1Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 351373
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Afilliations
1 School of Pharmaceutical and
Food Sciences. Universidad de
Antioquia, Medellín, Colombia.
*Corresponding
Karina Motato-Rocha
karina.motato@udea.edu.co
Received: 26 September 2022
Accepted: 27 March 2024
Published: 18 April 2024
Identification of microorganisms in wet
coffee fermentation Coffea arabica Var
Catimor and Castillo in Jardín, Antioquia-
Colombia, using culture-dependent
methods
Identificación de microorganismos en fermentación húmeda
de café Coffea arabica Var Catimor y Castillo en Jardín,
Antioquia-Colombia, usando métodos cultivo-dependientes
Karina Motato-Rocha1
* , María Orfilia Román-Morales1 , Valentina Gonzalez-Montero1 .
ABSTRACT
Background: Mild Colombian coffees are recognized worldwide for their high-quality coffee
cup. However, there have been some failures in post-harvest practices, such as coffee grain
fermentation. These failures could occasionally lead to defects and inconsistencies in quality
products and economic losses for coffee farmers. In Colombia, one of the fermentation
methods most used by coffee growers is wet fermentation, conducted by submerging the
de-pulped coffee beans for enough time in water tanks to remove the mucilage. Objectives:
We evaluated the effect of the water (g)/de-pulped coffee (g) ratio (I: 0/25, II: 10/25, III: 20/25)
and final fermentation time (24, 48, and 72 hours) on the total number of microbial groups. We
also identified microorganisms of interest as starter cultures. Methods: We used a completely
randomized experimental design with two factors; the effect of the water (g)/de-pulped coffee
(g) ratio (I: 0/25, II: 10/25, III: 20/25) and final fermentation time (24, 48, and 72 hours), for 9
treatments with two replicates. During the coffee fermentation (1,950 g), the pH and °Brix
were monitored. Total counts of different microbial groups (mesophiles, coliforms, lactic-acid
bacteria, acetic-acid bacteria, and yeasts) were performed. Various isolates of microorganisms
of interest as starter cultures (lactic-acid bacteria and yeasts) were identified using molecular
sequencing techniques. Results: 21 lactic-acid bacteria (LAB) isolates and 22 yeasts were
obtained from the different mini-batch fermentation systems. The most abundant lactic-
acid bacteria species found were Lactiplantibacillus plantarum (46%) and Levilactobacillus
brevis (31%). Pichia kluivery (39%) and Torulaspora delbrueckii (22%) were the most abundant
yeast species. Conclusion The studied factors did not have effect over the microorganism’s
development. The identified bacterial and yeasts species have potential as starter cultures
for better-quality coffees and in fermentation-related applications.
Keywords: Coffee Fermentation, Lactic-Acid Bacteria, Yeasts, Coffee arabica, Culture-
Dependent Methods.
ORIGINAL ARTICLE
Published 18 April 2024
Doi: https://doi.org/10.17533/udea.vitae.v31n1a351373
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Afilliations
1 School of Pharmaceutical and
Food Sciences. Universidad de
Antioquia, Medellín, Colombia.
*Corresponding
Karina Motato-Rocha
karina.motato@udea.edu.co
Received: 26 September 2022
Accepted: 27 March 2024
Published: 18 April 2024
Identification of microorganisms in wet
coffee fermentation Coffea arabica Var
Catimor and Castillo in Jardín, Antioquia-
Colombia, using culture-dependent
methods
Identificación de microorganismos en fermentación húmeda
de café Coffea arabica Var Catimor y Castillo en Jardín,
Antioquia-Colombia, usando métodos cultivo-dependientes
Karina Motato-Rocha1
* , María Orfilia Román-Morales1 , Valentina Gonzalez-Montero1 .
ABSTRACT
Background: Mild Colombian coffees are recognized worldwide for their high-quality coffee
cup. However, there have been some failures in post-harvest practices, such as coffee grain
fermentation. These failures could occasionally lead to defects and inconsistencies in quality
products and economic losses for coffee farmers. In Colombia, one of the fermentation
methods most used by coffee growers is wet fermentation, conducted by submerging the
de-pulped coffee beans for enough time in water tanks to remove the mucilage. Objectives:
We evaluated the effect of the water (g)/de-pulped coffee (g) ratio (I: 0/25, II: 10/25, III: 20/25)
and final fermentation time (24, 48, and 72 hours) on the total number of microbial groups. We
also identified microorganisms of interest as starter cultures. Methods: We used a completely
randomized experimental design with two factors; the effect of the water (g)/de-pulped coffee
(g) ratio (I: 0/25, II: 10/25, III: 20/25) and final fermentation time (24, 48, and 72 hours), for 9
treatments with two replicates. During the coffee fermentation (1,950 g), the pH and °Brix
were monitored. Total counts of different microbial groups (mesophiles, coliforms, lactic-acid
bacteria, acetic-acid bacteria, and yeasts) were performed. Various isolates of microorganisms
of interest as starter cultures (lactic-acid bacteria and yeasts) were identified using molecular
sequencing techniques. Results: 21 lactic-acid bacteria (LAB) isolates and 22 yeasts were
obtained from the different mini-batch fermentation systems. The most abundant lactic-
acid bacteria species found were Lactiplantibacillus plantarum (46%) and Levilactobacillus
brevis (31%). Pichia kluivery (39%) and Torulaspora delbrueckii (22%) were the most abundant
yeast species. Conclusion The studied factors did not have effect over the microorganism’s
development. The identified bacterial and yeasts species have potential as starter cultures
for better-quality coffees and in fermentation-related applications.
Keywords: Coffee Fermentation, Lactic-Acid Bacteria, Yeasts, Coffee arabica, Culture-
Dependent Methods.
ORIGINAL ARTICLE
Published 18 April 2024
Doi: https://doi.org/10.17533/udea.vitae.v31n1a351373
2Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 31 | Number 01 | Article 351373Karina Motato-Rocha, María Orfilia Román-Morales y Valentina Gonzalez-Montero
RESUMEN
Antecedentes: Los cafés suaves lavados colombianos son reconocidos a nivel mundial por su buena puntuación sensorial; sin
embargo, se han detectado fallas en las prácticas de postcosecha, como lo es la fermentación de los granos de café. Dichas
fallas pueden causar defectos y carecer de consistencia en la calidad del producto, ocasionando pérdidas económicas para los
caficultores. En Colombia, uno de los métodos más usados por los caficultores es la fermentación húmeda, la cual consiste en
sumergir los granos de café despulpado en tanques con agua por un período de tiempo que permita la remoción del mucílago.
Objetivos: La presente investigación evaluó la incidencia que tienen la proporción agua/granos despulpados de café (I: 0/25,
II: 10/25, III: 20/25) y el tiempo final de fermentación (24, 48 y 72 horas) en el recuento final de grupos microbianos. Por otra
parte, se identificaron taxonómicamente microorganismos de interés para su uso como cultivos iniciadores. Métodos: Mini-
lotes consistieron en café despulpado (1950 g) puesto en recipientes de plástico abiertos y sumergidos en agua. Se aplicó un
diseño experimental completamente aleatorizado de dos factores (proporción agua/ granos de café despulpado y tiempo)
a tres niveles, para un total de nueve tratamientos con dos replicas. Durante las fermentaciones de café (1,950 g), el pH y
los grados ºBrix, fueron monitoreados. Se realizaron los recuentos totales de los diferentes grupos microbianos: mesófilos,
coliformes, bacterias ácido-lácticas, bacterias ácido-acéticas y levaduras. Se identificaron molecularmente diferentes aislados
con potencial para ser usados como cultivos iniciadores (bacterias ácido-lácticas y levaduras). Resultados: Los resultados
obtenidos mostraron que no hubo diferencia estádisticamente significativa entre los tratamientos aplicados y el recuento final
de microorganismos. Un total de 21 aislados de bacterias ácido-lácticas (BAL) y 22 levaduras lograron obtenerse a partir de
los diferentes sistemas de fermentación en mini-lote. Las especies de bacterias ácido-lácticas con mayor porcentaje acorde a
su identificación taxonómica, corresponden a Lactiplantibacillus plantarum (46%), Levilactobacillus brevis (31%). Las especies
de levaduras con mayor porcentaje acorde a su identificación taxonómica corresponden a Pichia kluivery (39%) y Torulaspora
delbrueckii (22%). Conclusión Los factores estudiados no afectaron el crecimiento de ninguno de los grupos microbianos
presentes en la fermentacion del café. Las especies de microorganismos identificados tienen potencial para se usados como
cultivos starter o en aplicaciones dentro de las ciencias de fermentación.
Palabras clave: Fermentación de café, Bacterias Ácido-Lácticas, Levaduras, Coffee arabica, Métodos Cultivo-Dependientes.
INTRODUCTION
Coffee fermentation is a crucial step in the production
of high-quality coffee, and the microorganisms
involved in the process play a significant role in
determining the flavor and aroma of the final
product (1,2). The unique growing conditions and
fermentation methods used in Colombia coffee
production have contributed to the country’s
reputation establishing it as the third major coffee-
producing country (3,4). However, there have been
some challenges in the post-harvest practices,
particularly in the fermentation process, leading to
occasional defects and inconsistencies in quality.
These issues not only affect the coffee’s overall
quality but also result in economic losses for the
farmers (5).
The fermentation of coffee fruits aims to degrade the
mucilage by indigenous bacteria that feed on various
components of the pulp and mucilage. Microbial
diversity depends on many factors, including the
agricultural area, coffee variety, harvesting, and
coffee cherry storage (6,7). Producers and some
researchers only indicate fermentation for mucilage
removal but do not evaluate the impact on aroma
and flavor. In fermented green coffee, mucilage
removal must be effective and contribute to the
quality due to the biochemical changes resulting
from their degradation. Besides the impact on the
beverage quality, there are fermentation benefits
such as decreased fungi contamination (8).
During fermentation, there are physicochemical
changes in the grains, such as a reduction in the water
and simple sugars content and production of organic
acids, alcohols, enzymes (poly-galacturanase,
pectinlyase), and other metabolites. All these
compounds are precursors of the coffee bean’s
aroma and flavor, influencing the beverage’s final
sensory characteristics (5,9). Wet fermentation is one
of Colombia’s most widely used methods for coffee
fermentation. In this method, the skin and pulp are
removed mechanically, leaving some of the mucilage
adhered to the beans. These de-pulped coffees are
then transferred to water tanks, where they ferment
for 6 to 72 hours, depending on the environmental
temperature. The remaining mucilage is degraded
and solubilized. The beans are then removed from
the tanks and dried (9).
