Food Bioscience 47 (2022) 101640

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Impact of microbial self-induced anaerobiosis fermentation (SIAF) on

coffee quality
Marcela Caroline Batista da Mota a, Nádia Nara Batista a, Disney Ribeiro Dias b,
Rosane Freitas Schwan a, *
a
Department of Biology, Federal University of Lavras, Lavras, MG, Brazil
b
Food Science Department, Federal University of Lavras, Lavras, MG, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The fermentation of natural (NC) and pulped coffee (PC) was performed with a conventional method (platform)
Bioreactor and under self-induced anaerobic fermentation (SIAF). Of the 12 samples analyzed during the fermentation
Fermentative process process, the highest temperature was obtained by the SIAF method (30.5 ◦ C for NC and 29.67 ◦ C for PC) with 87
Specialty coffee
h of fermentation. Nonvolatile compounds (36 samples) were evaluated by high-performance liquid chroma­
Anaerobic conditions
tography. Fermentation in the SIAF method contributed to the maximum amount of citric acid (2.534 mg/g) in
Natural processing
pulped coffee and acetic acid (6.04 mg/g) and lactic acid (2.533 mg/g) in NC. Furan was the primary chemical
class detected, followed by ketones and pyrazines. All coffees (12 samples) were evaluated five times and
classified as specialty coffees (>80 points) following Specialty Coffee Association (SCA) protocols. The pulped
coffee processed by the SIAF method showed a 2.83-point increase in the sensory score compared to the con­
ventional method. Therefore, the SIAF method is accessible to producers, contributes to coffees with differen­
tiated sensory profiles, and increases beverage quality.

1. Introduction fundamental (Clay et al., 2016). Generally, conventional postharvest

Coffee is one of the primary traded commodities of high economic
impact. Brazil, Vietnam, and Colombia are the largest coffee-producing
countries, while the largest consuming and importing markets of coffee
are represented by European Union and the USA (ICO, 2021a; 2021b).
The growing coffee consumption has driven the market to seek new
technologies to improve quality beverages (Guimarães et al., 2019;
Samper et al., 2017).
In recent years, coffee fermentation has attracted much interest in
the producing areas due to the higher price that it can reach in the in­
ternational market. This process generates an array of chemical changes
essential for developing complex flavors in the beverage sensory profile
(Evangelista et al., 2015; Martins et al., 2020; Ribeiro et al., 2017; Silva
et al., 2000, 2013; Vilela et al., 2010).
Coffee quality starts on the farm and is guaranteed by numerous
factors, including crop management practices to achieve healthily,
quality production, free from pests, and diseases, with harvests carried
out at the correct time, and the fermentation drying period are
processing occurs in an uncontrolled environment that often leads to
stuck fermentation and a lack of coffee bean quality predictability since
several undesirable microbial metabolites can be produced and act as
off-flavor precursors (Masoud et al., 2005; Silva et al., 2013). Therefore,
bioreactors are an alternative to control the fermentative process and
improve coffee quality.
A bioreactor is a valuable tool that controls, enhances, optimizes the
fermentations process, reduces fermentation time, avoids microbial
contamination, and contributes to reproducibility (Figueroa-Hernández
et al., 2019). In addition, bioreactors are used in many industrial ap­
plications, such as biomass production, synthesis of metabolites, water,
waste treatment, enzyme production, foods, and tissue culture (Behera
et al., 2019). However, their performance depends on many design pa­
rameters, including cultivation, height, diameter, and operating condi­
tions (Angst & Kraume, 2006; Varley & Birch, 1999).
Using an open bioreactor or cement tank is typical for the wet
method in coffee processing. In the wet method, the skin and pulp are
mechanically removed from the coffee fruit transferred to water tanks

* Corresponding author. Federal University of Lavras, Department of Biology, Campus Universitário, 3037, 37200-900, Lavras, MG, Brazil.
E-mail addresses: mmmotacarol@gmail.com (M.C. Batista da Mota), nadia.nb@hotmail.com (N.N. Batista), diasdr@ufla.br (D.R. Dias), rschwan@ufla.br
(R.F. Schwan).

