fermentation

Article
Effect of Prolonged Fermentations of Coffee Mucilage with
Different Stages of Maturity on the Quality and Chemical
Composition of the Bean
Valentina Osorio Pérez 1, *, Cristina Inés Álvarez-Barreto 2 , Luis Gerónimo Matallana 2 , José Ricardo Acuña 1 ,
Luz Fanny Echeverri 1 and Luis Carlos Imbachí 1

1 National Coffee Research Center, Planalto, km. 4, Vía Antigua Chinchiná-Manizales,

Manizales 170009, Colombia
2 Departamento de Ingeniería, Universidad de Caldas, Manizales 170007, Colombia
* Correspondence: valentina.osorio@cafedecolombia.com

Abstract: The sensory quality of coffee begins in the plant tree, where the characteristics of the
fruits define the composition of the chemical precursors, which can be preserved or transformed
in stages such as mucilage fermentation, and are the basis for the beverage attributes. This study
evaluated three degrees of maturity and their comportment in fermentation under two temperatures
and two-time extensions, establishing their sensory and chemical characteristics through analytical
techniques such as liquid and gas chromatography. The effect of the prolongation time was evidenced
for oxalic, quinic, citric acids, glucose, and fructose in two of the three degrees of maturity evaluated.
The interaction of the process conditions increased the content of fructose and glucose in one of
the states, being more evident at 20 ◦ C. The treatments associated with the most advanced stage
Citation: Osorio Pérez, V.;
Álvarez-Barreto, C.I.; Matallana, L.G.;
of maturity and with higher temperature decreased the scores of five sensory attributes and the
Acuña, J.R.; Echeverri, L.F.; Imbachí, fructose content increased by 48.50% and the glucose content increased by 47.31%. Advanced stages
L.C. Effect of Prolonged of maturity preserve quality standards, but their performance can be differential in postharvest
Fermentations of Coffee Mucilage processes, especially in those that are beyond the standards, such as those involving prolongations in
with Different Stages of Maturity on different processes such as fermentation.
the Quality and Chemical
Composition of the Bean. Keywords: maturity; organic acids; fructose; glucose; quality
Fermentation 2022, 8, 519.
https://doi.org/10.3390/
fermentation8100519

Academic Editor: Amparo Gamero 1. Introduction

The quality of coffee is the result of the combination of multiple factors that converge in
Received: 29 August 2022
Accepted: 19 September 2022
the productive system of the crop, each of which generates a unique chemical composition
Published: 7 October 2022
of the bean that is the basis for the expression of the sensory attributes of coffee [1,2]. The
postharvest process begins with the selection of the fruit at the optimal state of maturity
Publisher’s Note: MDPI stays neutral and ends with the drying of the bean, generating both positive and negative effects on the
with regard to jurisdictional claims in
sensory characteristics of the coffee beverage. For many years, the research associated with
published maps and institutional affil-
this process was focused on the development of equipment and operational optimizations
iations.
and controls to avoid the appearance of defects. Today, this process has a new perspective,
and elements such as the degree of maturity and the biotechnological conditions of mucilage
removal take on increasing importance in the sense that they can generate changes in the
Copyright: © 2022 by the authors.
chemical composition of the coffee bean and finally be reflected in sensory quality [3,4].
Licensee MDPI, Basel, Switzerland. The mucilage of coffee fruit is a hydrogel composed mainly of water, sugars, and pectic
This article is an open access article substances [5]. This covers or envelops the seed, representing approximately 22% of the
distributed under the terms and weight of the pulped coffee. The amount depends largely on the degree of maturity of the
conditions of the Creative Commons grain [6]. The chemical composition of the mucilage in combination with yeasts, fungi, and
Attribution (CC BY) license (https:// bacteria present in the environment, on the surface of the fruit (phyllosphere microbiota),
creativecommons.org/licenses/by/ and in the equipment explains the natural occurrence of fermentation at room temperature
4.0/). without resorting to inoculations. Due to the high impact of the mucilage fermentation

Fermentation 2022, 8, 519. https://doi.org/10.3390/fermentation8100519 https://www.mdpi.com/journal/fermentation

Fermentation 2022, 8, 519 2 of 16

process on the sensory quality of coffee, numerous studies have emerged with the objective
of generating control and consistency in the fermentation process. An important area of
study includes the use of microbiological starter cultures [7–11]. Several authors have
evaluated the ability of different starter cultures to improve the quality of coffee obtained
by fermentation in wet, dry, and semidry processing methods, finding that coffees with
different sensory profiles can be obtained [4,8]. While there is no consensus [12,13], some
results show that the use of pure cultures for the fermentation process in coffee processing
promotes consistency and control. Regarding the fermentation process [11], other studies
show that the changes are not significant when compared with the sensory quality of the
coffee obtained by spontaneous fermentation [12,14].
The microorganisms naturally present during the postharvest process (or processing)
use the compounds of the peel and mucilage as substrates during fermentation. They
produce organic acids and other metabolites that can affect the sensory characteristics of the
beverage [15,16]. In the case of the fungal population during postharvest, they can affect
the quality of the grain by generating negative flavors and producing mycotoxins [17].
The main factors that contribute to the formation of volatile compounds in coffee are the
microbial metabolites and the inherent compounds of the beans, and these can be highly
variable depending on the region of production and the variety of coffee used [16,18].
Numerous elements influence the formation of the aroma of coffee, and there is a delicate
relationship between flavor and fermentation; with adequate controls, it can improve
sensory qualities [11]. This can be attributed to the composition of the aroma precursors
present in green coffee after fermentation. Numerous biological, chemical, and physical
factors are involved in this process; however, temperature and oxygen availability have
been referenced as the main external factors [19]. In the case of dry-processed coffee, the
microbiota is much more varied and complex than that found in wet fermentations [9].
The microbial activities that occur during fermentation depend on the physicochemical
properties of the peel and mucilage, and these conditions can be variable depending on
the initial conditions of the coffee fruits and the type and processing conditions of the
benefit. In this research, the effect of the state of maturity of the fruit on the spontaneous
fermentation of the mucilage was determined at two external temperatures of the process
and two prolonged fermentation times. This influence was determined in the variables of
sensory quality and in the chemical composition of green almond coffee.