For over a century, the wet method has been a
water-intensive process, as the name indicates, and
there was little concern for water consumption and
contamination (10). The constant market demand
for high-quality coffees and sustainable production
processes has prompted the exploration of the
environment and microbiological interactions in
wet coffee fermentation. This research aimed to
study cultivable and viable microbial communities
in the coffee from the wet fermentation process of
Coffea arabica varieties Catimor and Castillo found
at Finca La Antigua (Jardín, Antioquia, Colombia).
RESUMEN
Antecedentes: Los cafés suaves lavados colombianos son reconocidos a nivel mundial por su buena puntuación sensorial; sin
embargo, se han detectado fallas en las prácticas de postcosecha, como lo es la fermentación de los granos de café. Dichas
fallas pueden causar defectos y carecer de consistencia en la calidad del producto, ocasionando pérdidas económicas para los
caficultores. En Colombia, uno de los métodos más usados por los caficultores es la fermentación húmeda, la cual consiste en
sumergir los granos de café despulpado en tanques con agua por un período de tiempo que permita la remoción del mucílago.
Objetivos: La presente investigación evaluó la incidencia que tienen la proporción agua/granos despulpados de café (I: 0/25,
II: 10/25, III: 20/25) y el tiempo final de fermentación (24, 48 y 72 horas) en el recuento final de grupos microbianos. Por otra
parte, se identificaron taxonómicamente microorganismos de interés para su uso como cultivos iniciadores. Métodos: Mini-
lotes consistieron en café despulpado (1950 g) puesto en recipientes de plástico abiertos y sumergidos en agua. Se aplicó un
diseño experimental completamente aleatorizado de dos factores (proporción agua/ granos de café despulpado y tiempo)
a tres niveles, para un total de nueve tratamientos con dos replicas. Durante las fermentaciones de café (1,950 g), el pH y
los grados ºBrix, fueron monitoreados. Se realizaron los recuentos totales de los diferentes grupos microbianos: mesófilos,
coliformes, bacterias ácido-lácticas, bacterias ácido-acéticas y levaduras. Se identificaron molecularmente diferentes aislados
con potencial para ser usados como cultivos iniciadores (bacterias ácido-lácticas y levaduras). Resultados: Los resultados
obtenidos mostraron que no hubo diferencia estádisticamente significativa entre los tratamientos aplicados y el recuento final
de microorganismos. Un total de 21 aislados de bacterias ácido-lácticas (BAL) y 22 levaduras lograron obtenerse a partir de
los diferentes sistemas de fermentación en mini-lote. Las especies de bacterias ácido-lácticas con mayor porcentaje acorde a
su identificación taxonómica, corresponden a Lactiplantibacillus plantarum (46%), Levilactobacillus brevis (31%). Las especies
de levaduras con mayor porcentaje acorde a su identificación taxonómica corresponden a Pichia kluivery (39%) y Torulaspora
delbrueckii (22%). Conclusión Los factores estudiados no afectaron el crecimiento de ninguno de los grupos microbianos
presentes en la fermentacion del café. Las especies de microorganismos identificados tienen potencial para se usados como
cultivos starter o en aplicaciones dentro de las ciencias de fermentación.
Palabras clave: Fermentación de café, Bacterias Ácido-Lácticas, Levaduras, Coffee arabica, Métodos Cultivo-Dependientes.
INTRODUCTION
Coffee fermentation is a crucial step in the production
of high-quality coffee, and the microorganisms
involved in the process play a significant role in
determining the flavor and aroma of the final
product (1,2). The unique growing conditions and
fermentation methods used in Colombia coffee
production have contributed to the country’s
reputation establishing it as the third major coffee-
producing country (3,4). However, there have been
some challenges in the post-harvest practices,
particularly in the fermentation process, leading to
occasional defects and inconsistencies in quality.
These issues not only affect the coffee’s overall
quality but also result in economic losses for the
farmers (5).
The fermentation of coffee fruits aims to degrade the
mucilage by indigenous bacteria that feed on various
components of the pulp and mucilage. Microbial
diversity depends on many factors, including the
agricultural area, coffee variety, harvesting, and
coffee cherry storage (6,7). Producers and some
researchers only indicate fermentation for mucilage
removal but do not evaluate the impact on aroma
and flavor. In fermented green coffee, mucilage
removal must be effective and contribute to the
quality due to the biochemical changes resulting
from their degradation. Besides the impact on the
beverage quality, there are fermentation benefits
such as decreased fungi contamination (8).
During fermentation, there are physicochemical
changes in the grains, such as a reduction in the water
and simple sugars content and production of organic
acids, alcohols, enzymes (poly-galacturanase,
pectinlyase), and other metabolites. All these
compounds are precursors of the coffee bean’s
aroma and flavor, influencing the beverage’s final
sensory characteristics (5,9). Wet fermentation is one
of Colombia’s most widely used methods for coffee
fermentation. In this method, the skin and pulp are
removed mechanically, leaving some of the mucilage
adhered to the beans. These de-pulped coffees are
then transferred to water tanks, where they ferment
for 6 to 72 hours, depending on the environmental
temperature. The remaining mucilage is degraded
and solubilized. The beans are then removed from
the tanks and dried (9).
For over a century, the wet method has been a
water-intensive process, as the name indicates, and
there was little concern for water consumption and
contamination (10). The constant market demand
for high-quality coffees and sustainable production
processes has prompted the exploration of the
environment and microbiological interactions in
wet coffee fermentation. This research aimed to
study cultivable and viable microbial communities
in the coffee from the wet fermentation process of
Coffea arabica varieties Catimor and Castillo found
at Finca La Antigua (Jardín, Antioquia, Colombia).
3Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 351373
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
Results generated from this study will allow knowing
the diversity of viable microorganisms present in
coffee fermentation under natural environmental
conditions and the variables studied. These
microorganisms can be” starter cultures” used
in controlled fermentation processes to produce
Specialty Coffees in Colombia.
MATERIALS AND METHODS
Location and coffee treatment
C of fe e c he r r ie s f ro m “L a A nt ig ua’’ f ar m
(5.608263510940495, -75.81934403607546) located
at 1,850 m above sea level in Jardín, Antioquia,
Colombia, were hand-picked at the mature stage
(cherries) and then were classified (red cherries)
comparing the cherries with a color chart (color
codes: #fc4f59, #ef2b2d, #d62828, #af2626, #ef2b2d,
#cc2d30, #a03033, #ce1126, #af1e2d) according to
reference 11. They were de-pulped in a traditional
pulper (Vencedora Estrella no. 3 1/2) to obtain beans
with mucilage. The pH (UdeA device, Colombia),
were measured from the fresh de-pulped coffee
beans and ºBrix (Ultechnovo, USA) were measured
from the cherries, by squeezing a drop of juice from
cherries into a hand refractometer.
Coffee fermentation conditions
Our experimental unit was a mini-batch (plastic
containers without lids (13x30x20 cm)). We used nine
mini-batches with 18 Kg of de-pulped coffee cherries
(a mixture of C. arabica var. Catimor and Castillo).
Each one had a different water (g)/de-pulped coffee
(g) ratio (I: 0/25, II: 10/25, III: 20/25) and a final time
of fermentation (A: 24h, B:48h, C:72h). The pH was
registered through the fermentation process of the
pulped coffee beans. ºBrix was measured at the end
of each fermentation experiment. The experimental
design consisted of a three-by-two factorial design,
being the factors the water (g)/pulped coffee (g)
ratio and the fermentation time. Each experiment
was done by duplicate. Each experiment was done
by duplicate.
Microbial count and isolation of lactic-acid
bacterial and yeast
300 g of samples from each mini-batch were
collected in sterile plastic bags in three moments of
fermentation: t0 (beginning), t2 (half-time:12h, 24h,
36h), and t3 (final time: 24h, 48, 72h). Afterward, each
sample was stored at 4ºC and then transported in
ice boxes to the Food Microbiology laboratory at
Universidad de Antioquia, Medellín, Colombia. 10
g of each sample was added to a bottle containing
90 ml of saline-peptone water (0.1% bacteriological
peptone, Merck, USA). After mixing, six-fold dilutions
were prepared. Microorganisms count was carried
out using five different culture media and plating
in duplicate using 50 μl of dilutions 10 -4 and 10 -6 as
follows: mesophiles microorganisms in Plate count
agar (PCA, Microkit, Spain) and incubated at 32 ºC
for 72 h; acetic-acid bacteria (AAB) in Wallerstein Lab
nutrient agar (WL, Scharlau, Spain) and incubated at
37 ºC for 24 h; coliforms in Chromocult (Merck, USA)
and incubated at 37 ºC for 24 h; lactic-acid bacteria
(LAB) in Man, Rogosa y Sharpe agar (MRS, Merck,
USA) supplemented with Fluconazole 0.2 % p/v (La
santé, Colombia); yeasts and filamentous fungi in
Oxytetracycline-Glucose Yeast Extract agar (OGYE,
Oxoid, UK) supplemented with 80 mg gentamycin
(Genfar, Colombia) in 500 ml and incubated at 25
ºC for 72 h.
All colonies were counted and results were
expressed in log CFU/g (Colony Forming Units per
gram) and described by shape, color, height, and
edge of each isolate. Gram staining and catalase
tests were made. Yeasts and lactic-acid bacteria
colonies with different macroscopic and microscopic
characteristics were isolated, purified, and stored in
vials at -80 ºC in 20 % glycerol.
Molecular identification of acid-lactic bacteria
and yeast.
Molecular identification of lactic-acid bacteria (LAB)
and yeasts colonies isolates was performed by
MACROGEN (Korea) (12). The samples were sent
according to MACROGEN’s (Korea) preparation
guidelines (12). The amplification and sequencing
of the 16S ribosomal gene were done using the
universal primers: 785F (3’ GGA TTA GAT CCC TGG
TA 5’) and 907R (5’ CCG TCA ATT CCT TTR AGT TT
3’). Yeast molecular identification was performed
by amplifying and sequencing of 26S rDNA region
using the universal primers ITS4 (TCC TCC GCT TAT
TGA TAT GC) and ITS5 (GGA AGT AAA AGT CGT
AAC AA GG) for sequencing of 18S rDNA region.