https://doi.org/10.1016/j.fbio.2022.101640
Received 26 July 2021; Received in revised form 21 February 2022; Accepted 22 February 2022
Available online 25 February 2022
2212-4292/© 2022 Elsevier Ltd. All rights reserved.
M.C. Batista da Mota et al.
where a natural fermentation occurs over 6–72 h to degradation and
solubilization of remaining mucilage. Nevertheless, bioreactors have
also been used to process natural coffee, which coffee fruit is processed
wholly, and pulped coffee, which coffee fruit is processed without the
skin and pulp, in open and closed batches from polyethylene and
stainless-steel containers (Bressani et al., 2018; Martinez et al., 2017;
Martins et al., 2020).
The improvement in the quality of the coffee beverage is evidenced
when comparing the conventional method (the method used by the
farmers) with new processing technologies, such as the use of closed or
open bioreactors and starter cultures. For example, in open batches, the
aerobic fermentation associated with culture starters improves coffee
quality by 4 points (80–84) (Bressani et al., 2018; Martinez et al., 2017).
In closed batches, the coffees reached 87 points, increasing 5 points the
final score to coffee processed without starter culture (82 points) (Da
Mota et al., 2020). In microbial induced anaerobic fermentation (SIAF),
the production of metabolites (organic acids, alcohols, and volatile
compounds) are intensified, contributing to flavor formation and
reducing undesirable microorganisms, including fungi and bacteria
associated with food poisoning spoilage (Bressani et al., 2018; Da Mota
et al., 2020; Martinez et al., 2017; Massawe & Lifa, 2010).
Modification processing conditions can improve the performance
fermentative of indigenous microbiota, allowing novel sensory profiles.
The SIAF method can become an additional step for traditional on-farm
natural and pulped coffee processing. The self-induced anaerobic
fermentation (SIAF) is performed by the microbial metabolism that
utilizes the remaining oxygen for their metabolic reactions releasing
CO2, volatile and nonvolatile compounds. CO2 influences the production
of glycerol, ethanol, succinic acid, acetic acid, and lactic acid (Li et al.,
2015; Martins et al., 2020). Therefore, this work aimed to evaluate the
impact of the SIAF method on the chemical and sensory quality of nat­
ural and pulped coffee fermented by indigenous microbiota.
2. Materials and methods
2.1. Bioreactor and fermentation
The fermentation experiments were conducted in Três Pontas, Minas
Gerais, Brazil (latitude 21◦ 22′ S, longitude 45◦ 30′ W, and elevation of
950 m) (Álvares et al., 2013). Catuaí Amarelo variety coffee fruits har­
vested in July 2020 were manually selected. Natural coffee (NC), the
fruit is processed wholly, while pulped coffee (PC) the fruits are
Fig. 1. Design of bioreactor: (A) cover, (B) closing cover, (C) mesh, and (D) bioreactor drawer.
2
Food Bioscience 47 (2022) 101640
processed without the skin and pulp; natural coffee and pulped coffee
were processed by conventional and SIAF (self-induced anaerobioses
fermentation) method (Fig. S1). In the conventional method, the coffee
is harvested, depulped (only for pulped coffee processing), and taken to
a drying platform where the beans were fermented and dried up to 11%
moisture content for 15 days. In the SIAF method (self-induced
anaerobiosis fermentation), each bioreactor was filled with approxi­
mately 40 L of natural or pulped coffee. The coffee remained in the
bioreactor until the fermentation process lasted approximately 87 h.
Later the coffee was washed, pulped (only natural processing), and
taken to the drying platform until it reached 11–12% moisture content
during 15 days.
The bioreactors were manufactured in high-density polyethylene
with a maximum of 0.05 m3 (Picillo, São Paulo, Brazil). The bioreactors
were cylindrical (Fig. 1) with 53.79 cm height and 40.39 cm diameter.
To support self-induced anaerobic fermentation, the lid (40.39 cm in
diameter) was closed with a galvanized steel seal. In addition, an
aluminum screen was fixed 10 cm from the bottom to separate the coffee
from the exudate produced during the fermentation process.
All fermentations were performed in triplicate and monitored by
measuring the temperature (glass thermometer standard, - 20 to +
110 ◦ C) and soluble solids concentration (◦ Brix – refractometer, Lorben,
Brazil) once a day during all processing. One hundred grams of sample
was withdrawn at 0, 22, and 87 h of the process, corresponding to
approximately 18, 22, and 30 ◦ C in the bioreactor and 18, 22, and 25 ◦ C
in the conventional method. All samples were taken approximately 25
cm from the center of the fermenting coffee surface, placed in plastic
pots, and stored at − 20 ◦ C for chemical analysis. The green coffees (11%
moisture) were stored in bags at 15 ◦ C for three months, and 350 g of
coffee was roasted and analyzed chemical and sensory.
2.2. Analysis of carbohydrates, alcohols, and organic acids
Organic acids (malic, lactic, acetic, citric, and succinic acids), car­
bohydrates (sucrose, fructose, and glucose), and alcohols (glycerol and
ethanol) were extracted from 36 coffee samples and analyzed using a
liquid chromatography system (Shimadzu, model LC-10Ai, Shimadzu
Corp., Japan) equipped with a dual detection: ultraviolet-visible (UV-V)
detector (SPD 10Ai) and a refractive index detector (RID-10Ai). The
sample coffee (3 g) was ground into a fine powder and extracted twice
with 10 mL of Milli-Q water vortexing for 5 min at room temperature,
and each 10 mL homogenate was transferred to another tube. The tubes
M.C. Batista da Mota et al.
containing 20 mL of solution were centrifuged (12745 RCF, 10 min,
4 ◦ C), and the supernatant was separated from the precipitate. Only for
acids analysis, the supernatant pH was adjusted to 2.11 with perchloric
acid (200 mM) solution and re-centrifuged under the same conditions.
After that, the supernatant was filtered through a 0.22 μm cellulose
acetate membrane and stored at − 18 ◦ C until analysis.
A Shimadzu ion exclusion column (Shim-pack SCR-101H, 7.9 mm 30
cm) was operated at 50 ◦ C for acids, and perchloric acid (16 mM) was
used eluent at a flow rate of 0.6 mL/min. A Shim-pack SCR-101C column
was used for alcohol and carbohydrates and was operated at 80 ◦ C, with
ultrapure water used as the eluent at a flow rate of 0.6 mL/min. The
acids were detected via a UV detector (210 nm), while the alcohols and
carbohydrates were detected via RID. Individual compounds were
identified based on the retention time of standards injected using the
same conditions. The sample concentrations were determined using an
external calibration method, and calibration curves were constructed by
injecting different standards under the same conditions as the samples
