Article

Effect of Fermentation Duration on the Chemical Compounds

of Coffea arabica from Ultra Performance Liquid
Chromatography–Triple Quadrupole Mass Spectrometry and
Gas Chromatography–Mass Spectrometry Analysis During the
Washed Processing
Xiaojing Shen 1 , Qi Wang 2 , Tingting Zheng 3 , Biao Yuan 1 , Zhiheng Yin 1 , Kunyi Liu 2, * and Wenjuan Yuan 1, *

1 College of Science, Yunnan Agricultural University, Kunming 650201, China; 2013017@ynau.edu.cn (X.S.)
2 School of Wuliangye Technology and Food Engineering, Yibin Vocational and Technical College,
Yibin 644100, China; mumu202107@163.com
3 Pu’er Institute of Pu-erh Tea, Pu’er 665000, China
* Correspondence: ben.91@163.com (K.L.); 2018020@ynau.edu.cn (W.Y.)

Abstract: The washed process is one of the traditional post-harvest processes of coffee beans, which
include selective harvesting, flotation, pulping, submerged fermentation underwater, washing, and
drying operations. During the washed processing, fermentation underwater can remove coffee
mucilage and change metabolites by microorganisms. Therefore, coffee fermentation is a key factor
influencing coffee’s flavor. To compare the influence of fermentation duration in an open environment
of Coffea arabica in 48 h during the washed processing on the coffee’s flavor, the sensory characteristics
of the coffee at different fermentation durations were evaluated using the Specialty Coffee Association
Citation: Shen, X.; Wang, Q.; Zheng, of America (SCAA) cupping protocol. Moreover, ultra performance liquid chromatography–triple
T.; Yuan, B.; Yin, Z.; Liu, K.; Yuan, W. quadrupole mass spectrometry (UHPLC–MS/MS) and gas chromatography–mass spectrometry
Effect of Fermentation Duration on (GC–MS) were combined to analyze and compare the chemical compounds of coffee samples from
the Chemical Compounds of Coffea fermentation durations of 24 h (W24) and 36 h (W36) during the washed processing method. The
arabica from Ultra Performance Liquid results showed that W36 had the highest total cupping score with 77.25 in all different fermentation
Chromatography–Triple Quadrupole duration coffee samples, and 2567 non-volatile compounds (nVCs) and 176 volatile compounds (VCs)
Mass Spectrometry and Gas
were detected in W36 and W24 during the washed processing method. Furthermore, 43 differentially
Chromatography–Mass Spectrometry
changed non-volatile compounds (DCnVCs) and 22 differentially changed volatile compounds
Analysis During the Washed
(DCVCs) were detected in W36 vs. W24. Therefore, suitable fermentation duration in an open
Processing. Fermentation 2024, 10, 560.
https://doi.org/10.3390/
environment is beneficial to coffee flavor, judging by chemical compound changes. For the washed
fermentation10110560 primary processing of C. arabica from Yunnan, China, 36 h fermentation was the suitable fermentation
duration in an open environment, which presented potential value as the reference for washed coffee
Academic Editors: Alice Vilela and
processing in the food industry.
Xidong Ren

Received: 19 August 2024 Keywords: Coffea arabica; washed processing method; fermentation duration; coffee flavor
Revised: 21 October 2024
Accepted: 29 October 2024
Published: 31 October 2024
1. Introduction
Coffee, tea, and cocoa are the three top beverages in the world. Consumers are very
Copyright: © 2024 by the authors.
interested in the special coffee flavor. However, a cup of a high-quality coffee beverage is
Licensee MDPI, Basel, Switzerland. comprehensively affected by many factors, such as genetic attributes, growing conditions,
This article is an open access article harvesting, post-harvest coffee processing, storage, roasting, and the brewing steps of
distributed under the terms and coffee beverages [1–4]. Coffee beans are surrounded by skin, pulp, mucilage, parchment,
conditions of the Creative Commons and silver skin. Therefore, post-harvesting primary processing (wet, dry, or semi-dry
Attribution (CC BY) license (https:// processing) is the first step of coffee processing used to obtain green coffee beans [5]
creativecommons.org/licenses/by/ which can significantly influence coffee’s flavor [6,7]. Microbial communities and chemical
4.0/). compounds showed significant differences compared to primary processing [8,9]. For

Fermentation 2024, 10, 560. https://doi.org/10.3390/fermentation10110560 https://www.mdpi.com/journal/fermentation