2. Materials and Methods

2.1. Coffee Fruits
Coffee fruits of the species Coffea arabica L., variety Castillo® , from the department of
Caldas, Colombia, were collected with the method of pass retention [20], which ensured the
maximum concentration of mature coffee. A total of 400 kg of fruits were processed (work
unit), hydraulically classified to remove low-quality fruits, and subsequently separated
into three degrees of maturity; for this research, there were 8 work units. The first state was
associated with the red-orange color (EM1-a *: 25.16); the second state was associated with
the crimson color (EM2-a *: 17.55); and the third state was associated with the garnet color
(EM3-a *: 8.09). The chromatic coordinate a * refers to the average obtained for the 8 work
units by state of maturity estimated with the CIEL * a * b * color scale.
The experimental units were formed by the stage of maturity, and the fruits were
pulped with a 2500 horizontal machine with a circular sieve (Medellín, Colombia). Then,
80 kg of pulped coffee was taken by stage of maturity and divided for the four treatments
(20 kg) corresponding to the spontaneous fermentation process at two temperatures, 15 and
20 ◦ C, and two additional fermentation times. The prolongation of the fermentation was
established as follows: the fermentation process was monitored following the FermaestroTM
methodology [21]. This method verifies the exact moment when the degradation of the
mucilage is completed by observing changes in the density of the coffee during the process.
The methodology used a truncated cone-shaped perforated vessel, which is more sensitive
to slight changes in volume. The decrease in volume of pulped coffee increase in the
 FermaestroTM methodology [21]. This method verifies the exact moment when the degra-
dation of the mucilage is completed by observing changes in the density of the coffee dur-
ing the process. The methodology used a truncated cone-shaped perforated vessel, which
is more sensitive to slight changes in volume. The decrease in volume of pulped coffee
increase in the height of the empty space at the top of the appliance. When the height of
Fermentation 2022, 8, 519 3 of 16
the coffee in the device stabilizes, this indicates that the washing point has been reached.
After the end time defined by this device, the grains continued the fermentation process
for two additional times: 10 and 20 h. In this way, 12 treatments were configured (EM:
height of stage;
maturity the empty space at thet:top
T: temperature; of the appliance.
extension When the
time): EM1T15t10, height of the
EM1T15t20, coffee in
EM1T20t10,
the device stabilizes,
EM1T20t20, EM2T15t10,this indicates
EM2T15t20, that the washing point
EM2T20t10, has been reached.
EM2T20t20, EM3T15t10, Afterand
the
end time defined by this device, the grains continued the fermentation
EM3T20t20 (Figure 1). After fermentation, in 30 L plastic containers, the coffee was process for two
additional
washed, times: 10
classified, and
and 20 h.inIna this
dried way, 12static
mechanical treatments were with
layer dryer configured (EM:
indirect maturity
combustion,
stage; T: temperature; t: extension time): EM1T15t10, EM1T15t20, EM1T20t10,
propane gas as fuel and with an air flow rate of 100 m /min/t cps at 40 °C to a humidity
3 EM1T20t20,
EM2T15t10,
range of 10 toEM2T15t20,
12%. EM2T20t10, EM2T20t20, EM3T15t10, and EM3T20t20 (Figure 1).
AfterDuring
fermentation, in 30 L plastic
the development of thecontainers,
process, the coffeetemperature
internal was washed,ofclassified, and dried
pulped coffee vol-
in a mechanical static layer dryer with indirect combustion, propane
ume was monitored every four hours, and the pH, titratable acidity, and Brix degrees of gas as fuel and with
an air
the flow rate
mucilage of 100
were m3 /min/t
evaluated untilcps 40 ◦of
theatend C the
to atotal
humidity range ofprocess.
fermentation 10 to 12%.

Figure
Figure 1.
1. Methodology
Methodology of
of prolonged
prolonged fermentation treatments.

During the development

2.2. Temperature, of the
pH, Degrees Brix, process,
and internal
Titratable temperature of pulped coffee volume
Acidity
was For
monitored every four hours, and the pH, titratable acidity, and with
temperature monitoring, a stainless-steel probe thermometer Brix an
degrees of the
accuracy of
mucilage were evaluated until the end of the total fermentation process.
± 0.3 °C was used. For the determination of pH, a Mettler Toledo MP 230 pH device (Im
Langacher, Switzerland) was used. The Brix degrees were determined with the Belling-
2.2. Temperature, pH, Degrees Brix, and Titratable Acidity
ham Stanley RFM742 refractometer (Bradford, United Kingdom); results were compen-
satedForby
◦
temperature
temperature monitoring, a stainless-steel
at 20 °C, with a resolution probe
of 0.01thermometer
and accuracy with an accuracy
± 0.04. of
Titratable
± 0.3 C
acidity was
was used. For the
determined determination
by volumetric of pH,
titration a Mettler
with a basicToledo
reagent MP 230 pHuntil
(NaOH) device (Im
reach-
Langacher, Switzerland) was used. The Brix degrees were determined with the Bellingham
ing pH 8.3 in a Mettler Toledo DL 53 titrator (Im Langacher, Switzerland). The result is
Stanley RFM742 refractometer (Bradford, UK); results were compensated by temperature at
expressed in mg CaCO3/L of the solution. For pH and temperature, the electrode was in-
20 ◦ C, with a resolution of 0.01 and accuracy ± 0.04. Titratable acidity was determined by
troduced into the volume of pulped coffee; for Brix degrees and titratable acidity, a sample
volumetric titration with a basic reagent (NaOH) until reaching pH 8.3 in a Mettler Toledo
of the liquid mucilage was taken.
DL 53 titrator (Im Langacher, Switzerland). The result is expressed in mg CaCO3 /L of the
solution. For pH and temperature, the electrode was introduced into the volume of pulped
2.3. High-Performance Liquid Chromatography HPLC
coffee; for Brix degrees and titratable acidity, a sample of the liquid mucilage was taken.
The compounds of interest were obtained from ground green coffee using water type
I2.3.
as the solvent. Sugars
High-Performance wereChromatography
Liquid obtained by refluxing
HPLC for 30 min of 0.02 g of green coffee,
subsequent centrifugation at 10,000 rpm,
The compounds of interest were obtained and filtration
from of 1.00 mL
ground of the
green solution
coffee through
using water
0.45 μm filters. Organic acids were obtained by stirring 0.08 g of green
type I as the solvent. Sugars were obtained by refluxing for 30 min of 0.02 g of green coffee and sedi-
mentation at 20 °C centrifugation
coffee, subsequent for 15 min andatfiltration of 1.00
10,000 rpm, andmL of the solution
filtration of 1.00 mLthrough
of the 0.45 μm
solution
filters.
through In0.45
the separation
µm filters. and quantification
Organic acids wereof sugars, by
obtained an stirring
Alliance0.08
2690g HPLC
of greensystem
coffeecou-
and
pled
sedimentation at 20 ◦index
to a refractive detector
C for 15 2414
min and was used,
filtration and
of 1.00 mLtheof process was through
the solution performed0.45in
µma
filters. In the separation and quantification of sugars, an Alliance 2690 HPLC system
coupled to a refractive index detector 2414 was used, and the process was performed
in a Sugar Pack I column (Waters, 6.5 × 300 mm, 10 µm) (Connecticut, USA) at 85 ◦ C
using deionized water at a flow rate of 0.5 mL/min. Organic acids and alkaloids were
determined using a Waters 600E HPLC system coupled to a diode array detector (DAD-996)
(Connecticut, USA) with a Hi-Plex H column (Agilent, 7.7 × 300 mm, 8 µm) (California,
USA) at a temperature of 50 ◦ C and a flow of 0.5 mL/min with deionized water (acidified:
0.01 M H2 SO4 ) and detected at 210 nm. For the determination of alkaloids, a Symmetry C18
Fermentation 2022, 8, 519 4 of 16

column (Waters, 4.6 mm × 250 mm, 100 Å, 5 µm) (Connecticut, USA) was used at 35 ◦ C,
with a flow of 1.0 mL/min using a mixture of water, methanol, and acetic acid (59:40:1
v/v/v) and detection at 273 nm.

2.4. Gas Chromatography

The composition of free fatty acids present in the lipid fraction of green almond coffee
was determined by gas chromatography coupled to a mass selective detector (HP-8860-
MSD 6890) (California, USA) using the reference method AOAC 969.33. Free fatty acids
were esterified from 80 µL of lipid extract, which was esterified with 1 mL of a 20% boron
trifluoride methanolic solution at 80 ◦ C for 1 h. Free fatty acids were extracted from the
methanolic solution by two successive liquid–liquid extractions with 1 mL of hexane. The
samples were shaken and after 5 min, the two-phase separation was completed. The separa-
tion of the compounds was performed with an HP-MS column (5% phenylmethylsiloxane
30 × 250 µm × 0.25 µm) (California, USA) with a temperature ramp program of 20 ◦ C/min
from 90 to 260 ◦ C and a constant helium flow of 1.2 mL/min. The structural confirmation
of the fatty acids present in the sample was performed from the mass spectra obtained and
compared with databases (NIST2017).