(Evangelista et al., 2014b). The chemical compounds used as standard
were sucrose, fructose, and glucose (Sigma-Aldrich - Saint Luis, EUA);
citric and malic acids (Merck - Germany), lactic, and acetic (Sigma-­
Chemical - EUA), succinic acid (Sigma- Aldrich - Germany); ethanol, and
glycerol (Merck - Darmstadt, Germany). The standard curve concen­
tration range from 0.009 to 24 g/L. The areas obtained were plotted on a
linear curve, whose equation was used to estimate the concentration in
the sample. All samples were analyzed in triplicate.
2.3. Volatile compounds
The volatile compounds of 12 samples of coffee roasted powder (2 g)
were extracted using headspace solid-phase microextraction (HS-
SPME). First, a divinylbenzene/carboxen/polydimethylsiloxane (DVB/
CAR/PDMS) 50/30 mm SPME fiber (Supelco Co., Bellefonte, PA, USA)
was equilibrated at 60 ◦ C for 15 min. Subsequently, the samples were
exposed for 30 min to absorb the volatile constituents.
The volatile compounds were evaluated through gas
chromatography-mass spectrometry (GC-MS) using a Shimadzu GCMS-
QP2010 SE system equipped with a carbowax column (30 m × 0.25
mm i.d. × 0.25 μm film thickness, J & W Scientific, Folsom, CA, EUA).
The temperature program was described by Visintin et al. (2017). The
carrier gas was helium at a 0.7 mL/min flow rate. A splitless injector was
used. The mass detector was a quadrupole type with an electron impact
ionization system operated at 70 eV and 260 ◦ C. GC/MS solution soft­
ware (version 2.6) was used for compound identification and comparing
the peaks’ mass spectra with those in the NIST11 mass spectral library.
Linear retention indices relative to a mixture of n-alkanes were calcu­
lated according to Kovats retention index (RI). The internal standard
(4-Nonanol; 12.5 g/L) was added to each sample. The chromatogram of
the roasted coffee is represented in Fig. S2.
2.4. Sensory analysis
Sensory analysis of 12 coffee samples was performed with a panel of
six trained coffee tasters with Q-grade coffee certificates. The coffee
beans were prepared and roasted according to the Specialty Coffee As­
sociation (SCA) guidelines (SCA, 2008). The coffee was roasted in a pilot
roaster (Probat Leogap, TP2, Curitiba, Brazil) with a capacity of 150 g
and ground in an electric mill (Mahlkönig, Guatemala Lab, Germany).
Samples of 7.25 g per batch of coffee consisting of five replicates were
used during cup tasting. The methodology applied to evaluate coffee was
performed according to SCA standards and check-all-that-apply (CATA)
(Dooley et al., 2010). The tasters were asked to assess sensory attributes
and select those they considered appropriate to describe the coffee’s
aroma and flavor (Da Mota et al., 2020). Furthermore, an unstructured
trend scale was included to evaluate the sweetness, acidity, body,
astringency, bitterness, and finish.
3
Food Bioscience 47 (2022) 101640
2.5. Statistical analysis
The experiment was conducted in a completely randomized design.
The production of the nonvolatile compound was evaluated in a factorial
arrangement with two processes (natural and pulped coffee), two
methods (SIAF and conventional), three fermentation times (0, 27, and
87 h). Three repetitions were performed, totaling 36 experimental units.
Physicochemical (temperature and solid soluble), chemical (carbohy­
drate, alcohol, organic acid, and volatile compound), and sensory data
were analyzed statistically using variance analysis (ANOVA) followed by
the Scott-Knott test (p < 0.05) to determine significant differences
among treatments (Ferreira, 2011). The data were analyzed using SIS­
VAR® version 5.7 software (Lavras, Brazil). Pearson’s correlation
analysis was carried out further to explore the relationship between the
volatile and sensory scores.
3. Results
3.1. Physical-chemical parameters of the coffee
The microbial activity was monitored by the physical-chemical pa­
rameters of coffee beans, such as temperature and soluble solids (Fig. 2).
All treatments showed a significant increase in temperature and signif­
icantly reduced soluble solids during the fermentation process
(Table S1). Furthermore, significant differences were obtained between
soluble solids concentration and temperature treatments. At the begin­
ning fermentation, a higher soluble solid concentration (25.83 ◦ Brix)
was detected in natural coffee. During fermentation, the SIAF method
had the most significant increase in temperature, providing an increase
of 13 ◦ C to natural coffee and 6.89 ◦ C to pulped coffee, while in the
conventional method, the increased by 10.84 ◦ C for natural coffee and
1.48 ◦ C for pulped coffee. In addition, the SIAF method provided of
reduction more intense of the soluble solid concentration from 25.5
◦
Brix to 16.67 ◦ Brix for natural coffee and from 17.25 ◦ Brix to 11.53
◦
Brix for pulped coffee.
3.2. Sugars and fermentation products
The sugar, acid, and alcohol concentrations during coffee fermen­
tation are shown in Table 1. Sucrose, glucose, and fructose were the
primary sugars detected in the coffee beans.
High sugar concentrations were detected in natural coffee. At the
beginning of the fermentation, the conventional method’s natural coffee
contained 3.431 mg/g sucrose, 10.623 mg/g glucose, and 15.19 mg/g
fructose, and the pulped coffee from the conventional method contained
0.386 mg/g sucrose, 4.772 mg/g glucose, and 3.88 mg/g fructose. The
natural coffee for the SIAF method contained 4.466 mg/g sucrose, 9.806
mg/g glucose, 17.75 mg/g fructose, and pulped coffee, and the con­
centrations were 0.548 mg/g sucrose, 4.581 mg/g glucose, and 4.84 mg/
g fructose. The consumption of sugars and acid production was observed
during the fermentation of coffee in all treatments, and low sugar con­
centrations were detected at the end of fermentation.
At the end of fermentation, malic acid, ethanol, and glycerol were
not detected for any of the experiments. In the SIAF method with 22 h of
fermentation, citric acid, acetic acid, and malic acid were detected at
higher concentrations in natural coffee, while only citric was detected in
pulped coffee. Succinic acid maintained a constant concentration in the
first 22 h of pulped coffee fermentation. Lactic acid was produced at the
end of fermentation for both processes.
In the conventional process, a high concentration of acetic acid (8.96
mg/g NC and 3.37 mg/g PC) and malic acid (3.914 mg/g NC and 0.806
mg/g PC) were detected at the beginning fermentation for natural and
pulped coffee. Furthermore, lactic acid was not produced in this process.
SIAF and the conventional method influenced the concentration of
the nonvolatile compound. The concentrations of sucrose, fructose,
ethanol, and malic acid at the end of fermentation for natural and pulped
M.C. Batista da Mota et al. Food Bioscience 47 (2022) 101640