The microbial sequencing chromatograms were
depurated using Benchling (13). The sequences
were compared with yeasts and LAB references
from the NCBI GenBank using the Basic Local
Alignment Search Tool (BLAST) algorithms (14).
Finally, phylogenetic trees were made using MEGA
X software (15).
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
Results generated from this study will allow knowing
the diversity of viable microorganisms present in
coffee fermentation under natural environmental
conditions and the variables studied. These
microorganisms can be” starter cultures” used
in controlled fermentation processes to produce
Specialty Coffees in Colombia.
MATERIALS AND METHODS
Location and coffee treatment
C of fe e c he r r ie s f ro m “L a A nt ig ua’’ f ar m
(5.608263510940495, -75.81934403607546) located
at 1,850 m above sea level in Jardín, Antioquia,
Colombia, were hand-picked at the mature stage
(cherries) and then were classified (red cherries)
comparing the cherries with a color chart (color
codes: #fc4f59, #ef2b2d, #d62828, #af2626, #ef2b2d,
#cc2d30, #a03033, #ce1126, #af1e2d) according to
reference 11. They were de-pulped in a traditional
pulper (Vencedora Estrella no. 3 1/2) to obtain beans
with mucilage. The pH (UdeA device, Colombia),
were measured from the fresh de-pulped coffee
beans and ºBrix (Ultechnovo, USA) were measured
from the cherries, by squeezing a drop of juice from
cherries into a hand refractometer.
Coffee fermentation conditions
Our experimental unit was a mini-batch (plastic
containers without lids (13x30x20 cm)). We used nine
mini-batches with 18 Kg of de-pulped coffee cherries
(a mixture of C. arabica var. Catimor and Castillo).
Each one had a different water (g)/de-pulped coffee
(g) ratio (I: 0/25, II: 10/25, III: 20/25) and a final time
of fermentation (A: 24h, B:48h, C:72h). The pH was
registered through the fermentation process of the
pulped coffee beans. ºBrix was measured at the end
of each fermentation experiment. The experimental
design consisted of a three-by-two factorial design,
being the factors the water (g)/pulped coffee (g)
ratio and the fermentation time. Each experiment
was done by duplicate. Each experiment was done
by duplicate.
Microbial count and isolation of lactic-acid
bacterial and yeast
300 g of samples from each mini-batch were
collected in sterile plastic bags in three moments of
fermentation: t0 (beginning), t2 (half-time:12h, 24h,
36h), and t3 (final time: 24h, 48, 72h). Afterward, each
sample was stored at 4ºC and then transported in
ice boxes to the Food Microbiology laboratory at
Universidad de Antioquia, Medellín, Colombia. 10
g of each sample was added to a bottle containing
90 ml of saline-peptone water (0.1% bacteriological
peptone, Merck, USA). After mixing, six-fold dilutions
were prepared. Microorganisms count was carried
out using five different culture media and plating
in duplicate using 50 μl of dilutions 10 -4 and 10 -6 as
follows: mesophiles microorganisms in Plate count
agar (PCA, Microkit, Spain) and incubated at 32 ºC
for 72 h; acetic-acid bacteria (AAB) in Wallerstein Lab
nutrient agar (WL, Scharlau, Spain) and incubated at
37 ºC for 24 h; coliforms in Chromocult (Merck, USA)
and incubated at 37 ºC for 24 h; lactic-acid bacteria
(LAB) in Man, Rogosa y Sharpe agar (MRS, Merck,
USA) supplemented with Fluconazole 0.2 % p/v (La
santé, Colombia); yeasts and filamentous fungi in
Oxytetracycline-Glucose Yeast Extract agar (OGYE,
Oxoid, UK) supplemented with 80 mg gentamycin
(Genfar, Colombia) in 500 ml and incubated at 25
ºC for 72 h.
All colonies were counted and results were
expressed in log CFU/g (Colony Forming Units per
gram) and described by shape, color, height, and
edge of each isolate. Gram staining and catalase
tests were made. Yeasts and lactic-acid bacteria
colonies with different macroscopic and microscopic
characteristics were isolated, purified, and stored in
vials at -80 ºC in 20 % glycerol.
Molecular identification of acid-lactic bacteria
and yeast.
Molecular identification of lactic-acid bacteria (LAB)
and yeasts colonies isolates was performed by
MACROGEN (Korea) (12). The samples were sent
according to MACROGEN’s (Korea) preparation
guidelines (12). The amplification and sequencing
of the 16S ribosomal gene were done using the
universal primers: 785F (3’ GGA TTA GAT CCC TGG
TA 5’) and 907R (5’ CCG TCA ATT CCT TTR AGT TT
3’). Yeast molecular identification was performed
by amplifying and sequencing of 26S rDNA region
using the universal primers ITS4 (TCC TCC GCT TAT
TGA TAT GC) and ITS5 (GGA AGT AAA AGT CGT
AAC AA GG) for sequencing of 18S rDNA region.
The microbial sequencing chromatograms were
depurated using Benchling (13). The sequences
were compared with yeasts and LAB references
from the NCBI GenBank using the Basic Local
Alignment Search Tool (BLAST) algorithms (14).
Finally, phylogenetic trees were made using MEGA
X software (15).
4Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 31 | Number 01 | Article 351373Karina Motato-Rocha, María Orfilia Román-Morales y Valentina Gonzalez-Montero
Data analysis.
The experiment design was completely randomized
in a factorial arrangement of three different
fermentation times and three water(g)/de-pulped
coffee(g) ratios for 9 treatments with 2 replicates.
Analysis of variance (ANOVA) and Pearson correlation
were conducted using R software (16). P-values less
than 0.05 were considered statistically significant.
RESULTS
pH and ºBrix in the coffee fermentation process.
pH and ºBrix in de-pulped coffee during the
fermentation process are reported in Figure 1 and
Table 1.
Figure 1. pH during fermentation of different water/pulped coffee
ratios (I: 0/25, II: 10/25, III: 20/25) and final time of fermentation
(A: 24h, B:48h, C:72h).
The water(g)/de-pulped coffee(g) ratio influenced
the initial pH values of the mini-batch systems. A
low positive correlation of 0.30 (data not shown)
suggests a basic state in rich water media (mini-
batch III: 5.82, mini-batch II: 5.28, and mini-batch
I: 4.96). Additionally, the pH values in each system
gradually decreased during fermentation and
converged at the end of each process (mini-batch
III: 3.56, mini-batch II: 3.56, and mini-batch I: 3.52).
The value of °Brix for all mini-batch treatments
is presented in table 1. ºBrix of mature coffee
cherries were in the optimal range of 15 and 15.8,
corresponding to the optimum maturity of the
coffee fruit Colombia variety (17). Mini-batches
A and C started with slightly higher values but in
general, the data shows a decreasing behavior of the
concentration of mucilage sugars in all treatments.
Table 1. ºBrix media values of coffee processed with different
water/de-pulped coffee ratios and final time of fermentation (A:
24h, B:48h, C:72h).
A B C SD
Mature coffee cherry 15.8 15.0 15.6 0.4
I (0/25) 9.8 6.5 10.0 2.0
II (10/25) 5.0 2.8 3.5 1.1
III (20/25) 1.8 1.5 1.8 0.1
Microbiological counts and isolation of lactic-acid
bacteria and yeasts.
Bacterial and yeast populations in mini-batch
fermentations were quantified by plating on
selective media. Microbial counts of mesophilic
aerobic bacteria, coliforms, AAB, LAB, and yeast
found during coffee processing are illustrated in
Figure 2 to Figure 6.
Figure 2. Total count (CFU / g) of mesophilic microorganisms in
Plate count agar (PCA) medium during coffee fermentation (final
time A: 24h, B:48h, C:72h) and different water(g)/pulped coffee
(g) ratios (I: 0/25, II: 10/25, III: 20/25).
Figure 3. Coliforms total count (CFU / g) in Chromocult medium
(CHC) during coffee fermentation (final time A: 24h, B:48h, C:72h)
and different water(g)/pulped coffee(g) ratios (I: 0/25, II: 10/25,
III: 20/25)
Data analysis.
The experiment design was completely randomized
in a factorial arrangement of three different
fermentation times and three water(g)/de-pulped
coffee(g) ratios for 9 treatments with 2 replicates.
Analysis of variance (ANOVA) and Pearson correlation
were conducted using R software (16). P-values less
than 0.05 were considered statistically significant.
RESULTS
pH and ºBrix in the coffee fermentation process.
pH and ºBrix in de-pulped coffee during the
fermentation process are reported in Figure 1 and
Table 1.
Figure 1. pH during fermentation of different water/pulped coffee
ratios (I: 0/25, II: 10/25, III: 20/25) and final time of fermentation
(A: 24h, B:48h, C:72h).
The water(g)/de-pulped coffee(g) ratio influenced
the initial pH values of the mini-batch systems. A
low positive correlation of 0.30 (data not shown)
suggests a basic state in rich water media (mini-
batch III: 5.82, mini-batch II: 5.28, and mini-batch
I: 4.96). Additionally, the pH values in each system
gradually decreased during fermentation and
converged at the end of each process (mini-batch
III: 3.56, mini-batch II: 3.56, and mini-batch I: 3.52).
The value of °Brix for all mini-batch treatments
is presented in table 1. ºBrix of mature coffee
cherries were in the optimal range of 15 and 15.8,
corresponding to the optimum maturity of the
coffee fruit Colombia variety (17). Mini-batches
A and C started with slightly higher values but in
general, the data shows a decreasing behavior of the
concentration of mucilage sugars in all treatments.
Table 1. ºBrix media values of coffee processed with different
water/de-pulped coffee ratios and final time of fermentation (A:
24h, B:48h, C:72h).
A B C SD
Mature coffee cherry 15.8 15.0 15.6 0.4
I (0/25) 9.8 6.5 10.0 2.0
II (10/25) 5.0 2.8 3.5 1.1
III (20/25) 1.8 1.5 1.8 0.1
Microbiological counts and isolation of lactic-acid
bacteria and yeasts.