2.5. Total Chlorogenic Acids, Total Lipids, and Total Protein

The determination of the total content of chlorogenic acids was performed in a Beck-
man spectrophotometer (DU-650) (California, USA) using three wavelengths of 265, 328,
and 380 nm after extraction with aqueous methanol and purification with Carrez reagents.
For the quantification of the lipid fraction, extraction by Soxhlet was used, followed by the
rotary evaporation process according to the AOAC 945.16 analysis method. The determi-
nation of total protein was performed using the method based on the principle of Dumas
990.03 of AOAC, 2005, through combustion with a controlled oxygen supply, in which the
organic matter is destroyed at high temperatures, generating nitrogen and carbon gases,
which are quantified by an IR detection cell and a thermal conductivity cell. The protein
value was obtained by multiplying the percentage of nitrogen obtained from the elemental
analysis by a factor of 6.25.

2.6. Physical and Sensory Quality

The physical quality included the determination of the moisture content, the per-
centage of loss, defective grains, brocaded grains, black grains and vinegars, and healthy
almonds [22]. The sensory evaluation was performed by five certified Q-Grader tasters
following the SCA (Specialty Coffee Association) protocol. This methodology includes the
beverage preparation protocol: degree and roasting times (55–65 Agtron/SCA, 8–12 min),
coffee proportion, grinding granulometry, water temperature, and quality and analysis
temperatures. Ten sensory attributes were recorded: fragrance/aroma, flavor, residual
flavor, acidity, body, balance, uniformity, clean cup, sweetness, taste score, defects, and
total score.

2.7. Statistical Analysis

For each maturity stage, the response variables were analyzed with an analysis of
variance corresponding to a completely randomized experimental design model in a 2 × 2
factorial arrangement at a significance level α = 0.05. When the analysis of variance showed
the effect of treatments (p-value < 0.05), Duncan’s multiple comparison test (α = 0.05)
was performed on the treatments to identify the effect of temperatures and/or prolonged
fermentation times.

3. Results and Discussion

3.1. Temperature, pH, Degrees Brix, and Titratable Acidity
The comportment of the variables was monitored at four-hour intervals. The treat-
ments with an extension time of 10 h after estimating the washing point of the coffee with
Fermentation 2022, 8, 519 5 of 16

FermaestroTM had an average total processing time of 24 h, while for the extension of 20 h,
it was 36 h.
The process temperatures of 15 ◦ C and 20 ◦ C generated differences in the internal
temperature of the samples as the fermentation time of the coffee mucilage increased. The
twelve treatments began the process with an average temperature of 21.81 ◦ C, but the dif-
ference was evident between hours 16 and 24. For hours 16, 20, and 24, the treatments with
a process temperature of 15 ◦ C presented temperature averages of 19.67 ◦ C, 19.53 ◦ C, and
19.67 ◦ C, and the treatments with a process temperature of 20 ◦ C had average temperatures
of 23.29 ◦ C, 24.42 ◦ C, and 24.59 ◦ C, respectively. Correa et al., 2014 [23], reported an average
temperature of 21.2 ◦ C for a spontaneous fermentation of 20 h. The average temperatures
described above are part of the optimal range for the growth of most microorganisms. The
greatest difference between the treatments with process temperatures of 15 ◦ C and 20 ◦ C
was 4.92 ◦ C at hour 24, and this difference decreased to 3.78 ◦ C at hour 36. The mass was
explained by the exothermic process generated by microbial growth. The comportment of
the temperature is contrary to that reported by de Oliveira Junqueira et al., 2019 [7], where
the temperature had an initial average value of 28 ◦ C and decreased until hour 12 when it
stabilized, ending at hour 48 with an average value of 18 ◦ C.
The pH at time zero of fermentation presented an average value of 5.29 for all treat-
ments, similar to the value of 5.4 reported by de Melo Pereira et al., 2015 [12]. It shows a
strong decrease until hour 16 (Figure 2), reaching an average value of 4.38, then continues
to show decreases in its value, but less accentuated and reaches an average value of 3.96 at
hour 36. The pH of the coffee mucilage during fermentation of fruits with different stages
of maturity shows a negative linear comportment within the evaluated times. As in the tem-
perature in monitoring hours 16 and 24, a separation of the treatments was generated with
different process temperatures (15 and 20 ◦ C). The treatments with a process temperature
of 15 ◦ C had values of 4.56 and 4.14, and the treatments with a temperature of 20 ◦ C had
values of 4.19 and 3.99, respectively. At hour 36, these average values of the treatments with
different process temperatures were equal to a value of 3.96. The minimum pH value found
was 3.90, which was present in the EM1T20t20 treatment at hour 36, while the maximum
value of 5.41 was observed at time zero for these same treatments. The initial pH value and
the comportment coincide with that found by de Oliveira Junqueira et al., 2019 [7], where
the initial average value was 5.2 with a slight increase in hour 6 of the process followed by
a continuous decrease until hour 48, ending with an average value of 4.2. Avallon et al.,
2001 [24], reported that after fermentation, the mucilage cell assemblages with apparently
intact walls are separated from the parchment by the rupture of the walls of the first cell
layer. The acidification process changes the properties of the inner layer of the mucilage,
weakening the polysaccharide network and generating a change in its texture. For this
reason, pH values lower than 4.5 are used as a method to determine the end of the coffee
fermentation process [18]. Peñuela-Martínez et al., 2018 [25], developed fermentations with
temperature and pH control, finding an effect of their interaction; at lower temperatures,
there were higher values of sensory quality. Fermentation at a temperature of 17 ◦ C and
pH of 5 obtained a significantly higher score.
The degrees of Brix did not show significant changes during the fermentation of the
mucilage, starting with average values of 10.31 and increasing to average levels of 15.57,
and after hour 4, the values remained stable in a range from 15 to 17 until hour 36. At
time zero, the minimum average values of 9.26, 10.62, and 11.04 were presented for the
maturity stages EM1, EM2, and EM3. This difference in the initial values may be associated
with the lower amount of water in the mucilage in the EM3 state, which generates a
concentration of the total soluble solids generating a slightly higher value, possibly also
by hydrolytic processes of the pectin. The mucilage content in the EM1 and EM3 stages
of maturity are different; the maximum percentage is in the EM1 stage, with a value of
15.44%, and decreases as the stage of maturity increases until reaching a value of 10.07
with respect to the total weight of the fruit. The treatments with maturity stages EM1 and
EM3 presented the maximum value of degrees Brix at hour 16, with values of 16.45 and
Fermentation 2022, 8, 519 6 of 16

17.68, respectively. For stage EM2, the maximum value of 16.92 was observed at hour 20.
Although the comportment of the degrees of Brix is similar to that reported by Oliveira
Junqueira et al., 2019 [7], the initial value differs; these authors report a slight increase in
the first 12 h of fermentation with an initial value of approximately 5.3, which was half
of what was reported in this study; although, these authors do not report the degrees
Fermentation 2022, 8, x FOR PEER REVIEW 6 ofof
17
maturity of the fruits.

Figure 2. Average pH of mucilage of coffee with prolonged fermentation.