Fig. 2. Temperature ( 0, 22, and 87 h) and soluble solids concentration ( 0, 22, and 87 h) during the fermentation of natural coffee and pulped coffee.
Column (temperature) and character (soluble solid) followed by the same lowercase letters and capital letters among the treatment do not differ from Scott–Knott’s
test (p > 0.05). The bars represent the standard error of the mean. SIAF: self-induced anaerobic fermentation.

Table 1
Chemical compounds detected in green coffee.
Compounds Process SIAF (mg/g) Conventional (mg/g) SEM

Time (hour)

0 22 87 0 22 87

Sucrose NC 4.466a,A 1.328b,A ndc,A,* 3.431a,A 0.781b,A 0.025c,A,* 0.042

PC 0.548a,B ndb,B ndb,A,* 0.386a,B 0.371a,B ndb,A,*
Glucose NC 9.806 a,A 4.358b,A 0.448c,A 10.623a,A 3.371b,A 0.942c,A 0.079
PC 4.581a,B,* 1.152b,B 0.939b,B 4.772a,B,* 1.714b,B 0.089c,B
Fructose NC 17.75a,A 6.76b,A 1.31c,A,* 15.19a,A 5.75b,A 1.12c,A,* 0.11
PC 4.84a,B 0.21b,B 0.06c,B,* 3.88a,B 0.63b,B 0.01c,B,*
Glycerola NC 0.071 0.365 nd 0.064 0.017 nd 0.012
PC 0.055 0.304 nd 0.039 nd nd
Ethanol NC 2.953b,A 5.451a,A ndc,A,* 2.467a,A 0.535b,A 0.011c,A,* 0.078
PC 0.328b,B 2.047a,B ndc,A,* 0.241b,B 0.649a,A 0.002c,A,*
Acetic acid NC 10.55b,A 14.05a,A 6.04c,A 8.96a,A 1.15b,A 0.15c,A 0.14
PC 3.05a,B,* 0.93b,B,* 0.29c,B,* 3.37a,B,* 0.51b,B,* 0.06c,A,*
Citric acid NC 0.919b,A,* 2.108a,B,* 2.021c,B 0.745b,A,* 1.288a,A,* 0.273c,A 0.061
PC 0.784c,A,* 2.319b,A 2.534a,A 0.706b,A,* 1.109a,B 0.045c,B
Lactic acid NC ndb,A,* ndb,A 2.533a,A nda,A,* ndb,A ndc,A 0.071
PC ndc,A,* 0.432b,A 2.136a,B 0.101a,A,* nda,A nda,A
Malic acid NC 2.933b,A 3.063a,A nd,A,* 3.914a,A 1.155b,A ndc,A.* 0.041
PC 0.868a,B,* ndb,B ndb,A,* 0.806a,B,* 0.893a,B ndb,A,*
Succinic acid NC 6.08a,A 3.13b,A 4.25c,A 6.64a,A 4.48b,A 1.09c,A 0.11
PC 1.34a,B,* 1.39a,B 0.82b,B 1.53a,B,* 0.51b,B 0.11c,B

SEM: standard error of the mean.

NC: natural coffee; PC: pulped coffee.
SIAF: self-induced anaerobic fermentation.
Means followed by the same letter in line (lowercase) and in column (capital) for each fermentation time and process, respectively, do not differ from each other by the
Scott-Knott test (p > 0.05). * indicate that SIAF and conventional method, in the same time of fermentation, do not differ by the Scott-Knott test (p > 0.05).
a
Statistics results describe in the manuscript.

coffee did not differ between methods and acetic acid for pulped coffee.
The interaction between the time of fermentation and process
significantly influenced the glycerol concentration. High concentration
was detected in the SIAF method at 22 h of fermentation following by
0 and 87 h. The significant difference between the processes (natural
and pulped coffee) was obtained with just 22 h of fermentation.
3.3. Volatile compounds
A total of 60 compounds were detected via HS–SPME/GC (Table 2).
In particular, amide (1), acid (5), alcohol (3), aldehyde (1), furan (10),
hydrocarbon (1), ketone (12), lactone (4), phenol (1), pyran (2), pyr­
azine (7), pyridine (3), pyrrole (2), thiophene (1), and other (7) com­
pounds were identified.
Furan was the most abundant chemical class detected in roasted
coffees, followed by ketone and lactone, represented by 2-
furanmethanol; 4-hepten-3-one, 5 methyl, and butyrolactone. The
SIAF method modified the concentrations of volatiles in the natural
coffee and pulped coffee. Acid, lactone, phenol, pyran, pyrazine, pyri­
dine, thiophene, and others increase during the SIAF method in natural
coffee. In the conventional method, natural coffee showed high alde­
hyde, furan, and pyrrole concentrations.
Among the volatile aromatic compounds detected, many were pro­
duced by coffee fruit, such as butanoic acid, 3-methyl; 2-furanmethanol,
acetate; 5-methyl-2-furancarboxaldehyde; 2-furanmethanol; maltol;
1,2-cyclopentanedione, 3-methyl; furfural acetone; and ethanone, 1-(2-
furanyl)-. SIAF method further increased the concentrations of these
compounds.
2-furanmethanol was detected in high concentration in all fermen­
tations. In the conventional method, benzyl alcohol, pentanoic acid, 4-
oxo-; and 2-butanone, 1-(acetyloxy)-were found for processed NC and
PC. Moreover, 2-pentanedione, 3-methyl-; and bis (2-furfuryl) disulfide

4
M.C. Batista da Mota et al. Food Bioscience 47 (2022) 101640

Table 2
Volatile compounds identified in roasted coffee processed by the conventional and SIAF method in natural and pulped coffee.
Volatile compounds Sensory Perception RI RT Relative area (%) SEM
(min)
Natural coffee Pulped coffee