Bacterial and yeast populations in mini-batch
fermentations were quantified by plating on
selective media. Microbial counts of mesophilic
aerobic bacteria, coliforms, AAB, LAB, and yeast
found during coffee processing are illustrated in
Figure 2 to Figure 6.
Figure 2. Total count (CFU / g) of mesophilic microorganisms in
Plate count agar (PCA) medium during coffee fermentation (final
time A: 24h, B:48h, C:72h) and different water(g)/pulped coffee
(g) ratios (I: 0/25, II: 10/25, III: 20/25).
Figure 3. Coliforms total count (CFU / g) in Chromocult medium
(CHC) during coffee fermentation (final time A: 24h, B:48h, C:72h)
and different water(g)/pulped coffee(g) ratios (I: 0/25, II: 10/25,
III: 20/25)
5Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 351373
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
Figure 4. Acetic-acid bacteria total count (CFU / g) of in WL
medium during coffee fermentation (final time A: 24h, B:48h,
C:72h), and different water/pulped coffee ratios (I: 0/25, II: 10/25,
III: 20/25).
Figure 5. LAB total count (CFU / g) in MRS medium during coffee
fermentation (final time A: 24h, B:48h, C:72h), and different water/
pulped coffee ratios (I: 0/25, II: 10/25, III: 20/25).
Figure 6. Yeasts total count (CFU/ g) in OGYE medium during
coffee fermentation (final time A: 24h, B:48h, C:72h), and different
water/pulped coffee ratios (I: 0/25, II: 10/25, III: 20/25).
Table 2 presents the statistical analysis data
(P-values) of the effect of each factor and both
factors combined (water/de-pulped coffee ratio
and fermentation final time) over the counts in each
microbial group.
Table 2. P- values obtained of statistical evaluation of water/
de-pulped coffee ratio and final time fermentation factors over
microbiological counts p- values.
Water/de-pulped
coffee Time Combined effect
of factors*
Mesophiles 0.7084 0.1245 0.9809
Coliforms 0.415 0.308 0.744
Yeasts 0.821 0.128 0.513
LAB 0.1084 0.0213 a 0.3712
AAB 0.0538 0.0176 a 0.0933
a p-value significance level >= 0.05
* Effect of Water/de-pulped coffee and Time on microbial counts
All bacterial and yeast isolates were identified
according to 16S rRNA and ITS gene sequencing,
respectively. Taxonomic identification of the
isolates was determined by aligning LAB and yeast
sequences and the species with a 99-100% identity
percentage (18) were selected. The amount of
species of lactic-acid bacteria and yeast isolates
present in coffee fermentation are shown in Figure
7 and Figure 8. The percentages are the quantity of
successfully recovered isolates, meaning viable and
cultivable microorganisms.
The isolation of different lactic-acid bacteria
(LAB) species corresponds to the first 48 hours of
fermentation mini-batches A and B and water/pulped
coffee ratios of I: 0/25 and II: 10/25. The most abundant
LAB isolates correspond to Lactiplantibacillus
plantarum (46%), and Levilactobacillus brevis (31%).
Lactiplantibacillus pentosus, Lacticaseibacillus
rhamnosus and Lactobacillus sp. had approximately
the same recuperation; 8% for the first two species
and 7% for Lactobacillus
46%
7%
31%
8%
8%
LAB species percentage
Lactiplantibacillus plantarum
ID: 99% (A.I, A.II, B.II)
Lactobacillus sp.ID: 99% (A.I)
Levilactobacillus brevisID:
99% (A.I, A.II)
Lacticaseibacillus rhamnosus
ID: 99% (B.III)
Lactiplantibacillus pentosus
ID: 99% (A.I, A.II)
Figure 7. Species of lactic-acid bacteria present in coffee
fermentation at final time (A: 24h, B:48h, C:72h), and different
water/de-pulped coffee ratios (I: 0/25, II: 10/25, III: 20/25).
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
Figure 4. Acetic-acid bacteria total count (CFU / g) of in WL
medium during coffee fermentation (final time A: 24h, B:48h,
C:72h), and different water/pulped coffee ratios (I: 0/25, II: 10/25,
III: 20/25).
Figure 5. LAB total count (CFU / g) in MRS medium during coffee
fermentation (final time A: 24h, B:48h, C:72h), and different water/
pulped coffee ratios (I: 0/25, II: 10/25, III: 20/25).
Figure 6. Yeasts total count (CFU/ g) in OGYE medium during
coffee fermentation (final time A: 24h, B:48h, C:72h), and different
water/pulped coffee ratios (I: 0/25, II: 10/25, III: 20/25).
Table 2 presents the statistical analysis data
(P-values) of the effect of each factor and both
factors combined (water/de-pulped coffee ratio
and fermentation final time) over the counts in each
microbial group.
Table 2. P- values obtained of statistical evaluation of water/
de-pulped coffee ratio and final time fermentation factors over
microbiological counts p- values.
Water/de-pulped
coffee Time Combined effect
of factors*
Mesophiles 0.7084 0.1245 0.9809
Coliforms 0.415 0.308 0.744
Yeasts 0.821 0.128 0.513
LAB 0.1084 0.0213 a 0.3712
AAB 0.0538 0.0176 a 0.0933
a p-value significance level >= 0.05
* Effect of Water/de-pulped coffee and Time on microbial counts
All bacterial and yeast isolates were identified
according to 16S rRNA and ITS gene sequencing,
respectively. Taxonomic identification of the
isolates was determined by aligning LAB and yeast
sequences and the species with a 99-100% identity
percentage (18) were selected. The amount of
species of lactic-acid bacteria and yeast isolates
present in coffee fermentation are shown in Figure
7 and Figure 8. The percentages are the quantity of
successfully recovered isolates, meaning viable and
cultivable microorganisms.
The isolation of different lactic-acid bacteria
(LAB) species corresponds to the first 48 hours of
fermentation mini-batches A and B and water/pulped
coffee ratios of I: 0/25 and II: 10/25. The most abundant
LAB isolates correspond to Lactiplantibacillus
plantarum (46%), and Levilactobacillus brevis (31%).
Lactiplantibacillus pentosus, Lacticaseibacillus
rhamnosus and Lactobacillus sp. had approximately
the same recuperation; 8% for the first two species
and 7% for Lactobacillus
46%
7%
31%
8%
8%
LAB species percentage
Lactiplantibacillus plantarum
ID: 99% (A.I, A.II, B.II)
Lactobacillus sp.ID: 99% (A.I)
Levilactobacillus brevisID:
99% (A.I, A.II)
Lacticaseibacillus rhamnosus
ID: 99% (B.III)
Lactiplantibacillus pentosus
ID: 99% (A.I, A.II)
Figure 7. Species of lactic-acid bacteria present in coffee
fermentation at final time (A: 24h, B:48h, C:72h), and different
water/de-pulped coffee ratios (I: 0/25, II: 10/25, III: 20/25).
6Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 31 | Number 01 | Article 351373Karina Motato-Rocha, María Orfilia Román-Morales y Valentina Gonzalez-Montero
39%
5%6%
22%
11%
6%
11%
Yeast species percentage
Pichia kluyveri ID: 99% (B.I)
Kurtzmaniella quercitrusa ID:
99% (B.II)
Kazachstania exigua ID: 99%
(B.II)
Torulaspora delbrueckii ID:
99% (C.I)
Hanseniaspora uvarum ID:
99% (B.II, C.III)
Kazachstania gamosporaD:
99% (C.I)
Pichia sp ID: 99% (B.II,
C.III)
Figure 8. Species of yeast present in coffee fermentation at final
time (A: 24h, B:48h, C:72h), and different water/de-pulped coffee
ratios (I: 0/25, II: 10/25, III: 20/25).
Genetic relationships between representative
isolates of lactic-acid bacteria and yeasts in coffee
fermentation are represented in phylogenetic trees
(Figures 9 and 10). The phylogenetic trees were built
to establish the evolutionary distance within each
group of LAB and yeasts identified in this study and
primally assess if they could be safe to be used as
starter cultures in other fermentations or if they have
been reported as species generally recognized as
safe (GRAS) in the National Center for Biotechnology
Information (NCBI) database.
Figure 9. Phylogenetic relationship between the identified lactic acid bacteria from wet coffee fermentation and other 16S rRNA
sequences of published strains.
39%
5%6%
22%
11%
6%
11%
Yeast species percentage
Pichia kluyveri ID: 99% (B.I)
Kurtzmaniella quercitrusa ID:
99% (B.II)
Kazachstania exigua ID: 99%
(B.II)
Torulaspora delbrueckii ID:
99% (C.I)
Hanseniaspora uvarum ID:
99% (B.II, C.III)
Kazachstania gamosporaD:
99% (C.I)
Pichia sp ID: 99% (B.II,
C.III)
Figure 8. Species of yeast present in coffee fermentation at final
time (A: 24h, B:48h, C:72h), and different water/de-pulped coffee
ratios (I: 0/25, II: 10/25, III: 20/25).
Genetic relationships between representative
isolates of lactic-acid bacteria and yeasts in coffee
fermentation are represented in phylogenetic trees
(Figures 9 and 10). The phylogenetic trees were built
to establish the evolutionary distance within each
group of LAB and yeasts identified in this study and
primally assess if they could be safe to be used as
starter cultures in other fermentations or if they have
been reported as species generally recognized as
safe (GRAS) in the National Center for Biotechnology
Information (NCBI) database.
Figure 9. Phylogenetic relationship between the identified lactic acid bacteria from wet coffee fermentation and other 16S rRNA
sequences of published strains.
7Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 351373
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
DISCUSSION
pH in coffee fermentation process.
According to the results shown in figure 1, the
water(g)/de-pulped coffee(g) ratio influenced the
initial pH values of the mini-batch systems. The
decrease in the pH during coffee bean fermentation
has been reported as the degradation of mucilage’s
components (e.g., pectic substances, complex
sugars, amino acids, and proteins) (19) into simpler
sugars due to the action of microorganisms (20).
ºBrix in the coffee fermentation process.