Figure 2. Average pH of mucilage of coffee with prolonged fermentation.

Thetitratable
The degrees of Brix did
acidity notcoffee
of the showmucilage
significant changes
during during thehad
fermentation fermentation of the
a positive linear
mucilage, starting
comportment; with average
the average at timevalues
zero for of 10.31 and increasing
all treatments was 862.16to average
(mg CaCO levels of 15.57,
3 /L), and
andaverage
the after hour value4, the
wasvalues
6148.46remained
in hour stable in a range
36. Similar to thefrom 15 totemperature
internal 17 until hourof36. theAtmass
time
zero, the minimum average values of 9.26, 10.62, and
and the pH as time passes, a separation of the treatments is generated under the two11.04 were presented for the ma-
turity stages EM1, EM2, and EM3. This difference in the initial
conditions of process temperature, and this differentiation is more evident beginning in values may be associated
with12.
hour theThelower amount values
maximum of water in the
were mucilage
found at hour in36;
thefor
EM3 thestate, which associated
treatments generates awith con-
acentration of the total soluble
process temperature of 15 C,◦ solids
this generating a slightlyand
value was 5600.60, higher value,of
for those ◦
possibly
20 C, alsoit wasby
hydrolytic
6696.32. Theprocesses of the pectin.
greatest difference betweenThe mucilage
treatments content in the EM1
with different and temperatures
process EM3 stages of
(15 ◦ C andare
maturity 20 ◦different; the maximum
C) was evidenced at hourpercentage
24 with a value is in ofthe1589.15
EM1 stage,
in favor with a value of
of treatments
15.44%,
with and decreases
a process temperatureas the 20 ◦ C.ofThe
of stage maturity increasesofuntil
comportment reaching
acidity implies a value of 10.07
a continuous
with respect to the total weight of the fruit. The treatments
accumulation of galacturonic acids from the hydrolysis of pectin and is much more evident with maturity stages EM1 and
EM3the
than presented
decreasethe maximum
in pH, value it
and although ofpresents
degrees negative
Brix at hourlinear16, with values in
comportment ofadvanced
16.45 and
17.68, of
stages respectively.
fermentation, Forthe
stage EM2, are
changes the less
maximum
markedvalue of 16.92 was
in comparison withobserved
titratableat acidity.
hour 20.
Although the comportment of the degrees of Brix is similar to that reported by Oliveira
3.2. PhysicaletQuality
Junqueira al., 2019 [7], the initial value differs; these authors report a slight increase in
For the
the first 12 hphysical quality variables,
of fermentation with an initial the analysis
value ofofapproximately
variance showed no effect
5.3, which was ofhalf
treat-
of
ments according to the F test (α = 0.05) for maturity stage,
what was reported in this study; although, these authors do not report the degrees of ma- process temperature, and
prolonged
turity of the fermentation
fruits. time. The average moisture content of the samples ranged from
11.10 The
to 11.68%,
titratablewhich implied
acidity of thethat
coffee themucilage
water activity
duringwas in the range
fermentation hadofa0.62–0.63. The
positive linear
percentage
comportment; of decline had anataverage
the average time zero value
for of
all 18.31%,
treatments andwas the minimum
862.16 (mgand CaCOmaximum
3/L), and
values of 18.15
the average valueandwas18.49, respectively,
6148.46 in hour 36. were found
Similar in the
to the EM2T15t10
internal temperatureand EM1T15t20
of the mass
treatments. With respect to the defective kernel, the maximum
and the pH as time passes, a separation of the treatments is generated under the two content of defective beans
con-
was presented by the EM3T20t20 treatment with a value of 3.64%,
ditions of process temperature, and this differentiation is more evident beginning in hour the general average of
the treatments was 2.68%, and the fermentation prolongation
12. The maximum values were found at hour 36; for the treatments associated with a pro- times of 10 and 20 h were
2.43% and 2.92%, respectively.
cess temperature of 15 °C, thisThe valuestatewasof 5600.60,
maturityand withforthethose
highest
of 20content of defective
°C, it was 6696.32.
beans was EM3,
The greatest with a value
difference between of 3.05%;
treatmentsin thiswith
same state, the
different minimum
process value of 1.91%
temperatures (15 °C
was found in the treatment with a temperature of 15 ◦ C and a prolonged time of 10 h.
and 20 °C) was evidenced at hour 24 with a value of 1589.15 in favor of treatments with a
The average
process value of the
temperature of 20black grains
°C. The and vinegars
comportment was 0.28%,
of acidity andathe
implies maximum
continuous value
accumu-
lation of galacturonic acids from the hydrolysis of pectin and is much more evident than
the decrease in pH, and although it presents negative linear comportment in advanced
stages of fermentation, the changes are less marked in comparison with titratable acidity.
Fermentation 2022, 8, 519 7 of 16

by state of maturity was found in EM3, with a value of 0.35%. In all maturity stages, the
treatments associated with a temperature of 20 ◦ C and an extension time of 20 h reported
maximum values of blacks and vinegars of 0.39, 0.34, and 0.54 in stages EM1, EM2, and
EM3, respectively, which may involve grain pigmentation associated with longer process
times due to possible darkening reactions caused by the enzymatic degradation of phenolic
compounds. The average of brocaded grains was 3.12%, and the maximum value was
found in EM3T20t10 with a value of 4.13%. The average value of the percentage of healthy
almonds is estimated at 74% with respect to dry parchment coffee, and higher values
indicate a better physical quality. The percentages of healthy almonds were 77.04, 76.75,
and 75.87 for the stages of maturity EM1, EM2, and EM3, respectively. The maximum value
of 77.55% was reported for the EM1T20t10 treatment, and the minimum value of 75.31%
was reported for the EM3T20t10 treatment.

3.3. Organic Acids

The major organic acids found in green almond coffee were citric, quinic, malic,
and acetic acids, with average contents (g/kg) of 10.62, 5.85, 4.85, and 2.59, respectively
(Figure 3). The analysis of variance did not show a significant effect of acetic, lactic, malic,
and succinic acids, which is different from that reported by De Bruyn et al., 2017 [26],
where the prolongation of the fermentation time generates a proportional increase in
the concentrations of acetic acid, ethanol, glycerol, glucuronic acid, lactic acid, mannitol,
and succinic acid. With respect to succinic acid, it is similar to that described by Elhalis
et al., 2020 [27], where the concentrations remain relatively constant during this stage.
Acetic acid had an average value of 2.59 g/kg, with minimum and maximum values
of 2.10 and 3.62 in treatments EM3T20t20 and EM2T15t10, respectively. The maximum
average was evidenced in the treatments associated with the state of maturity EM2, with
a value of 2.74. Lactic acid presented an average content of 0.31 g/kg, and the maximum
average value was reported in the treatments associated with the state of maturity EM3
and prolongation times of 20 h, with values close to 0.32. De Melo Pereira et al., 2020 [18],
report that the abundant sugar content present in the mucilage of the coffee fruit, which
includes pentoses, hexoses, and polysaccharides, is the primary source of carbon and
energy for the growth of lactic acid bacteria, but that under the conditions of carbon and
environmental limitation acid, these homofermentative species can change to a mixed
acid metabolism so that, according to the process conditions, different compositions of
organic acids can be found. De Carvalho et al., 2018 [28], did not observe differences in
the concentrations of citric and succinic acids in the samples with inoculated fermentation
treatments and with spontaneous fermentation. However, they reported approximately
double the concentration of lactic acid in the process inoculated with the Lactobacillus
species. The average content of malic acid was 4.85 g/kg, and the average values of the
treatments increased with more advanced stages of maturity, with values of 4.57, 4.90, and
5.07 for the states. EM1, EM2, and EM3, respectively. The average value of succinic acid
was 1.62 g/kg, the minimum was 1.45 in treatment EM1T15t10, and the maximum was 1.79
in treatment EM3T15t20. Succinic acid is naturally present in coffee, and the translocation
of this acid occurs from the pulp to the bean with the help of microbial activity and can
cause changes in the attributes of the beverage [1]. De Oliveira Junqueira et al., 2019 [7],
reported in the first description of the predominant bacteria in spontaneous fermentation
that Leuconostoc and Pichia nakasei were dominant, and the metabolic activity of these
microbial groups resulted mainly in the production of lactic acid and acetaldehyde. These
authors found a concentration of 2.76 g/L organic acids, which were not modified after the
fermentation processes, indicating that the microbial activity and the drying process do not
interfere with the composition of the main compounds within the coffee.
 of the predominant bacteria in spontaneous fermentation that Leuconostoc and Pichia nak-
asei were dominant, and the metabolic activity of these microbial groups resulted mainly
in the production of lactic acid and acetaldehyde. These authors found a concentration of
2.76 g/L organic acids, which were not modified after the fermentation processes, indicat-
Fermentation 2022, 8, 519
ing that the microbial activity and the drying process do not interfere with the8composi-
of 16