SIAF Conventional SIAF Conventional

Amide
Acetamide, N-(2-methoxyphenyl)- – 1512 18 0.07391b nd nd 0.11718a 0.00095

Acid
Formic acid Unpleasant flavor a 637 9.3 0.69451a ndb ndb ndb 0.00017
Propanoic acid Acida 1528 9.9 0.9721d 1.4063c 1.6279a 1.6151b 0.0023
Butanoic acid, 3-methyl- Sweaty, acid b 1543 11.7 4.2123b 3.0135d 4.0836c 4.3615a 0.0069
Pentanoic acid, 4-oxo- Caramel and acid a 1043 18.8 nda 0.037a nda 0.071a 0.040
a
N-Hexadecanoic acid Rancid and acid flavor 1354 23.5 0.11691a ndb ndb ndb 0.00038
Total acid 5.996a 4.456c 5.711b 6.047a 0.035

Alcohol
1,6-octadien-3-ol, 3,7-dimethyl- Floral a 1236 10.1 0.08749a ndb ndb ndb 0.00031
Benzyl alcohol Sweet, chocolate, fruity and floral a 1884 14.2 ndc 0.13571b ndc 0.20616a 0.00057
3-mercapto-3-methylbutanol – 1489 11.7 0.02812a 0.01841c 0.02101b 0.01524d 0.00050
Total alcohol 0.1156a 0.1541a 0.0210a 0.2214a 0.0011

Aldehyde
Pentanal – 707 13.5 0.27576b 0.30244a 0.16013d 0.25381c 0.00032

Furan
2(5H)-furanone, 3-methyl – 1267 12.3 0.213287a 0.09575d 0.12385c 0.19543b 0.00067
2,5-dimethyl-4-hydroxy-3(2H)-furanone – 2063 16.1 1.5173b 1.0542d 1.2082c 1.7036a 0.0020
2-furanmethanol, acetate Soft, and floral flavor b 1521 9.6 3.589d 6.936a 5.469c 6.176b 0.011
2-furancarboxaldehyde, 5-methyl- Spicy, and slightly caramelized 998 10.3 19.401a 18.479b 18.240b 18.016b 0.054
flavor b
2-furanmethanol Mild, and slightly caramelized 1501 11.6 37.593d 45.388b 45.724a 41.541c 0.078
flavor e
4-methyl-5H-furan-2-one – 1754 14.4 0.13857c 0.07525d 0.17191a 0.15339b 0.00028
Furan, 2,2’-[oxybis(methylene)]bis- – 1178 15.2 0.24076a 0.17335a 0.08872a 0.14702a 0.000042
Furan, 2,2′ -methylenebis- – 1232 10.6 0.20932a 0.21032a ndb ndb 0.00051
Benzofuran, 2,3-dihydro- – 1326 19.2 0.10538a 0.04547c 0.04002d 0.05259b 0.00059
2,5-furandione, 3 methyl – 982 12.4 0.11427a 0.05741b 0.04220d 0.05280c 0.00057
Total furan 63.122d 72.516a 71.108b 68.038c 0.092

Hydrocarbon
Tetramethylbenzene Odor Hazelnut a 1145 18.6 0.33991a 0.17477d 0.19769c 0.22486b 0.00036

Ketone
2-pentanedione, 3-methyl- – 998 12.5 ndb 0.55086a ndb ndb 0.00062
3,4-dymethyl-2-pentanone – 818 12.6 ndb ndb ndb 0.53637a 0.00030
1,2-cyclopentanedione, 3-methyl- Caramela 1057 13.8 2.18714a 1.67288b 1.66620b 1.82347b 0.00028
Furfural acetone Mild, woody flavor, and spices like 1460 13.5 0.24032a 0.12982 0.11950d 0.14592b 0.00038
cinnamon a
Ethanone, 1-(2-hydroxy-5-methylphenyl)- Nuts, floral, and fruity flavor b 1972 16.3 0.08957a ndb ndb ndb 0.00028
Ethanone, 1-(2-furanyl)- – 1987 9.2 0.7182d 1.4154b 1.2734c 1.7370a 0.00022
Ethanone,1-(3-methylpyraziny) – 1123 13.6 1.24751a ndb ndb ndb 0.00025
2-pyrrolidinone – 1086 16.6 0.61a nda 0.24a nda 0.12
2,3-hexonedione – 784 18.4 0.31669a 0.17917d 0.18359c 0.21404b 0.00051
2-propanone, 1-hydroxy- Sweet, Caramel, and acida 1290 9.7 ndc 0.17372b ndc 0.18993a 0.00048
4-hepten-3-one, 5 methyl – 1660 10.5 0.45759a ndd 0.20063c 0.36905b 0.00022
2-butanone, 1-(acetyloxy)- – 1526 11.8 2.97683a 2.89039b 2.71818c 2.64215d 0.00044
Total ketone 8.84a 7.01a 6.40a 7.66a 0.11

Lactone
c
Butyrolactone Sweet flavor 834 11.2 4.4858b 3.3488c 5.0604a 3.0234d 0.0072
2(5H)-furanone – 834 12.7 1.18919c 1.15441d 1.26992a 1.22721b 0.0019
2(3H)-furanone, 5-acetyldihydro- – 967 16.2 0.61483a 0.47506c ndd 0.50522b 0.00040
2-hydroxy-gamma-butyrolactone – 931 17.3 0.2175b 0.1852c 0.1697d 1.6432a 0.0017
Total lactone 6.507a 5.163c 6.499a 6.399b 0.010

Phenol
Phenol, 2-methoxy- – 2027 13.9 1.1342a 0.8297c 0.8113d 0.8598b 0.0012

Pyran
4H-pyran-4-one, 2,3-dihydro-3,5- – 1136 18.2 0.63797b 0.34684c 0.229101d 0.73858a 0.00080
dihydroxy-6-methyl-
b a b c c
Maltol Fruity and Caramel 1131 15.4 2.38667 1.98797 1.60861 1.61622 0.0024
Total pyran 3.0246a 2.3348c 1.8996d 2.3548b 0.0031

