee
beans to coffee cupping.
Acids, alcohols, aldehydes, ketones, esters, hydrocarbons,
and other compounds (phenols, alkanes, and alkaloids) were
classified in the study (Tables 3, 4). The volatile compounds
predominantly found in initial fermentation belong to alco-
hols, aldehydes, and ketones. These compounds are pro-
duced during coffee processing due to the pectin and sugars
degeneration, resulting in a decrease in pH (De Bruyn et al.
2017).
The presence of crucial flavor marker phenethyl alcohol
before inoculation in this study indicates that it might have
been produced after bean collection, observing the same
results in Martinez et al. (2019) wet fermentation. Apart
from the above compound, other compounds such as Benzyl
alcohol, Hexadecanoic acid, Henecoisane, and 2-Pentade-
canone, 6,10,14-trimethyl- were also present in the same
study, indicating that its production is due to the same factor
that its proper of the coffee variety Catuaí vermelho.
The volatile compounds predominantly found in roasted
coffee belong to furans aldehydes and ketones, and esters
(Table 4). The main groups (acids, alcohols, aldehydes,
ketones, and esters) can be correlated with contributions to
the citric, herbaceous, caramel-like, musty, mushroom-like,
or fruity flavors that are desirable in coffee beverages (Fla-
ment 2002; Czerny and Grosch 2000; Gonzaléz-Rios et al.
2007). Like yeasts (Flament and Bessière-Thomas 2002;
Sunarharum et al. 2014; Bressani et al. 2019), Bacillus and
Pantoea genus (Martinez et al. 2019), LAB also produced
essential compounds for the volatile profile and coffee cup
(Wang et al. 2019a, b; Pothakos et al. 2020). A high percent-
age of pyrazines and pyridines in roasted beans is a posi-
tive indicator because they are responsible for producing
attributes that influence the cup through Maillard’s reactions
(Wang et al. 2019b). Moreover, these compound groups are
commonly found in coffee fermentation.
Conclusion
The use of selected starter bacteria in wet coffee fermenta-
tion is an alternate means of producing coffee with higher
quality and distinct aromas. It is possible to direct the
coffee flavor precursors using different strains of bacte-
ria. Leuconostoc mesenteroides CCMA 1105 and Lacto-
bacillus plantarum CCMA 1065 gave better results con-
cerning acid and volatile compound production in green
13
World Journal of Microbiology and Biotechnology (2020) 36:186
coffee. Future work should be conducted to evaluate these
strains in different coffee varieties and the sensory profiles
produced.
Acknowledgements  The authors thank the Brazilian agencies, Con-
selho Nacional de Desenvolvimento Científico e Tecnológico do Brasil
(CNPQ), Fundação de Amparo à Pesquisa do Estado de Minas Gerais
(FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) for financial support. We also thank Daterra farm,
Patrocínio, Minas Gerais, Brazil, for collecting the samples.
Compliance with ethical standards 
Conflict of interest  The authors declared that there is no conflict of
interest.
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