tion of the main compounds within the coffee.

Figure 3. Average
Figure 3. Averageorganic
organicacid
acid content ofcoffee
content of coffeebeans
beans with
with prolonged
prolonged mucilage
mucilage fermentation.
fermentation.

ForFor
thethe oxalic
oxalic andquinic
and quinicacids
acidsinin the
the EM1 stage
stage of
ofmaturity
maturityandandforfor
the citric
the acid
citric acid in
in the EM2 stage, the analysis of variance showed a significant effect
the EM2 stage, the analysis of variance showed a significant effect on the fermentationon the fermentation
prolongation
prolongation time
time (Figure4).
(Figure 4).Oxalic
Oxalicacidacidininthe
theEM1
EM1state
statepresents
presents aa reduction
reduction inin its
its con-
concentration of 42.85% and quinic acid an increase of 10.93% when going from 10 to 20 h
centration of 42.85% and quinic acid an increase of 10.93% when going from 10 to 20 h of
of prolongation of the process. The citric acid in the state of maturity EM2 shows a decrease
prolongation of the process. The citric acid in the state of maturity EM2 shows a decrease
of 3.82%. The state of maturity EM3 showed no effect on the contents of organic acids in
of 3.82%. The state
green almond of maturity
coffee. De BruynEM3 et al.,showed
2017 [26],noreport
effectthat
on green
the contents of organic
coffee beans processedacids in
green almond coffee. De Bruyn et al., 2017 [26], report that green coffee
wet contain higher concentrations of citric acid than green coffee beans processed dry. They beans processed
wetalso
contain higher
reported concentrations
that mucilage of citric
changes are acid
reflected in than green coffee
the endosperm, wherebeans processed
they found high dry.
They also reported that mucilage changes are reflected in the endosperm, where
concentrations of microbial metabolites. Contrary to what was reported by Elhalis et al., they
2020 [27], who described relatively stable levels of quinic acid during
found high concentrations of microbial metabolites. Contrary to what was reported by fermentation, this
research
Elhalis reported
et al., an increase
2020 [27], in the staterelatively
who described of maturitystable
EM1, which
levels may be associated
of quinic with fer-
acid during
the degradation of total chlorogenic acids.
mentation, this research reported an increase in the state of maturity EM1, which may be
associated
3.4. Total with
Lipidsthe
anddegradation of total chlorogenic acids.
Free Fatty Acids
For the lipid fraction of green almond coffee, the analysis of variance did not show
a significant effect on the state of maturity or the interaction of the temperature of the
process and the time of mucilage fermentation. Table 1 shows the average values per
treatment of palmitic, linoleic, oleic, stearic, and arachidic fatty acids. The average value of
the percentage of lipids was 10.50, the minimum was 10.27 in the EM1T15t20 treatment,
and the maximum was 10.79 in the EM3T20t20 treatment.
Fermentation 2022, 8, 519 9 of 16
Fermentation 2022, 8, x FOR PEER REVIEW 9 of 17