Pyrazine
Pyrazine, 2-methyl-6-(1-propenyl)-, (E)- – 1156 12.7 ndb ndb ndb 0.10382a 0.00019
Pyrazine, 2-ethyl-3,5-dimethyl- Coffee flavor a 1356 9 0.31142a ndb ndb ndb 0.00011
2-acetyl-3-methylpyrazine – 1081 12.1 2.2066a 1.2679a 1.5272a 1.7488a 0.0026
1-(6-methyl-2-pyrazinyl)-1-ethanone Popcorn flavor b 1093 12.1 1.4517a 0.8989d 1.0137c 1.2123b 0.0020
1,4-diazine Sweet flavorc 725 13.5 0.37640a 0.30921b 0.26894c 0.29012d 0.00049
(continued on next page)

5
M.C. Batista da Mota et al. Food Bioscience 47 (2022) 101640

Table 2 (continued )
Volatile compounds Sensory Perception RI RT Relative area (%) SEM
(min)
Natural coffee Pulped coffee

SIAF Conventional SIAF Conventional

Pyrazine – 1415 6.3 1.1078a 0.76373c 0.67618d 0.78369b 0.00076

3-methyl-2-pyrazinylmethanol – 1506 14.8 0.18a nd 0.03c 0.14b 0.00043
Total Pyrazine 5.6381a 3.2397d 3.5180c 4.2765b 0.0048

Pyridine
Pyridine – 1195 10.3 0.18351a ndb ndb ndb 0.00025
2(1H)-quinalinone, 3 methyl – 1267 11.8 0.09368a ndb ndb ndb 0.00013
4(H)-pyridine, N-acetyl- – 1721 12.2 1.67091a 1.16211d 1.21066c 1.44149b 0.0019
Total pyridine 1.9481a 1.1621d 1.2107c 1.4415b 0.0019

Pyrrole
d
Pyrrole Roasted flavor 768 9 nda 0.22257a 0.24468a 0.44149a 0.00084
1H-pyrrole-2-carboxaldehyde, 1-methyl- Mild flavor a 2030 11 1.96a 1.75b 1.69c 1.06d 0.0022
Total pyrrole 1.9631b 1.9773a 1.9376c 1.5227d 0.0028
Thiophene
2-Thiophenemethanol – 1024 14.9 0.30704a 0.22431d 0.23432b 0.23123c 0.00071

Other
N-hexyl acetate – 870 18.3 0.31068a 0.26159b ndd 0.24263c 0.00047
Bis (2-furfuryl) disulfide – 1534 18.9 ndb 0.11701a ndb ndb 0.00041
Pantolactone Caramel a 1987 20.6 0.05685b 0.06421a ndc 0.06557a 0.00049
Diacetyl sulphyde – 1128 15.9 0.21145a ndc 0.11575b ndc 0.00033
Furfuryl methyl disulfide – 1234 17.2 0.02384b ndc ndc 0.04596a 0.00057
c
Tetradecan Oily, almost odorless 734 15.3 ndb ndb 0.04980a nd 0.00019
Caffeine – 1854 28 0.11686a ndb 0.11614a ndb 0.00034
Total other 0.7197a 0.4528b 0.2917d 0.3542c 0.0018

nd: not detected.

SIAF: self-induced anaerobic fermentation.
SEM: standard error of the mean, RI: retention index, RT: retention time.
Means value followed by the same lowercase letter in line does not differ from each other by the Scott-Knott test (p > 0.05).
a
Flament (2001).
b
Czerny and Grosch (2000).
c
Eom and Jung (2013).
d
López-Galilea et al. (2006).
e
Osada and Shibamoto (2006), Lee et al., (2017).

was detected only in processed NC, whereas 3,4-dimethyl-2-pentanone; differences were obtained in acidity and score (Table 3). All coffees were
and pyrazine, 2-methyl-6-(1-propenyl)-, (E)- was detected in the sample evaluated as specialty coffees (score >80). The SIAF method stood out
processed by PC. by high acidity (7.51) and score (84.83) obtained from natural coffee
Flavor compounds such as 5-methyl-2-furancarboxaldehyde; 2-fur­ and pulped coffee. The body was correlated in the score (0.968) and
anmethanol; maltol; butanoic acid, 3-methyl; propanoic acid; 1,2-cyclo­ sweetness (0.835) of coffee beverages. Furthermore, the sweetness was
pentanedione, methyl; furfural acetone; 1 ethanone, 1-(2-hydroxy-5- correlated with acidity (0.991) (Fig. 3).
methylphenyl)-; 2,3-hexanedione; butyrolactone; and l-1H-pyrrole-2- Volatile compounds influence the sensory attributes. Furthermore,
carboxaldehyde, 1-methy; were detected in all fermentative processes. score (- 0.944), body (- 0.845) and aftertaste (- 0.850) were correlated
with aldehyde (Fig. 3). Score (0.802) and body (0.823) were correlated
with lactone.
3.4. Sensorial analysis
4. Discussion
The sensorial analysis is one of the main parameters used for eval­
uating the quality of the coffee. According to the SCA methodology, an Conventional postharvest methods do not always result in specialty
assessment of the coffee beverages was performed, and significant coffees, but new methods such as bioreactors and inoculation of mi­
croorganisms have been investigated (Bressani et al., 2018; Da Mota
Table 3 et al., 2020; Martinez et al., 2017; Martins et al., 2020).
Scores of sensory attributes obtained for natural and pulped coffee processed by High-density polyethylene is a relatively inexpensive material
the SIAF and conventional method. resistant to impact damage at room temperature and most chemical
SIAF Conventional SEM attacks; it also provides an excellent gas barrier (Selke & Hernandez,
2001). After capping the bioreactor, gas exchange is limited, and an
Natural Pulped Natural Pulped
coffee coffee coffee coffee anaerobic environment gradually forms during the fermentation pro­
cess. In addition, carbon dioxide (CO2) is one yeast metabolism
Sweetness 7.81a 7.51a 6.39a 6.45a 0.56
Acidity 7.51a 6.68b 5.15c 5.10c 0.24
by-products, and its concentration can be harmful to growth and mi­
Body 6.60a 7.54a 4.69a 5.69 0.61 crobial metabolism (Li et al., 2015), contributing to controlling the
Aftertaste 4.83a 6.36a 5.03a 6.00a 0.55 fermentation process.
Score 82.33b 84.83a 79.92c 82.00b 0.36 The microbial CO2 production during the fermentative process in the
SEM: standard error of the mean. sealed bioreactor system is not enough to inhibit microorganisms’ per­
SIAF: self-induced anaerobic fermentation. formance fermentatively, but the cell division can be inhibited when the
Means value followed by the same lowercase letter in line do not differ from each endogenous CO2 pressure reaches 40 psi (Li et al., 2015). Furthermore,
other by the Scott-Knott test (p > 0.05).