The standard deviation (SD) shows a low variability
of the ºBrix throughout time (Table 1). Regardless of
the water (g)/de-pulped coffee (g) ratio used in the
mini-batch fermentation, the ºBrix decreased at
the end of all the experiments since microorganisms
consumed sugars to develop their metabolic
processes. The aforementioned is in agreement
with other studies (8). For example, Pantoja López.
(21) reported a ºBrix exponential decline in a
study conducted in Huila, Colombia, and Betancur
Henao.(22) obtained the same results in southwest
Antioquia, Colombia.
Microbiological counts and isolates of lactic-acid
bacteria and yeasts.
In table 2 is observed that the fermentation time
factor had influence over the LAB and AAB counts,
but the combination of the factors; time and water/
de-pulped coffee level ratio, did not show there was
a significant amount difference in microbial counts
(P-value > 0.05).
The viable and cultivable counts of the different
microbial groups analyzed in the wet fermentation
were between 10 5 and 10 6 log CFU/g of fermented
coffee (Figure 2 to Figure 6). As no treatment
exceeds at least 3 logarithms above or below the
counts it is concluded that there are no statistically
significant differences
Figure 10. Phylogenetic relationship between the identified yeasts from wet coffee fermentation and other ITS sequences of
published strains.
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
DISCUSSION
pH in coffee fermentation process.
According to the results shown in figure 1, the
water(g)/de-pulped coffee(g) ratio influenced the
initial pH values of the mini-batch systems. The
decrease in the pH during coffee bean fermentation
has been reported as the degradation of mucilage’s
components (e.g., pectic substances, complex
sugars, amino acids, and proteins) (19) into simpler
sugars due to the action of microorganisms (20).
ºBrix in the coffee fermentation process.
The standard deviation (SD) shows a low variability
of the ºBrix throughout time (Table 1). Regardless of
the water (g)/de-pulped coffee (g) ratio used in the
mini-batch fermentation, the ºBrix decreased at
the end of all the experiments since microorganisms
consumed sugars to develop their metabolic
processes. The aforementioned is in agreement
with other studies (8). For example, Pantoja López.
(21) reported a ºBrix exponential decline in a
study conducted in Huila, Colombia, and Betancur
Henao.(22) obtained the same results in southwest
Antioquia, Colombia.
Microbiological counts and isolates of lactic-acid
bacteria and yeasts.
In table 2 is observed that the fermentation time
factor had influence over the LAB and AAB counts,
but the combination of the factors; time and water/
de-pulped coffee level ratio, did not show there was
a significant amount difference in microbial counts
(P-value > 0.05).
The viable and cultivable counts of the different
microbial groups analyzed in the wet fermentation
were between 10 5 and 10 6 log CFU/g of fermented
coffee (Figure 2 to Figure 6). As no treatment
exceeds at least 3 logarithms above or below the
counts it is concluded that there are no statistically
significant differences
Figure 10. Phylogenetic relationship between the identified yeasts from wet coffee fermentation and other ITS sequences of
published strains.
8Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 31 | Number 01 | Article 351373Karina Motato-Rocha, María Orfilia Román-Morales y Valentina Gonzalez-Montero
LAB has been recognized as an integral component
of coffee processing in most coffee-producing
countries (23–25). The LAB growth is because
of adaptability to the environment and stress
factors of coffee processing, such as pH variation,
sugar availability, and competition with other
microorganisms (23,26,27). L AB species can
catabolize pentoses and hexoses in coffee pulp into
a vast range of end-metabolites, including lactate,
acetate, CO2, and ethanol, via the phosphoketolase
or pentose phosphate pathway (28).
The results revealed that the most prevalent yeast
species were Pichia kluivery, accounting for 39% of
the isolates, followed by Torulaspora delbrueckii
at 22%. Hanseniaspora uvarum, Kurtzmaniella
quercistrusa, and Kazachstania gamospora were
also detected, although at lower amounts of 11%,
5% and 6%, respectively.
The presence of Pichia kluivery, Torulaspora
delbrueckii and Hanseniaspora uvarum, as the
dominant yeast isolates suggests their potential
role in driving the fermentation process and
influencing the final coffee product. The use of these
yeast species as starter cultures has been studied
due to their association with desirable sensory
characteristics and are recognized for their ability
to contribute to the development of distinct flavors
and aromas, thereby playing a crucial role in the
production of specialty coffee (2,29,30).
Lactic acid bacteria were isolated from the first 48
hours while yeasts were isolated after 48 and up to
72 hours. LAB are well-adapted to the pre-existing
environmental conditions; therefore, it is common
to find LAB species instead of yeasts in the initial
hours of coffee fermentation(25,31). The microbial
community is known to vary in composition and
initial abundance during this process, and the
succession of bacterial and fungal species is
complex because their behavior depends on the
time and environmental factors (32–34).
A phylogenetic tree analysis was conducted to examine
the genetic relationships among representative
isolates of lactic acid bacteria (LAB) species belonging
to the Lacticaseibacillus, Lactiplantibacillus, and
Levilactobacillus genera (Figure 9)
The phylogenetic tree revealed distinct clusters
representing each genus. The Lacticaseibacillus and
Lactiplantibacillus cluster comprised species within
the plantarum group lactobacilli. These clusters
showed a close evolutionary relationship to other
lactobacillus groups commonly used as starter
cultures in fermentations (35), antimicrobials (36,37),
and the next generation of probiotics (38,39). This
suggests a shared genetic heritage and functional
similarities among these lactobacilli.
The Levilactobacillus cluster represented a group
of lactobacilli known for their leavening potential
(40). Multiple species within this genus have been
identified in type sourdoughs used as leavening
agents. The presence of Levilactobacillus highlights
the diversity and adaptability of these lactobacilli in
different fermentation environments (40).
Understanding the genetic relationships among
these species contributes to our knowledge of
their diversity, evolutionary history, metabolic
capabilities, and potential applications. It provides
a framework for further investigation into the
functional properties, ecological roles, and their
impact on fermentation and product quality.
The yeast isolates identified in this research
belong to five different genera (Figures 8 and 10),
namely Torulaspora, Kazachstania, Kurtzmaniella,
Hanseniaspora, and Pichia. Of particular interest is
the isolation of Torulaspora delbrueckii, which has
recently been reported in metagenomic studies
conducted in Colombia (41) and has also been
investigated in Latin America (42). This highlights the
presence and potential significance of T. delbrueckii
in coffee fermentation processes. Yeast species from
the genus Kazachstania, including Kazachstania
gamospora, have been documented in both
Colombia (43) and Rwanda (44). This suggests that
these yeasts are not limited to a specific geographic
region and may have a broader distribution in
coffee-growing areas.
Coffee processing steps have been found to harbor
a diverse range of yeast species from various
genera such as Pichia, Candida, Saccharomyces,
and Torulaspora (45). The presence of these
yeast species broadens the understanding of the
diversity and distribution of yeast species in coffee
processing.
The analysis of LAB and yeasts in coffee fermentation
provides valuable insights into the diversity and
functional attributes of these microorganisms;
including the production of flavor-active compounds
and the establishment of desirable fermentation
profiles. Further investigations into the metabolic
pathways and interactions of these microorganisms
will contribute to optimizing coffee fermentation
processes and enhancing the quality of the final
coffee product.
LAB has been recognized as an integral component
of coffee processing in most coffee-producing
countries (23–25). The LAB growth is because
of adaptability to the environment and stress
factors of coffee processing, such as pH variation,
sugar availability, and competition with other
microorganisms (23,26,27). L AB species can
catabolize pentoses and hexoses in coffee pulp into
a vast range of end-metabolites, including lactate,
acetate, CO2, and ethanol, via the phosphoketolase
or pentose phosphate pathway (28).
The results revealed that the most prevalent yeast
species were Pichia kluivery, accounting for 39% of
the isolates, followed by Torulaspora delbrueckii
at 22%. Hanseniaspora uvarum, Kurtzmaniella
quercistrusa, and Kazachstania gamospora were
also detected, although at lower amounts of 11%,
5% and 6%, respectively.
The presence of Pichia kluivery, Torulaspora
delbrueckii and Hanseniaspora uvarum, as the
dominant yeast isolates suggests their potential
role in driving the fermentation process and
influencing the final coffee product. The use of these
yeast species as starter cultures has been studied
due to their association with desirable sensory
characteristics and are recognized for their ability
to contribute to the development of distinct flavors
and aromas, thereby playing a crucial role in the
production of specialty coffee (2,29,30).
Lactic acid bacteria were isolated from the first 48
hours while yeasts were isolated after 48 and up to
72 hours. LAB are well-adapted to the pre-existing
environmental conditions; therefore, it is common
to find LAB species instead of yeasts in the initial
hours of coffee fermentation(25,31). The microbial
community is known to vary in composition and
initial abundance during this process, and the
succession of bacterial and fungal species is
complex because their behavior depends on the
time and environmental factors (32–34).
A phylogenetic tree analysis was conducted to examine
the genetic relationships among representative
isolates of lactic acid bacteria (LAB) species belonging
to the Lacticaseibacillus, Lactiplantibacillus, and
Levilactobacillus genera (Figure 9)
The phylogenetic tree revealed distinct clusters
representing each genus. The Lacticaseibacillus and
Lactiplantibacillus cluster comprised species within
the plantarum group lactobacilli. These clusters
showed a close evolutionary relationship to other
lactobacillus groups commonly used as starter
cultures in fermentations (35), antimicrobials (36,37),
and the next generation of probiotics (38,39). This
suggests a shared genetic heritage and functional
similarities among these lactobacilli.
The Levilactobacillus cluster represented a group
of lactobacilli known for their leavening potential
(40). Multiple species within this genus have been
identified in type sourdoughs used as leavening
agents. The presence of Levilactobacillus highlights
the diversity and adaptability of these lactobacilli in
different fermentation environments (40).
Understanding the genetic relationships among
these species contributes to our knowledge of
their diversity, evolutionary history, metabolic
capabilities, and potential applications. It provides
a framework for further investigation into the
functional properties, ecological roles, and their
impact on fermentation and product quality.