Oxalic acid Quinic Acid Citric acid

EM1-Prolongation time (h) EM1-Prolongation time (h) EM2-Prolongation time (h)
10 0.14 A 10 5.03 B 10 10.97 A
20 0.08 B 20 5.58 A 20 10.55 B
Figure 4. Effect of mucilage fermentation prolongation time on organic acids. Uncommon letters
Figure
imply4. Effect of mucilage
a difference fermentation
in averages according toprolongation
Duncan’s test time on organic acids. Uncommon letters
(α = 0.05).
imply a difference in averages according to Duncan’s test (α = 0.05).
3.4. Total Lipids and Free Fatty Acids
Average
Table 1.For content
the lipid of lipids,
fraction freealmond
of green fatty acids, protein,
coffee, alkaloids,ofand
the analysis total chlorogenic
variance acidsa for
did not show
significant
coffee effect on
with different the state
stages of maturity
of maturity or the interaction
and prolonged of the
fermentation temperature
of the mucilage. of the pro-
cess and the time of mucilage fermentation. Table 1 shows the average values per treat-
Palm * Lino of
ment * palmitic,
Olei * linoleic,
Steaoleic,
* Arac * and arachidic fatty acids. The average value of the
stearic,
Lip * Prot * Caf * Theob Trig * CGA *
(C16:0) (C18:2) (C18:1) (C18:0) (C20:0)
percentage of lipids was 10.50, the minimum was 10.27 in the EM1T15t20 treatment, and
(%) (%) (%) * (%) (%) (%)
(%) (%)
the maximum (%)
was 10.79 (%) (%)
in the EM3T20t20 treatment.
EM1T15t10 10.39 41.32 34.37 10.19 9.57 4.56 13.77 1.09 0.02 0.87 3.90
EM1T15t20 10.27 40.73 Table
34.331. Average
10.76content9.61
of lipids, free fatty acids,
4.57 protein, alkaloids,
13.75 1.09 and total chlorogenic
0.02 0.87 acids3.94
for
EM1T20t10 10.56 40.84 34.65with different
coffee 10.50 stages9.60 4.41
of maturity 13.74 fermentation
and prolonged 1.10 0.02mucilage.
of the 0.87 3.82
EM1T20t20 10.29 40.13 34.77 10.95 9.62 4.53 13.75 1.09 0.02 0.87 3.81
EM2T15t10 10.40 Lino *
Lip40.67
* Palm34.90
* 10.42 9.50*
Olei 4.51 *
Stea 13.83
Arac * 1.12 Prot * Caf0.02* Theob 0.86 3.91
Trig * CGA *
EM2T15t20 10.63 40.58 34.42 (C18:2)
10.83 9.42 4.75 13.87 1.12 0.02 0.86 3.74
(%) (C16:0) (%) (C18:1) (%) (C18:0) (%) (C20:0) (%) (%) (%) * (%) (%) (%)
EM2T20t10 10.43 40.59 34.94 10.55
(%) 9.31 4.61 13.79 1.14 0.02 0.88 3.75
EM2T20t20 10.46
EM1T15t10 41.17 41.32
10.39 34.39 34.37 10.23 9.47
10.19 4.73
9.57 13.64
4.56 1.16
13.77 1.09 0.02 0.02 0.89 3.83
0.87 3.90
EM3T15t10 10.75 41.13 34.20 10.54 9.47 4.65 14.02 1.16 0.02 0.89 3.80
EM1T15t20 10.27 40.73 34.33 10.76 9.61 4.57 13.75 1.09 0.02 0.87 3.94
EM3T15t20 10.61 41.36 34.24 10.30 9.50 4.60 13.80 1.14 0.02 0.88 3.84
EM1T20t10
EM3T20t10 10.44 10.56
41.62 40.84
33.88 34.65 10.37 10.50
9.40 9.60
4.73 4.41
13.65 13.74 1.10
1.14 0.02 0.02 0.880.87 3.82
3.62
EM1T20t20
EM3T20t20 10.79 10.29
42.25 40.13
33.03 34.77 10.22 10.95
9.65 9.62
4.85 4.53
14.02 13.75 1.09
1.16 0.02 0.02 0.880.87 3.81
3.65
EM2T15t10 10.40 40.67 34.90 10.42 9.50 4.51 13.83 1.12 0.02 0.86 3.91
* For the compounds in Table 1, the analysis of variance showed no treatment effect. The fatty acid composition is
EM2T15t20 10.63 40.58
reported as a percentage of the lipid fraction. The content of lipids, 13.87
34.42 10.83 9.42 4.75 protein, 1.12
alkaloids,0.02 0.86
and total 3.74 acids
chlorogenic
EM2T20t10 10.43 on the
40.59 weight of the
34.94 sample.
10.55 9.31 4.61 13.79 1.14 0.02 0.88 3.75
EM2T20t20 10.46 41.17 34.39 10.23 9.47 4.73 13.64 1.16 0.02 0.89 3.83
EM3T15t10 10.75 41.13 Among the fatty 10.54
34.20 acids present9.47 in coffee, linoleic
4.65 acid is the
14.02 main0.02
1.16 fatty acid,
0.89 followed
3.80 by
EM3T15t20 10.61 palmitic
41.36 acid.
34.24The waxes
10.30of almond coffee
9.50 originate
4.60 in the epicarp of the
13.80 1.14 0.02 0.88 3.84fruit and represent
EM3T20t10 10.44 between 0.06
41.62 and 0.1%
33.88 of normally
10.37 roasted coffee
9.40 4.73 [29].13.65
For the average
1.14 0.02 values
0.88 of free
3.62 fatty
EM3T20t20 10.79 acids
42.25 per treatment,
33.03 palmitic
10.22 acid was
9.65 the predominant
4.85 fatty acid, followed
14.02 1.16 0.02 0.88 3.65 by linoleic,
oleic,
* Forstearic, and arachidic
the compounds in Table acids. The analysis
1, the analysis of variance
of variance showed no did not show
treatment a significant
effect. effect
The fatty acid
ofcomposition
the state of
is reported as a percentage of the lipid fraction. The content of lipids, protein, alkaloids,the
maturity, the temperature of the process, the time of prolongation of
fermentation, or theacids
and total chlorogenic interaction of theofprevious
on the weight the sample.factors on the percentage composition of
free fatty acids (Table 1). Palmitic acid presented an average value of 41.03%, and the
maximum average value was found in the treatment associated with a process temperature
of 20 ◦ C, extension time of 20 h, and state of maturity EM3 with a value of 41.59%. Linoleic
acid presented an average value of 34.34%, and the minimum average value was associated
Fermentation 2022, 8, 519 10 of 16

with the fermentation treatments performed with the state of maturity EM3 (33.83%). Oleic
acid showed a tendency to decrease its values as the treatments were associated with higher
levels of fruit maturity, which were 10.60%, 10.51%, and 10.35% in the EM1, EM2 and EM3
states, respectively. Stearic fatty acids presented an average value for all treatments of
9.50%, and the average value of the treatments with the longest extension time increased
from 9.47% to 9.54% after 10 to 20 additional hours of fermentation of the mucilage.
This comportment was similar for the arachidic fatty acid, whose average values of the
treatments with an extension time of 10 h ranged from 4.57% to 4.67% at 20 h. Garrett et al.,
2016 [30], described increases in the levels of the compounds palmitic acid, linoleic acid,
and stearic acid in regions of the endosperm attacked by insects, suggesting that perhaps
these compounds have a direct implication in the coffee bean–insect interaction. In this
study, the lipids and the composition of free fatty acids, which are some of the compounds
responsible for the texture and body of coffee, were not modified by the treatments; the
expression of these genes could have been associated with a protective effect.

3.5. Total Crude Protein and Alkaloids

The average protein content of the treatments was 13.80% (Table 1), and the lowest
average contents were observed in the treatments with a process temperature of 20 ◦ C
and an extension time of 10 h, with values of 13.76 and 13.79, respectively. The treatments
associated with the state of maturity EM3 presented the maximum average value of 13.87%.
Bressani et al., 2020 [31], reported that higher protein concentrations in coffee are corre-
lated with increases in pyrazine, characterized by nutty, almond, and sweet flavors, and
with an increase in sucrose degradation during roasting. These authors used different
processing methods, finding differences in the types of proteins, describing an increase in
the concentration of proteins in natural dry coffee and a decrease in semi-dry coffee. In
this investigation, no changes in the total protein content were identified since different
processing methods were not contrasted, nor were inoculations of pulped coffee used. This
content was not affected by the stages of maturity of the fruit, prolonged fermentation
times, or temperature.
Alkaloids and grain proteins, although usually defined as responsible for the bitter
taste, have a fundamental role in the chemical reactions responsible for the aroma and
characteristic flavor of coffee [26]. The former were not modified by the different conditions
of prolonged fermentation of the coffee mucilage. Caffeine showed a tendency to increase
its contents in the treatments associated with advanced stages of maturity, with values
of 1.09%, 1.13%, and 1.14% for states EM1, EM2, and EM3, respectively. The average
percentage of theobromine in the treatments was 0.021, and the lowest average value was
reported for the treatments associated with the state of maturity EM3, with an average
value of 0.019. The average percentage of trigonelline in the treatments was 0.87, and unlike
theobromine, the highest average value was reported for the treatments associated with
the state of maturity EM3, with an average value of 0.88. The mean alkaloid content of the
treatments is shown in Table 1.

3.6. Total Chlorogenic Acids

Total chlorogenic acids showed a tendency to decrease their content in the treatments
associated with advanced stages of maturation, taking values of 3.86%, 3.80%, and 3.72%
for states EM1, EM2, and EM3, respectively, which for the state of maturity EM2 involved
the increase in quinic acid by prolonging the fermentation time. As reported by Zhang et al.,
2019 [32], total chlorogenic acids decrease slightly in standard and extended fermentations.
Lower average values were also observed at the highest process temperatures, 3.85% and
3.74% for 15 ◦ C and 20 ◦ C, respectively.