6
M.C. Batista da Mota et al.
Fig. 3. Pearson’s correlation (p < 0.5) between volatile compounds and the beverage sensory attributes.
the CO2 has an essential effect on the pathways of oxaloacetic acid
formation from pyruvic acid and ribose-5-phosphate formation from
glucose-6-phosphate, influencing glycerol, succinic acid, and acetic acid
production. In the NC SIAF method, the glycerol concentration increased
by 414% and acetic acid by 33.17% with 22 h of fermentation.
Under anaerobic conditions, glycerol production is stimulated and
plays as protecting yeast from high osmotic pressure, maintaining cell
redox balance, and providing precursors for the synthesis of phospho­
lipids (Quirós et al., 2013).
The fermentation process is one factor that impacts coffee quality,
and when properly conducted, it increases the probability of standard­
izing the quality of the beverage and decreases the risk of economic loss
for producers (Huch & Franz, 2015). Biotic and abiotic factors, pro­
cessing method, and geographic position interfere in the microbial
community’s fermentative performance and fermentation time,
affecting the metabolism of primary compounds and their conversion to
secondary compounds that will be perceived in the sensory analysis (De
Bruyn et al., 2017). Coffee pulp is a nutrient-rich substrate and offers
essential compounds required for microbial growth, such as sucrose,
glucose, fructose, citric acid, malic acid, and succinic acid (Silva, 2014).
In addition, pectolytic enzymes break down pectin complex chains,
releasing sugars used by microorganisms (Esquivel & Jiménez, 2012).
Monitoring the coffee fermentation process is essential to guarantee
that every quality potential of fruit is developed and remains in coffee
beans. One of the parameters to be monitored during the fermentation
process is temperature. The increase in temperature is evidence of
proper fermentation (Córdoba-Castro & Guerrero-Fajardo, 2016; Quin­
tero & Molina, 2015). This increase is due to the intense metabolic ac­
tivity of microorganisms that can be explained due to exothermic
reaction that occurs during the conversion of sugars and pectic substrate
of mucilage to alcohols and organic acids, and it was obtained in both
fermentation (Correa et al., 2014). However, temperature control is
essential, as an excessive increase in temperature accompanied by the
odor of acetic acid indicates the entry of oxygen into the system, and
excessive production of this acid is not desirable in coffee (Evangelista
et al., 2014). Coffee fermentation in closed bioreactors showed signifi­
cantly higher temperatures than the conventional method providing a
better process standardization. In the conventional method, the heat
generated by metabolic reactions is dissipated into the environment,
explaining the lower temperatures in this treatment.
Additionally, the processing of the coffees also interferes with the
fermentative performance of microorganisms. For example, in pulped
coffees, the removal of the husk contributes to a reduction in the pulp
concentration and the microbial community (Evangelista et al., 2014a,
2014b; Martins et al., 2020). Microbial metabolites can be produced in
different proportions, such as citric acid and lactic acid, showing the
maximum proportion in PC and acetic acid in NC fermented by the SIAF
7
Food Bioscience 47 (2022) 101640
method.
The production of citric acid results from carbohydrate metabolism
mainly by yeasts. Carbohydrates such as sucrose, glucose, and fructose
are the preferred sources of sugars for these microorganisms (Taylor
et al., 2019; Vandenberghe et al., 1999). The yeast metabolism can be
observed during the fermentation of coffee, in which the concentration
of sugars presents decreased over the fermentation time. Glucose, for
example, decreased around 95% in the NC in the SIAF method with 87 h
of fermentation, and sucrose was consumed entirely. The synthesis of
citric acid results from the first stage of the Krebs cycle, in which the
pyruvate resulting from glucose degradation will undergo oxidative
decarboxylation and carboxylation, forming Acetyl-CoA oxaloacetic
acid. These intermediate products suffer the action of the enzyme citrate
synthase and some cofactors that trigger the reaction, causing the
accumulation of citric acid and initiating the tricarboxylic cycle
(Angumeenal & Venkappayya, 2013; Vandenberghe et al., 1999, 2018).
Lactic acid is produced mainly by lactic acid bacteria that prefer the
use of glucose. The lactic acid production can occur by homo­
fermentative lactic acid bacteria under that use aldolase enzymes and
produce lactic acid as the main final product or by heterofermentative
that can convert pentose sugars into lactic acid and by-products (e.g.,
acetic acid) via phosphoketolase (Abdel-Rahman et al., 2013; Eiteman &
Ramalingam, 2015). These acids contributed to coffee beverages’ tart­
ness, buttery, fruity, and floral aromas (Vandenberghe et al., 2018;
Wang et al., 2019). Among these attributes, fruity and floral aromas
were observed during the cup tasting, mainly in the natural cone from
the SIAF method.
The anaerobic conditions established in the bioreactors provided an
ideal environment for lactic acid bacteria development, allowing their
rapid growth and leading to an acidification of the environment.
Moreover, the high proportion of acetic acid and reduced lactic acid may
have occurred due to the transformation of lactic acid into acetic acid by
some heterofermentative lactic acid bacteria during the controlled
fermentation of natural coffee (Elferink et al., 2001).
The SIAF method contributes to yeast and bacterial growth (Martinez
et al., 2017). Synergistic interaction between LAB and yeast favors its
predominance during the fermentative process. Therefore, yeast growth
in fermented foods is favored by the acidifying environment created by
LAB. The yeast can provide growth factors, such as vitamins and soluble
nitrogen compounds that stimulate lactic acid bacteria growth (Adesu­
lu-Dahunsi et al., 2020). However, when poorly conducted, the growth
of detrimental microorganisms is observed, directly affecting the flavor