The yeast isolates identified in this research
belong to five different genera (Figures 8 and 10),
namely Torulaspora, Kazachstania, Kurtzmaniella,
Hanseniaspora, and Pichia. Of particular interest is
the isolation of Torulaspora delbrueckii, which has
recently been reported in metagenomic studies
conducted in Colombia (41) and has also been
investigated in Latin America (42). This highlights the
presence and potential significance of T. delbrueckii
in coffee fermentation processes. Yeast species from
the genus Kazachstania, including Kazachstania
gamospora, have been documented in both
Colombia (43) and Rwanda (44). This suggests that
these yeasts are not limited to a specific geographic
region and may have a broader distribution in
coffee-growing areas.
Coffee processing steps have been found to harbor
a diverse range of yeast species from various
genera such as Pichia, Candida, Saccharomyces,
and Torulaspora (45). The presence of these
yeast species broadens the understanding of the
diversity and distribution of yeast species in coffee
processing.
The analysis of LAB and yeasts in coffee fermentation
provides valuable insights into the diversity and
functional attributes of these microorganisms;
including the production of flavor-active compounds
and the establishment of desirable fermentation
profiles. Further investigations into the metabolic
pathways and interactions of these microorganisms
will contribute to optimizing coffee fermentation
processes and enhancing the quality of the final
coffee product.
9Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 351373
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
CONCLUSIONS
This research study aimed to know the influence of
fermentation time and water on the development
of viable microorganisms. Firstly, the study
revealed that no treatment had an effect on the
different microbial groups without controlling other
environmental factors such as temperature and
anaerobiosis. The identification of diverse bacterial
and fungal species, including Pichia, Torulaspora,
and Lactiplantibacillus, emphasizes their potential
as starter cultures in controlled coffee fermentations
and their relevance in fermentation technology and
other biotechnological applications. The findings in
this study are important for the future production
of sustainable high-qualit y cof fees and the
development of starter cultures that have the
potential of developing distinctive flavor profiles
and aromatic compounds.
CONFLICTS OF INTEREST
The authors declare that they have no conflict of interest.
ACKNOWLEDGMENTS
We want to acknowledge the support provided
by the administration of La Antigua farm, for
allowing the development of this research in
their facilities, especially to coffee farmer Sergio
Echeverr y. Laboratorio de Café del Ser vicio
Nacional de Aprendizaje, SENA (Centro de los
Recursos Renovables Naturales, La Salada). We also
thank the Research Development Committee at
Universidad de Antioquia (Comité para el Desarrollo
de la investigación de la Universidad de Antioquia,
CODI) for the funding of this research. Finally, we
are grateful to Statistic Professors Guillermo Correa
and Kevin Hidalgo for their ongoing collaboration
and commitment.
AUTHORS´ CONTRIBUTIONS.
Karina E. Motato-Rocha: Conception and design of the
work, data collection, data analysis and interpretation,
drafting and critical revision of the article, and final
approval of the version to be published.
Valentina González-Montero: Conception and
design of the work, data collection, data analysis,
and interpretation, drafting the article.
María O Román-Morales: Conception and design of
the work, critical revision of the article, final approval
of the version to be published.
REFERENCES
1. Jiyuan Zhang S, De Bruyn F, Pothakos V, Torres J, Falconi C,
Moccand C. Following Coffee Production from Cherries to Cup:
Microbiological and Metabolomic Analysis of Wet Processing of
Coffea arabica. Appl Environ Microbiol. 2019;15;85(6):02635-18.
DOI: https://doi.org/10.1128/AEM.02635-18
2. Elhalis H, Cox J, Damian F, Zhao J. Microbiological and Chemical
Characteristics of Wet Coffee Fermentation Inoculated with
Hansinaspora uvarum and Pichia kudriavzevii and Their Impact
on Coffee Sensory Quality. Frontiers in Microbiology 2021; 12.
DOI: https://doi.org/10.3389/fmicb.2021.713969.
3. Bermudez S, Voora V, Larrea C. Coffee prices and sustainability
sustainable commodities marketplace series. International
Institute for Sustainable Development [Internet]. 2019 Jun [cited
2023 Jun 9]. Available from: https://www.iisd.org/system/files/
publications/ssi-global-market-report-coffee.pdf
4. ICO. Coffee Market report [Internet]. 2023 Feb [cited 2023 Mar 5];
Available from: https://www.icocoffee.org/documents/cy2022-23/
cmr-0223-e.pdf
5. Haile M, Kang WH. The Role of Microbes in Coffee Fermentation
and Their Impact on Coffee Quality. J Food Qual. 2019; 2019:6.
DOI: https://doi.org/10.1155/2019/4836709
6. Veloso TGR, da Silva M de CS, Cardoso WS, Guarçoni RC,
Kasuya MCM, Pereira LL. Effects of environmental factors on
microbiota of fruits and soil of Coffea arabica in Brazil. Sci Rep.
2020;10(1):14692. DOI: https://doi.org/10.1038/s41598-020-
71309-y
7. Peñuela AE, Zapata AD, Durango DL. Performance of different
fermentation methods and the effect on coffee quality (Coffea
arabica L.). Coffee Sci [Internet]. 2018 Aug [cited 2021 Nov
21]; 4:465 –76. Available from: http://www.sbicafe.uf v.br/
handle/123456789/11118
8. Cardoso WS, Agnoletti BZ, de Freitas R, de Abreu Pinheiro
F, Pereira LL. Biochemical Aspects of Coffee Fermentation.
Food Engineering Series. 2021;149–208. DOI: https://doi.
org/10.1007/978-3-030-54437-9_4
9. Bastian F, Hutabarat OS, Dirpan A, Nainu F, Harapan H, Emran TB,
Simal-Gandara J. From Plantation to Cup: Changes in Bioactive
Compounds during Coffee Processing. Foods. 2021; 10(11):2827.
DOI: https://doi.org/10.3390/foods10112827
10. Ijanu EM, Kamaruddin · M A, Norashiddin FA. Coffee processing
wastewater treatment: a critical review on current treatment
technologies with a proposed alternative. 2020;10:11. DOI:
https://doi.org/10.1007/s13201-019-1091-9
11. Logorapid. Tabla de códigos Pantone y RGB. https://www.
logorapid.com/pantone https://www.logorapid.com/pantone
2021 (accessed 2020 August 17).
12. Macrogen Online Sequencing Order System. https://dna.
macrogen.com/pageLinkDnaSys.do?layout=page_sub&link=/
support/retrieveGuideCes#none 2023 (accessed 2023 May 16).
13. Benchling. Benchling Biology Software. San Francisco, CA 94107.
https://benchling.com 2022. (accessed 2023 May 16).
14. NCBI. BLAST: Basic Local Alignment Search Tool. https://blast.
ncbi.nlm.nih.gov/Blast.cgi 2021 (accessed 2023 May 16).
15. Stecher G, Tamura K, Kumar S. Molecular Evolutionary Genetics
Analysis (MEGA) for macOS. Society for Molecular Biology and
Evolution. https://www.megasoftware.net/pdfs/Stecher_et_
al_2020.pdf 2020 (accessed 2022 May 2)
16. R Core Team. R: A language and environment for statistical
computing. https://www.r-project.org/ 2021 (accessed 2022
February 13).
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
CONCLUSIONS
This research study aimed to know the influence of
fermentation time and water on the development
of viable microorganisms. Firstly, the study
revealed that no treatment had an effect on the
different microbial groups without controlling other
environmental factors such as temperature and
anaerobiosis. The identification of diverse bacterial
and fungal species, including Pichia, Torulaspora,
and Lactiplantibacillus, emphasizes their potential
as starter cultures in controlled coffee fermentations
and their relevance in fermentation technology and
other biotechnological applications. The findings in
this study are important for the future production
of sustainable high-qualit y cof fees and the
development of starter cultures that have the
potential of developing distinctive flavor profiles
and aromatic compounds.
CONFLICTS OF INTEREST
The authors declare that they have no conflict of interest.
ACKNOWLEDGMENTS
We want to acknowledge the support provided
by the administration of La Antigua farm, for
allowing the development of this research in
their facilities, especially to coffee farmer Sergio
Echeverr y. Laboratorio de Café del Ser vicio
Nacional de Aprendizaje, SENA (Centro de los
Recursos Renovables Naturales, La Salada). We also
thank the Research Development Committee at
Universidad de Antioquia (Comité para el Desarrollo
de la investigación de la Universidad de Antioquia,
CODI) for the funding of this research. Finally, we
are grateful to Statistic Professors Guillermo Correa
and Kevin Hidalgo for their ongoing collaboration
and commitment.
AUTHORS´ CONTRIBUTIONS.
Karina E. Motato-Rocha: Conception and design of the
work, data collection, data analysis and interpretation,
drafting and critical revision of the article, and final
approval of the version to be published.
Valentina González-Montero: Conception and
design of the work, data collection, data analysis,
and interpretation, drafting the article.
María O Román-Morales: Conception and design of
the work, critical revision of the article, final approval
of the version to be published.
REFERENCES
1. Jiyuan Zhang S, De Bruyn F, Pothakos V, Torres J, Falconi C,
Moccand C. Following Coffee Production from Cherries to Cup:
Microbiological and Metabolomic Analysis of Wet Processing of
Coffea arabica. Appl Environ Microbiol. 2019;15;85(6):02635-18.
DOI: https://doi.org/10.1128/AEM.02635-18
2. Elhalis H, Cox J, Damian F, Zhao J. Microbiological and Chemical
Characteristics of Wet Coffee Fermentation Inoculated with
Hansinaspora uvarum and Pichia kudriavzevii and Their Impact
on Coffee Sensory Quality. Frontiers in Microbiology 2021; 12.
DOI: https://doi.org/10.3389/fmicb.2021.713969.
3. Bermudez S, Voora V, Larrea C. Coffee prices and sustainability
sustainable commodities marketplace series. International
Institute for Sustainable Development [Internet]. 2019 Jun [cited
2023 Jun 9]. Available from: https://www.iisd.org/system/files/
publications/ssi-global-market-report-coffee.pdf
4. ICO. Coffee Market report [Internet]. 2023 Feb [cited 2023 Mar 5];
Available from: https://www.icocoffee.org/documents/cy2022-23/
cmr-0223-e.pdf
5. Haile M, Kang WH. The Role of Microbes in Coffee Fermentation
and Their Impact on Coffee Quality. J Food Qual. 2019; 2019:6.