3.7. Sugars
The treatments associated with the EM1 state had average values of glucose, fructose,
and sucrose of 0.80, 1.07, and 89.80 (g/kg), respectively, and those of the EM2 state had
Fermentation 2022, 8, 519 11 of 16

values of 0.71, 1.02, and 88.57, respectively, and treatments associated with the EM3 state
had values of 0.83, 1.07, and 86.28, respectively. For the maturity stages EM1 and EM3,
the analysis of variance showed a significant effect at 5% on the contents of glucose and
fructose by the time of mucilage fermentation after 10 to 20 h of prolongation. In the EM1
stage of maturity, the fructose content increased from 0.71 (B) to 1.43 (A), and glucose
increased from 0.55 (B) to 1.03 (A) and in the EM3 stage of maturity. The fructose content
increased from 0.69 (B) to 1.34 (A), and the glucose content increased from 0.49 (B) to 0.93
(A). In the EM1 stage of maturity, the fructose content increased by 50.34%, and the glucose
content increased by 46.60%. In the EM3 stage of maturity, the fructose content increased by
48.50% and the glucose content increased by 47.31%; the hydrolysis of sucrose influenced
the predominance of glucose and fructose [33]. For the state of maturity EM2, the analysis
of variance showed the effect of the interaction of the process temperature and the prolonga-
tion time on the content of fructose and glucose (Figure 5). De Bruyn et al., 2017 [26], define
differences in the concentrations of fructose and glucose due to the type of processing:
wet or dry. Additionally, they show that the anoxia of the grains can trigger germination,
which generates a response of consumption among other carbohydrates, which is even
more intense during prolonged fermentation. Coffee beans subjected to anoxia consume
carbohydrates continuously through glycolysis, which causes the concentration of sucrose
in the endosperm to decrease, which was evident in this study because the sucrose content
of the treatments associated with prolonged fermentation for 10 h presented an average
of 89.01 g/kg, while those associated with 20 h, that is, 10 additional hours, presented
an average value of 87.43 g/kg [26]. In comparison with mucilage in the grain, fewer
noticeable changes are produced in the concentrations of metabolites; after fermentation,
they report significant decreases in the concentrations of fructose, glucose, sucrose, and
caffeine. However, Elhalis et al., 2020 [27], found that during fermentation, reducing sugars
and lactic acid accumulated inside the grains. For the state of maturity EM2, the effect of
the interaction of the process temperature and the prolongation time was evident for the
content of fructose and glucose, and the increase in these simple sugars was12more
Fermentation 2022, 8, x FOR PEER REVIEW of 17 evident
in the treatments with a process temperature of 20 C. ◦

EM2-Process temperature (°C) and prolongation time (h)

Fructose Glucose
10 20 10 20
15 0.74 0.85 0.49 0.69
20 0.66 1.83 0.50 1.17
Figure 5. Effect of prolonged mucilage fermentation on coffee sugars in the EM2 maturity stage
Figure 5. Effect of prolonged mucilage fermentation on coffee sugars in the EM2 maturity stage
according to Duncan’s test (α = 0.05).
according to Duncan’s test (α = 0.05).
3.8. Sensory Quality
The sensory quality was determined following the SCA cupping protocol, which in-
volves the individual determination of attributes, and the summation configures the total
score. In this study, scores lower than 80 points imply the presence of sensory defects. A
total of 96 samples were evaluated, associated with 12 treatments and 8 experimental
Fermentation 2022, 8, 519 12 of 16

3.8. Sensory Quality

The sensory quality was determined following the SCA cupping protocol, which
involves the individual determination of attributes, and the summation configures the total
score. In this study, scores lower than 80 points imply the presence of sensory defects. A
total of 96 samples were evaluated, associated with 12 treatments and 8 experimental units,
of which 15.63% had sensory defects. Three treatments associated with the maturity stage
EM1 (EM1T15t20, EM1T20t10, and EM1T20t20) presented sensory defects in an experimen-
tal unit, the first two with earthy defects and the third with fermented defects. In the EM2
and EM1 stages, three treatments showed defects in an experimental unit: the EM2T20t20
ferment and the EM2T15t10 and EM2T20t10 earthy treatments. In the state of maturity
EM3, the highest percentage of sensory defects was present at 9.38%. The EM3T20t10 treat-
ment had ferment sensory defects in two experimental units. The EM3T20t20 treatment
presented fermented sensory defects in seven of the eight experimental units analyzed
(87.5%) and was the treatment with the greatest negative effect on quality. Treatments with
the same process temperatures and prolongation times of the fermentation process in the
stages of maturity EM1 and EM2 did not present defects with the same frequency as the
treatment associated with EM3. Do Carmo et al., 2020 [34], evaluated 6, 12, 18, 24, 30, and
36 h of fermentation and found the lowest values of sensory score in treatments 30 and 36,
reporting a green astringency associated with changes in the cell membranes of the grains,
which determined that the excess fermentation was detrimental to the quality of the drink
and the physiological quality.
The analysis of variance for the treatments associated with the maturity stages EM1
and EM2 showed no effect on the prolongation time, process temperature, or their interac-
tion on the different sensory attributes or on the total SCA score. For the total score, the
treatments associated with ME1 obtained an average value of 81.35 with a maximum of
83.92, and those associated with ME2 obtained an average value of 82.05 with a maximum
of 83.83. The maximum values obtained coincide with the maximum value of 84 points
reported by Do Carmo et al., 2020 [34], for the treatment of 18 h of fermentation. De
Carvalho et al., 2018 [28], reported scores higher than 80 and obtained the highest scores in
the sensory attributes of aroma, flavor, acidity, body, and balance in inoculated treatments
compared to spontaneous fermentation, while sweetness, cleanliness, and uniformity were
statistically similar for both treatments, possibly due to the absence of sensory defects.
However, de Melo Pereira et al., 2015 [12], reached a score of 89 points for the treatment
with spontaneous fermentation, which was equal to the inoculated score; however, they
reported that the use of the strain favored distinctive characteristics of vanilla flavor and
floral aromas.
In this research, the tasters identified 337 sensory descriptors of flavor that were
classified into categories: caramel-sweet, chocolate, citrus, spices, floral, fruity, dried, and
red fruits. Most descriptors (42.73%) corresponded to the candy-sweet group, 17.51% to
chocolate, and 14.54% to nuts. The frequency of descriptors of the caramel-sweet group
increased in the treatments associated with the different stages of maturity, going from
36.84%, 44.25%, and 49.45% in EM1, EM2, and EM3, respectively. The fruit group increased
its frequency from 5.76% to 10.94% by increasing the process temperature from 15 to 20 ◦ C,
while the dried fruits decreased from 18.60% to 10.33% due to the increase in time from 10
to 20 h.
The analysis of variance showed an effect in the treatments of the state of maturity
EM3. Increasing the prolongation time from 10 to 20 h generated an effect on the acidity
attribute, decreasing from an average value of 7.26 to 6.79. Increasing the temperature of
the fermentation process of the mucilage with fruits of the state of maturity EM3, which
was the most advanced stage evaluated, showed a negative effect, since a decrease in the
scores of five attributes of the eleven evaluated was generated. The temperature of the
process had an effect on the attributes of fragrance/aroma, residual flavor, acidity, balance,
and taste score by reducing their values from 7.62 to 7.40, 7.31 to 6.60, 7.41 to 6.65, 7.36 to
6.62, and 7.36 to 6.63, respectively. These decreases are associated with the values obtained
 The analysis of variance showed an effect in the treatments of the state of maturity
EM3. Increasing the prolongation time from 10 to 20 h generated an effect on the acidity
attribute, decreasing from an average value of 7.26 to 6.79. Increasing the temperature of
the fermentation process of the mucilage with fruits of the state of maturity EM3, which
was the most advanced stage evaluated, showed a negative effect, since a decrease in the
Fermentation 2022, 8, 519 13 of 16
scores of five attributes of the eleven evaluated was generated. The temperature of the
process had an effect on the attributes of fragrance/aroma, residual flavor, acidity, balance,
and taste score by reducing their values from 7.62 to 7.40, 7.31 to 6.60, 7.41 to 6.65, 7.36 to
in6.62,
theand 7.36 to 6.63,
EM3T20t20 respectively.
treatment, These
which decreases
are are associated
in the lowest segmentwith ofthe
thevalues obtained
evaluation scale due
in the EM3T20t20 treatment, which are in the lowest segment of the evaluation
to sensory defects. The interaction of the process temperature (15 C and 20 ◦ C) ◦ scale duewith the
to sensory defects.
fermentation The interaction
prolongation of the
time (10 andprocess
20 h) temperature
had an effect(15on°Cthe
andflavor
20 °C)attributes,
with the clean
fermentation prolongation time (10 and 20 h) had an effect on the flavor attributes, clean
cup, and total SCA score according to the F test (α = 0.05) (Figure 6).
cup, and total SCA score according to the F test (α = 0.05) (Figure 6).