and aroma of coffee beverages (Haile & Kang, 2019). The bioreactor’s
positive impact is reflected in the perceived sensory attributes, such as
sweetness, acidity, and body, mainly associated with coffee quality
improvement.
Postharvest techniques influence the profile of metabolites and the
M.C. Batista da Mota et al.
sensory quality of the coffee (Borrella et al., 2015; De Bruyn et al.,
2017). There must be equilibration between volatile and nonvolatile
compounds present in the seed (Watson et al., 2016). One criterion
consumers use to accept food products such as coffee is its aroma
(Pérez-Martínez et al., 2008). Numerous biochemical reactions occur
(Maillard reactions, pyrolysis, Strecker degradation), generating various
volatile compounds (Marek et al., 2020).
The profile and concentration of volatile compounds differed be­
tween natural and pulped coffee processing. For example, in pulped
coffee, low concentrations of volatile compounds were produced for this
coffee, characterizing it as a soft beverage.
In the SIAF method, maltol (fruity and caramel flavor) and 1,2-cyclo­
pentanedione, 3-methyl- (caramel flavor) were detected, approximately
1.2 times as much of concentration in natural coffee than in the other
treatments (Czerny & Grosch, 2000).
Furan was the primary chemical class detected in roasted coffee
because it is related to flavor. In particular, 2-furanmethanol was the
most abundant compound in coffee, and it was detected 2.6 times more
than found by Caporaso et al. (2018). A similar result was obtained by
Martinez et al. (2021) for the Catuai variety and contributed to caramel,
sweet, bitter, coffee, astringent, and burnt flavors besides being able to
exhibit antioxidative activity (Bressani et al., 2021; Caporaso et al.,
2018; Da Mota et al., 2020; Osada & Shibamoto, 2006).
In the conventional method, benzyl alcohol was detected, giving the
beverage a fruity, slightly floral flavor (Bobo-García et al., 2015). Vol­
atile acids such as butanoic and propanoic acid were detected in SIAF,
and conventional coffee undergoes a degradation process during roast­
ing and synthesizes important volatile groups belonging to the groups of
pyridines and pyrrole that enhance the flavor of coffee (Sunarharum
et al., 2014).
In the SIAF method, the NC showed an intense acidity, sweetness,
and medium body, whereas the PC exhibited less acidity, lingering
sweetness, and a dense body. Despite the enhanced sweetness and
acidity of the SIAF-method NC, the body was one of the correlated at­
tributes for the final score. The increase in the body was correlated with
a decrease in the aldehyde concentration detected in the roasted coffee
(Swiegers & Pretorius, 2005). Therefore, the decreased aldehyde con­
centration contributed to the quality and tasting profile (Toledo et al.,
2016) since unsaturated aldehydes in roasted coffee can negatively
impact the flavor characterized as green coffee odor (Flament, 2001).
Among the lactones, butyrolactone was the compound detected in
greater abundance and seems to influence both the body and the final
beverage score. In the SIAF method, butyrolactone concentration
increased 1.34 times for natural coffee and 1.67 for pulped coffee. In
addition, fermented coffees have been shown to increase the concen­
tration of butyrolactone by up 5 times (Lee et al., 2017).
When well conducted, coffee fermentation in closed containers en­
hances the removal of sugars from the coffee pulp and favors the syn­
thesis of flavor-related molecules, making it an alternative to the
conventional method. This new fermentation model gives homogeneous
and superior quality coffees in the most demanding markets. Further­
more, high-density polyethylene bioreactors are accessible to farmers,
and this self-induced anaerobic fermentation system ensures that the
fermented coffee yields a higher quality final beverage.
5. Conclusions
Coffee fermentation in a closed batch system without water was
successfully performed. The coffee beans’ microbial activity promoted a
self-induced anaerobic fermentation system, with an increased temper­
ature and a decreased soluble solids content, indicating that fermenta­
tion was well conducted. The SIAF method provided the highest lactic
acid concentration in fermented coffee and increased the coffee
beverage SCA scores by up to 2 points. Furthermore, SIAF modified the
concentration of volatile compounds, and the sensory score correlated
with the aldehyde concentration. Therefore, the bioreactor’s
8
Food Bioscience 47 (2022) 101640
fermentation process under anaerobic conditions is accessible to pro­
ducers and contributes to obtaining coffees with differentiated sensory
profiles and increased beverage quality.
Authors statement
Marcela Caroline Batista da Mota- Conducting a research and
investigation process, specifically performing the experiments in
Microbiology lab. Wrote the first draft of the manuscript. Nadia Nara
Batista - Formulation and plan the project, critical review, commentary,
or revision – of the manuscript. Disney Ribeiro Dias - Conceptualization,
Methodology, critical review, commentary, or revision – of the manu­
script. Rosane Freitas Schwan - Ideas; formulation or evolution of
overarching research goals and aims. Financial resources and final
critical review of the manuscript pre and pos submission.
Funding sources
This work was supported by the Brazilian agencies Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG),
and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) for financial support. We also thank Syngenta (NuCoffee) and
the coffee producers, where the experiment was performed.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
The authors thank the pos graduate colleagues who help with this
work in the field – D.C. Souza, A.P.P. Bressani, S.J. Martinez, T.S. Per­
eira, J.M.C. Palumbo, M.G.C.P. Miguel, P.V. Fernandes, and E. Schwan.
The authors gratefully acknowledge the anonymous referees for their
comments and suggestions for improving this manuscript’s quality.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fbio.2022.101640.
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