DOI: https://doi.org/10.1155/2019/4836709
6. Veloso TGR, da Silva M de CS, Cardoso WS, Guarçoni RC,
Kasuya MCM, Pereira LL. Effects of environmental factors on
microbiota of fruits and soil of Coffea arabica in Brazil. Sci Rep.
2020;10(1):14692. DOI: https://doi.org/10.1038/s41598-020-
71309-y
7. Peñuela AE, Zapata AD, Durango DL. Performance of different
fermentation methods and the effect on coffee quality (Coffea
arabica L.). Coffee Sci [Internet]. 2018 Aug [cited 2021 Nov
21]; 4:465 –76. Available from: http://www.sbicafe.uf v.br/
handle/123456789/11118
8. Cardoso WS, Agnoletti BZ, de Freitas R, de Abreu Pinheiro
F, Pereira LL. Biochemical Aspects of Coffee Fermentation.
Food Engineering Series. 2021;149–208. DOI: https://doi.
org/10.1007/978-3-030-54437-9_4
9. Bastian F, Hutabarat OS, Dirpan A, Nainu F, Harapan H, Emran TB,
Simal-Gandara J. From Plantation to Cup: Changes in Bioactive
Compounds during Coffee Processing. Foods. 2021; 10(11):2827.
DOI: https://doi.org/10.3390/foods10112827
10. Ijanu EM, Kamaruddin · M A, Norashiddin FA. Coffee processing
wastewater treatment: a critical review on current treatment
technologies with a proposed alternative. 2020;10:11. DOI:
https://doi.org/10.1007/s13201-019-1091-9
11. Logorapid. Tabla de códigos Pantone y RGB. https://www.
logorapid.com/pantone https://www.logorapid.com/pantone
2021 (accessed 2020 August 17).
12. Macrogen Online Sequencing Order System. https://dna.
macrogen.com/pageLinkDnaSys.do?layout=page_sub&link=/
support/retrieveGuideCes#none 2023 (accessed 2023 May 16).
13. Benchling. Benchling Biology Software. San Francisco, CA 94107.
https://benchling.com 2022. (accessed 2023 May 16).
14. NCBI. BLAST: Basic Local Alignment Search Tool. https://blast.
ncbi.nlm.nih.gov/Blast.cgi 2021 (accessed 2023 May 16).
15. Stecher G, Tamura K, Kumar S. Molecular Evolutionary Genetics
Analysis (MEGA) for macOS. Society for Molecular Biology and
Evolution. https://www.megasoftware.net/pdfs/Stecher_et_
al_2020.pdf 2020 (accessed 2022 May 2)
16. R Core Team. R: A language and environment for statistical
computing. https://www.r-project.org/ 2021 (accessed 2022
February 13).
10Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 31 | Number 01 | Article 351373Karina Motato-Rocha, María Orfilia Román-Morales y Valentina Gonzalez-Montero
17. Marín López S, Arcilla Pulgarín J, Montoya Restrepo E, Oliveros
Tascón C. Relación entre el estado de madurez del fruto del
café y las características de beneficio rendimiento y calidad de
la bebida. Cenicafé [Internet]. 2004 Jun. [cited 2021 Mar 29];
54(4):297-315. 2003. Available from: https://www.cenicafe.org/
es/publications/arc054%2804%29297-315.pdf
18. BLAST Glossary - BLAST® Help - NCBI Bookshelf. https://www.
ncbi.nlm.nih.gov/books/NBK62051/ 2023 (accessed 2023 Jun 12).
19. Orrego D, Zapata-Zapata AD, Kim D. Optimization and Scale-
Up of Coffee Mucilage Fermentation for Ethanol Production.
Energies. 2018; 11(4):786. DOI: ht tps://doi.org/10.3390/
en11040786
20. Sulaiman I, Hasni D. Microorganism growth profiles during
fermentation of Gayo Arabica wine coffee. IOP Conf Ser
Ear th Environ Sci [Internet]. 2022 Jan 1 [cited 2022 May
8];951(1):012076. Available from: https://iopscience.iop.org/
article/10.1088/1755-1315/951/1/012076
21. Pantoja López F, Rojas Gutiérrez PA, Oriana L, Macias M, Sofía
E, Quinayás T. Estudio de algunas variables en el proceso de
fermentación de café y su relación con la calidad de taza en el
sur de Colombia. Cienc. Tecnol [Internet]. 2015 Jul [cited 2020
Sep 2]. Vol. 3, Available from: https://repositorio.sena.edu.co/
handle/11404/6735?show=full
22. Betancur Henao NY, Motato Rocha KE. Procesos de fermentación
natural de café y su relación con el perfil sensorial final taza.
Métodos de seguimiento y control para pequeños caficultores
en el suroeste antioqueño [Tesis Work]. [Medellín, Colombia]:
Universidad de Antioquia; 2020.
23. de Melo Pereira G V., da Silva Vale A, de Carvalho Neto DP,
Muynarsk ES, Soccol VT, Soccol CR. Lactic acid bacteria: what
coffee industry should know? Vol. 31, Current Opinion in Food
Science. Elsevier; 2020; 31:1–8. DOI: https://doi.org/10.1016/J.
COFS.2019.07.004
24. Leong KH, Chen YS, Pan SF, Chen JJ, Wu HC, Chang YC. Diversity
of lactic acid bacteria associated with fresh coffee cherries
in Taiwan. Curr Microbiol. 2014;68(4):440–7. DOI: https://doi.
org/10.1007/s00284-013-0495-2
25. Pothakos V, De Vuyst L, Zhang SJ, De Bruyn F, Verce M, Torres
J. Temporal shotgun metagenomics of an Ecuadorian coffee
fermentation process highlights the predominance of lactic acid
bacteria. Curr Res Biotechnol. 2020;2:1–15. DOI: https://doi.
org/10.1016/j.crbiot.2020.02.001
26. Mayo B, Aleksandrzak-Piekarczyk T, Fernández M, Kowalczyk
M, Álvarez-Martín P, Bardowski J. Updates in the Metabolism
of L ac tic Acid Bac teria. Biotechnology of L ac tic Acid
Bacteria: Novel Applications. 2010; 3–33. DOI: https://doi.
org/10.1002/9780813820866.ch1
27. de Oliveira Junqueira, A, de Melo Pereira, G., Coral Medina,
J. First description of bacterial and fungal communities in
Colombian coffee beans fermentation analysed using Illumina-
based amplicon sequencing. Sci Rep 2019;9(1):1–10. DOI: https://
doi.org/10.1038/s41598-019-45002-8.
28. Bintsis T. Lactic acid bacteria as starter cultures: An update in
their metabolism and genetics. AIMS Microbiol. 2018;4(4):665-
684. DOI: https://doi.org/10.3934/microbiol.2018.4.665
29. Wang C, Sun J, Lassabliere B, Yu B, Liu SQ. Coffee flavour
modification through controlled fermentations of green coffee
beans by Saccharomyces cerevisiae and Pichia kluyveri: Part I.
Effects from individual yeasts. Food Res Int. 2020;136:109588.
DOI: https://doi.org/10.1016/j.foodres.2020.109588
30. da Mota, M.C., Batista, N.N., Rabelo, M.H., Ribeiro, D.E., Borém,
F.M., & Schwan, R.F. Influence of fermentation conditions on the
sensorial quality of coffee inoculated with yeast. Food research
international. 2020;136, 109482. DOI: https://doi.org/10.1016/j.
foodres.2020.109482
31. Silva CF, Batista LR, Abreu LM, Dias ES, Schwan RF. Succession of
bacterial and fungal communities during natural coffee (Coffea
arabica) fermentation. Food Microbiol . 2008;25(8):951–7. DOI:
https://doi.org/10.1016/j.fm.2008.07.003
32. Pino A, Espinosa Y, Cabrera E. Characterization of the Rhizosphere
Bac terial Microbiome and Cof fee Bean Fermentation in
the Castillo-Tambo and Bourbon Varieties in the Popayán-
Colombia Plateau. BMC Plant Biol. 2023;23,217. DOI: https://doi.
org/10.1186/s12870-023-04182-2
33. Jiyuan Zhang S, De Bruyn F, Pothakos V, Contreras GF, Cai Z,
Moccand C. Influence of Various Processing Parameters on
the Microbial Community Dynamics, Metabolomic Profiles,
and Cup Qualit y During Wet Cof fee Processing. Front
Microbiol. 2019;10,2621:1–24. DOI: https://doi.org/10.3389/
fmicb.2019.02621
34. Peñuela-Martínez AE, Velasquez-Emiliani AV, Angel CA. Microbial
Diversity Using a Metataxonomic Approach, Associated with
Coffee Fermentation Processes in the Department of Quindío,
Colombia. Fermentation. 2023; 9(4):343. DOI: https://doi.
org/10.3390/fermentation9040343
35. Ferreira I, de Sousa Melo D, Menezes AGT, Fonseca HC,
de Assis BBT, Ramos CL. Evaluation of potentially probiotic
yeasts and Lactiplantibacillus plantarum in co-culture for the
elaboration of a functional plant-based fermented beverage.
Food Research International. 2022 Oct 1;160:111697. DOI: https://
doi.org/10.1016/j.foodres.2022.111697
36. Kawahara A, Zendo T, Mat susaki H. Identification and
characterization of bacteriocin biosynthetic gene clusters found
in multiple bacteriocins producing Lactiplantibacillus plantarum
PUK6. J Biosci Bioeng. 2022;133(5):444-451. DOI: https://doi.
org/10.1016/j.jbiosc.2022.01.008.
37. Vougiouklaki D, Tsironi T, Papaparaskevas J, Halvatsiotis P,
Houhoula D. Characterization of Lacticaseibacillus rhamnosus,
Levilactobacillus brevis and Lactiplantibacillus plantarum
Metabolites and Evaluation of Their Antimicrobial Activity against
Food Pathogens. Appl. Sci. 2022; 12(2):660. DOI: https://doi.
org/10.3390/app12020660
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39. Liu DM, Huang Y Y, Liang MH. Analysis of the probiotic
characteristics and adaptability of lactiplantibacillus plantarum
DMDL 9010 to gastrointestinal environment by complete genome
sequencing and corresponding phenotypes. LWT. 2022;158,
113129-. DOI: https://doi.org/10.1016/j.lwt.2022.113129
40. Zheng J, Wittouck S, Salvetti E, Franz CMAP, Harris HMB,
Mattarelli P. A taxonomic note on the genus Lactobacillus:
Description of 23 novel genera, emended description of the genus
Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and
Leuconostocaceae. Int J Syst Evol Microbiol. 2020;70(4):2782-
2858. DOI: https://doi.org/10.1099/ijsem.0.004107
41. Cruz-O’Byrne R, Gamez-Guzman A, Piraneque-Gambasica
N, Aguirre-Forero S. Genomic sequencing in Colombian
coffee fermentation reveals new records of yeast species.