EM3-Process temperature (°C) and extension time (h)

Flavor Clean cup SCA total score
10 20 10 20 10 20
15 7.34 7.41 15 10.00 10.00 15 81.74 81.89
20 7.06 6.20 20 7.50 1.25 20 75.11 57.83
Figure 6. Effect of prolonged mucilage fermentation on the sensory quality of coffee according to
Figure 6. Effect
Duncan’s test (α of prolonged mucilage fermentation on the sensory quality of coffee according to
= 0.05).
Duncan’s test (α = 0.05).

The state of maturity of coffee is a determining condition in fermentation, since states

such as EM3 where the mucilage content is lower with respect to the state of maturity
EM1 can consistently generate a negative effect on quality by causing defects such as
fermentation. As reported by Avallone et al., 2001 [35], the initial microflora of fermentation
is abundant and varied; aerobic is predominant and more heterogeneous when there
is a higher water content in the process. The acidic conditions prevailing at the end of
fermentation favor the development of yeasts, which could be responsible for the alcoholic
flavor of the coffee beverage after overfermentation. In the case of the state of maturity
EM3, a lower mucilage content of 10.07% compared to the state of EM1 with a value of
15.44% was explained by a lower water content associated with the dehydration that the
fruit undergoes. The last stages of maturation could favor the growth of yeasts.
For the variables evaluated, a backward regression analysis was performed to identify
the set of variables that significantly influenced the dependent variable defined as the
total SCA score. Through individual contrast (of the t or the F) and as a result of 37 initial
variables, there are 10 that ultimately influence the total sensory score SCA (Figure 7).
With these selected variables, the Pearson correlation coefficient was determined, and its
significance was evaluated according to the t-test (α = 0.05).
 undergoes. The last stages of maturation could favor the growth of yeasts.
For the variables evaluated, a backward regression analysis was performed to iden-
tify the set of variables that significantly influenced the dependent variable defined as the
total SCA score. Through individual contrast (of the t or the F) and as a result of 37 initial
variables, there are 10 that ultimately influence the total sensory score SCA (Figure 7).
Fermentation 2022, 8, 519 14 of 16
With these selected variables, the Pearson correlation coefficient was determined, and its
significance was evaluated according to the t-test (α = 0.05).

Heatmap of
Figure 7. Heatmap ofquality
qualityvariables
variablescorrelated with
correlated thethe
with total SCA
total score.
SCA Significant
score. codes:
Significant 0 ‘***’ 0.001
codes: 0
‘**’ 0.01 ‘*’ 0.05.
'***’ 0.001 '**' 0.01 '*’ 0.05.

4. Conclusions
4. Conclusions
The quality
The quality of
of coffee
coffee is
is the
the result
result of
of the
the combination
combination of
of multiple
multiple factors
factors that
that converge
converge
in the production system, each of which generates a unique expression of the chemical
in the production system, each of which generates a unique expression of the chemical
composition of the bean that is the basis for the generation of the sensory attributes of the
composition of the bean that is the basis for the generation of the sensory attributes of the
beverage. This research evaluated the characteristics of the coffee bean to define the effect
beverage. This research evaluated the characteristics of the coffee bean to define the effect
of the maximum level of maturity of the fruit on the fermentation of the mucilage. The
of the maximum level of maturity of the fruit on the fermentation of the mucilage. The
results indicated that the three stages of ripening evaluated showed no effect of temperature
and prolongation time on the physical quality variables, nor on acetic, lactic, malic and
succinic acids, free fatty acids, lipids, protein, caffeine, theobromine, trigonelline, and
total chlorogenic acids. The treatments associated with maturity stages EM1 and EM2
showed no effect on the sensory profile due to prolongation time, processing temperature,
or their interaction. For oxalic and quinic acids at maturity stage EM1 and for citric acid at
stage EM2, a significant effect of fermentation prolongation time was observed. Maturity
stage EM3 had no effect on the contents of organic acids in green almond coffee. Maturity
stages EM1 and EM3 showed increases in the contents of glucose and fructose due to the
fermentation time. The maturity stage EM2 presented an effect of the interaction of process
temperature and prolongation time for fructose and glucose content. The treatments at
maturity stage EM3 showed an interaction effect of process temperature and fermentation
extension time on flavor, clean cup, and total SCA scores. This implies a differential
response of the coffee fruit to fermentation time and temperature conditions depending
on its degree of maturity. Consistent with what has been reported by different authors,
the fermentation of mucilage generates conditions that favor changes within the grain in
organic acids and sugars such as glucose and fructose. It is important to recognize that the
source of grain changes occurs in two ways: there is a contribution from external processes,
Fermentation 2022, 8, 519 15 of 16

but these, in turn, generate conditions during fermentation that induce grain responses
to them, generating exosmosis or endosmosis. The final response of the grain against
the profile of its chemical composition and quality is also the result of the endogenous
metabolism of the endosperm under conditions of anoxia during fermentation and not
exclusively of the transfer of microbial metabolites to the interior of the grain during the
fermentation of the mucilage. This research defines the degree of maturity of the fruit as
a process variable since it establishes the initial conditions of the process with respect to
the amount and composition of the substrate for microbial activities. Advanced stages
of maturity have a quality that preserves the standards of commercialization, but their
comportment is different in the different stages of postharvest, such as fermentation, where
prolonged times and high temperatures consistently affect the sensory attributes. For this
reason, it is important to know the configuration of the maturity stages of the initial mass to
correctly define the conditions of the subsequent postharvest processes to avoid damaging
the integrity of the grain.

Author Contributions: Conceptualization, formal analysis, investigation, data curation, writing—original

draft preparation, V.O.P.; review and editing, C.I.Á.-B., L.G.M., and J.R.A.; review and formal analysis,
L.F.E. and L.C.I. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by National Coffee Research Center (Cenicafé) (Crossref Funder
ID 100019597).
Informed Consent Statement: Written informed consent has been obtained from the coffee sensory
analysts to publish this paper.
Data Availability Statement: Data is contained within the article.
Acknowledgments: The authors gratefully acknowledge to principals Hernando Duque and Álvaro
Gaitán of Cenicafé; the colleagues Jenny Pabón, Claudia Gallego, Paola Calderón, Wilson Vargas,
Carlos Gonzalo Mejía, José Farid López, John Félix Trejos, Rubén Medina, and Esther C. Montoya;
and the support staff Víctor Castañeda and Benjamín Sánchez.
Conflicts of Interest: The authors declare no conflict of interest.

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