Food Biosci. 2023;1;52:102415. DOI: https://doi.org/10.1016/j.
fbio.2023.102415.
42. Cassimiro DM de J, Batista NN, Fonseca HC, Naves JAO, Dias
DR, Schwan RF. Coinoculation of lactic acid bacteria and yeasts
increases the quality of wet fermented Arabica coffee. Int J Food
Microbiol. 2022; 16; 369:109627. DOI: https://doi.org/10.1016/j.
ijfoodmicro.2022.109627
17. Marín López S, Arcilla Pulgarín J, Montoya Restrepo E, Oliveros
Tascón C. Relación entre el estado de madurez del fruto del
café y las características de beneficio rendimiento y calidad de
la bebida. Cenicafé [Internet]. 2004 Jun. [cited 2021 Mar 29];
54(4):297-315. 2003. Available from: https://www.cenicafe.org/
es/publications/arc054%2804%29297-315.pdf
18. BLAST Glossary - BLAST® Help - NCBI Bookshelf. https://www.
ncbi.nlm.nih.gov/books/NBK62051/ 2023 (accessed 2023 Jun 12).
19. Orrego D, Zapata-Zapata AD, Kim D. Optimization and Scale-
Up of Coffee Mucilage Fermentation for Ethanol Production.
Energies. 2018; 11(4):786. DOI: ht tps://doi.org/10.3390/
en11040786
20. Sulaiman I, Hasni D. Microorganism growth profiles during
fermentation of Gayo Arabica wine coffee. IOP Conf Ser
Ear th Environ Sci [Internet]. 2022 Jan 1 [cited 2022 May
8];951(1):012076. Available from: https://iopscience.iop.org/
article/10.1088/1755-1315/951/1/012076
21. Pantoja López F, Rojas Gutiérrez PA, Oriana L, Macias M, Sofía
E, Quinayás T. Estudio de algunas variables en el proceso de
fermentación de café y su relación con la calidad de taza en el
sur de Colombia. Cienc. Tecnol [Internet]. 2015 Jul [cited 2020
Sep 2]. Vol. 3, Available from: https://repositorio.sena.edu.co/
handle/11404/6735?show=full
22. Betancur Henao NY, Motato Rocha KE. Procesos de fermentación
natural de café y su relación con el perfil sensorial final taza.
Métodos de seguimiento y control para pequeños caficultores
en el suroeste antioqueño [Tesis Work]. [Medellín, Colombia]:
Universidad de Antioquia; 2020.
23. de Melo Pereira G V., da Silva Vale A, de Carvalho Neto DP,
Muynarsk ES, Soccol VT, Soccol CR. Lactic acid bacteria: what
coffee industry should know? Vol. 31, Current Opinion in Food
Science. Elsevier; 2020; 31:1–8. DOI: https://doi.org/10.1016/J.
COFS.2019.07.004
24. Leong KH, Chen YS, Pan SF, Chen JJ, Wu HC, Chang YC. Diversity
of lactic acid bacteria associated with fresh coffee cherries
in Taiwan. Curr Microbiol. 2014;68(4):440–7. DOI: https://doi.
org/10.1007/s00284-013-0495-2
25. Pothakos V, De Vuyst L, Zhang SJ, De Bruyn F, Verce M, Torres
J. Temporal shotgun metagenomics of an Ecuadorian coffee
fermentation process highlights the predominance of lactic acid
bacteria. Curr Res Biotechnol. 2020;2:1–15. DOI: https://doi.
org/10.1016/j.crbiot.2020.02.001
26. Mayo B, Aleksandrzak-Piekarczyk T, Fernández M, Kowalczyk
M, Álvarez-Martín P, Bardowski J. Updates in the Metabolism
of L ac tic Acid Bac teria. Biotechnology of L ac tic Acid
Bacteria: Novel Applications. 2010; 3–33. DOI: https://doi.
org/10.1002/9780813820866.ch1
27. de Oliveira Junqueira, A, de Melo Pereira, G., Coral Medina,
J. First description of bacterial and fungal communities in
Colombian coffee beans fermentation analysed using Illumina-
based amplicon sequencing. Sci Rep 2019;9(1):1–10. DOI: https://
doi.org/10.1038/s41598-019-45002-8.
28. Bintsis T. Lactic acid bacteria as starter cultures: An update in
their metabolism and genetics. AIMS Microbiol. 2018;4(4):665-
684. DOI: https://doi.org/10.3934/microbiol.2018.4.665
29. Wang C, Sun J, Lassabliere B, Yu B, Liu SQ. Coffee flavour
modification through controlled fermentations of green coffee
beans by Saccharomyces cerevisiae and Pichia kluyveri: Part I.
Effects from individual yeasts. Food Res Int. 2020;136:109588.
DOI: https://doi.org/10.1016/j.foodres.2020.109588
30. da Mota, M.C., Batista, N.N., Rabelo, M.H., Ribeiro, D.E., Borém,
F.M., & Schwan, R.F. Influence of fermentation conditions on the
sensorial quality of coffee inoculated with yeast. Food research
international. 2020;136, 109482. DOI: https://doi.org/10.1016/j.
foodres.2020.109482
31. Silva CF, Batista LR, Abreu LM, Dias ES, Schwan RF. Succession of
bacterial and fungal communities during natural coffee (Coffea
arabica) fermentation. Food Microbiol . 2008;25(8):951–7. DOI:
https://doi.org/10.1016/j.fm.2008.07.003
32. Pino A, Espinosa Y, Cabrera E. Characterization of the Rhizosphere
Bac terial Microbiome and Cof fee Bean Fermentation in
the Castillo-Tambo and Bourbon Varieties in the Popayán-
Colombia Plateau. BMC Plant Biol. 2023;23,217. DOI: https://doi.
org/10.1186/s12870-023-04182-2
33. Jiyuan Zhang S, De Bruyn F, Pothakos V, Contreras GF, Cai Z,
Moccand C. Influence of Various Processing Parameters on
the Microbial Community Dynamics, Metabolomic Profiles,
and Cup Qualit y During Wet Cof fee Processing. Front
Microbiol. 2019;10,2621:1–24. DOI: https://doi.org/10.3389/
fmicb.2019.02621
34. Peñuela-Martínez AE, Velasquez-Emiliani AV, Angel CA. Microbial
Diversity Using a Metataxonomic Approach, Associated with
Coffee Fermentation Processes in the Department of Quindío,
Colombia. Fermentation. 2023; 9(4):343. DOI: https://doi.
org/10.3390/fermentation9040343
35. Ferreira I, de Sousa Melo D, Menezes AGT, Fonseca HC,
de Assis BBT, Ramos CL. Evaluation of potentially probiotic
yeasts and Lactiplantibacillus plantarum in co-culture for the
elaboration of a functional plant-based fermented beverage.
Food Research International. 2022 Oct 1;160:111697. DOI: https://
doi.org/10.1016/j.foodres.2022.111697
36. Kawahara A, Zendo T, Mat susaki H. Identification and
characterization of bacteriocin biosynthetic gene clusters found
in multiple bacteriocins producing Lactiplantibacillus plantarum
PUK6. J Biosci Bioeng. 2022;133(5):444-451. DOI: https://doi.
org/10.1016/j.jbiosc.2022.01.008.
37. Vougiouklaki D, Tsironi T, Papaparaskevas J, Halvatsiotis P,
Houhoula D. Characterization of Lacticaseibacillus rhamnosus,
Levilactobacillus brevis and Lactiplantibacillus plantarum
Metabolites and Evaluation of Their Antimicrobial Activity against
Food Pathogens. Appl. Sci. 2022; 12(2):660. DOI: https://doi.
org/10.3390/app12020660
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11Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 351373
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
43. Cruz-O’byrne R, Piraneque-Gambasica N, Aguirre-Forero S.
Microbial diversity associated with spontaneous coffee bean
fermentation process and specialty coffee production in northern
Colombia. Int J Food Microbiol. 2021;354:109282. DOI: https://
doi.org/10.1016/j.ijfoodmicro.2021.109282
44. Hale AR, Ruegger PM, Rolshausen P, Borneman J, Yang J in.
Fungi associated with the potato taste defect in coffee beans
from Rwanda. Botanical Studies 2022;63(1):1–8. DOI: https://doi.
org/10.1186/s40529-022-00346-9
45. de Carvalho Neto DP, de Melo Pereira GV, Tanobe VOA, Soccol VT,
da Silva BJG, Rodrigues C. Yeast Diversity and Physicochemical
Characteristics Associated with Coffee Bean Fermentation from
the Brazilian Cerrado Mineiro Region. Fermentation. 2017; 3;1:11.
DOI: https://doi.org/10.3390/fermentation3010011
Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods
43. Cruz-O’byrne R, Piraneque-Gambasica N, Aguirre-Forero S.
Microbial diversity associated with spontaneous coffee bean
fermentation process and specialty coffee production in northern
Colombia. Int J Food Microbiol. 2021;354:109282. DOI: https://
doi.org/10.1016/j.ijfoodmicro.2021.109282
44. Hale AR, Ruegger PM, Rolshausen P, Borneman J, Yang J in.
Fungi associated with the potato taste defect in coffee beans
from Rwanda. Botanical Studies 2022;63(1):1–8. DOI: https://doi.
org/10.1186/s40529-022-00346-9
45. de Carvalho Neto DP, de Melo Pereira GV, Tanobe VOA, Soccol VT,
da Silva BJG, Rodrigues C. Yeast Diversity and Physicochemical
Characteristics Associated with Coffee Bean Fermentation from
the Brazilian Cerrado Mineiro Region. Fermentation. 2017; 3;1:11.
DOI: https://doi.org/10.3390/fermentation3010011