Fermentation 2024, 10, 560 2 of 13

example, levels of fructose, glucose, arabinose, and other free low-molecular-weight sugars
in green coffee beans undergoing wet processing were lower than in those undergoing dry
processing [10]. However, levels of glutamic acid and aspartic acid in green coffee beans
undergoing wet processing were higher than in those undergoing dry processing [11].
Moreover, microbial species were different compared across different primary processes.
For bacteria, Leuconostoc, Lactiplantibacillus, Klebsiella, and Weissela were the main bacteria
in wet processing, while Enterobacter, Bacillus, Tatumella, and Pseudomonas were the main
bacteria in dry processing [12].
Coffea arabica and Coffea robusta are two popular marked coffee species worldwide [13].
Dry processing is often used for C. robusta, while wet processing is often used for C. ara-
bica [5]. Washed or wet processing, a traditional post-harvest coffee processing technique,
includes de-pulping, fermentation, washing, drying, and other main operations [2]. Fer-
mentation is a critical operation, as it can remove the pulp and mucilage by enzymes from
the coffee fruit and microflora from the environment, and since mucilage is composed
of protein, this reduces sugar, pectates, and ash [14,15]. However, traditional coffee fer-
mentation is carried out in a developed environment; microbial communities often are
influenced by environmental factors, such as the coffee region, temperature, altitude, pH,
and so on. Therefore, the control of fermentation conditions is a prerequisite for improv-
ing coffee quality, such as suitable fermentation duration, processing type, application of
soaking, etc. [2,16]. For example, a long fermentation duration would produce positive
fruity and acid notes along with negative cereal and floral notes in the coffee flavor [2]. Fer-
mentation leads to the change in volatile compounds related to coffee flavor [15]. Previous
studies have showed that 143 non-volatile compounds were differentially changed in a
36 h fermentation compared to non-fermentation [6].
To provide a reference for further understanding the influence of fermentation on
coffee’s flavor during the washed processing method, the Specialty Coffee Association
of America (SCAA) cupping protocol was used to evaluate coffee flavor and characteris-
tics by ten attributes (fragrance/aroma, flavor, aftertaste, acidity, body, balance, overall
impression, uniformity, sweetness, and clean cup). Furthermore, ultra performance liq-
uid chromatography–triple quadrupole mass spectrometry (UHPLC–MS/MS) and gas
chromatography–mass spectrometry (GC–MS) were combined to analyze and compare the
differences of non-volatile compounds and volatile compounds at different fermentation
durations of coffee beans in this paper. Then, the different changed compounds after
24 h vs. 36 h durations in C. arabica from Yunnan province were analyzed.

2. Materials and Methods

2.1. Materials and Chemical Standards
The raw materials were mature coffee cherries from C. arabica, which were harvested
and collected from Pu-er City, Yunnan Province, China. According to the washed processing
method, the mature coffee cherries were processed, followed by sorting in water, de-
pulping of the exocarp, and fermentation under water. A total of 1 kg of de-pulping coffee
was spontaneously fermented under 1 L of sterile water (the pH was 6.8, the content of
dissolved oxygen was 0.17 mg/L) at room temperature ranging from 18 to 25 ◦ C in an open
environment. During fermentation under the water, four coffee samples from different
fermentation durations were collected at fermentation times of 12 h, 24 h, 36 h, and 48 h,
respectively. An unfermented control (UC) sample was taken at the start of the fermentation.
Then, these five coffee samples were washed and dried under the sun to obtain green coffee.
Finally, these green coffee beans were roasted using a IKWA Pro V3 coffee bean roaster
(IKAWA Ltd., London, UK) under medium roasting conditions (10–15 min roasting time,
210–220 ◦ C roasting temperature) to obtain roasted coffee beans by certified professionals
from Anke Coffee Limited Company (Kunming, China). The roasted coffee samples were
stored at −20 ◦ C for the subsequent analysis. To clearly distinguish the different coffee
samples, they were marked as UC, W12 (fermentation duration of 12 h), W24 (fermentation
duration of 24 h), W36 (fermentation duration of 36 h), and W48 (fermentation duration
Fermentation 2024, 10, 560 3 of 13

of 48 h), respectively. The chemical reagents including methyl alcohol, acetonitrile, and
propyl alcohol were high-performance liquid-chromatography-grade and purchased from
Fisher Co., Ltd. (Shanghai, China).

2.2. Cupping Analysis

The cupping analysis of five coffee samples was conducted based on the Specialty
Coffee Association of America (SCAA) cupping protocol. Ten attributes (fragrance, flavor,
aftertaste, acidity, body, balance, uniformity, sweetness, cleanliness, and score) were cate-
gorized and scored based on quality by nine certified professionals (five males and four
females, aged 18 to 35 years) with expertise in cupping analysis from Anke Coffee Limited
Company (Kunming, China) [6]. Each coffee sample was independently given a triplicate
score and its coffee characteristics described by a professional under blind conditions. In
brief, uniformity, sweetness, and clean cup were used to reflect the absence of defects with
10 points. Other attributes were scored based on their quality on a scale of 6 to 10 points at
intervals of 0.25 points. Simultaneously, the characteristics of fragrance, aroma, aftertaste,
and body were assessed in the detailed description.

2.3. Analysis of Non-Volatile Compounds by UHPLC–MS/MS

According to the result of the cupping analysis, non-volatile compounds (nVCs) from
W24 and W36 were analyzed using UHPLC–MS/MS. In order to extract non-volatile com-
pounds to 50 mg of roasted coffee powder sample, 0.4 mL of 80% methanol was added.
Then the mixture was centrifugated at 13,000× g for 15 min at 4 ◦ C to obtain the super-
natant. A total of 2 µL of supernatant was injected into a UHPLC-Q Exactive system
(Thermo Fisher Scientific, Bremen, Germany) with HSS T3 C18 column (2.1 × 100 mm,
1.8 µm; Waters Corporation, Milford, MA, USA) at a 0.4 mL/min flow rate [17,18]. A
mixture of (A) 0.1% formic acid in water: acetonitrile (95: 5, v/v) and (B) 0.1% formic acid in
acetonitrile: isopropanol: water (47.5: 47.5: 5, v/v) was the mobile phase under the gradient
elution: 0–5% B for 0–0.1 min, 5–25% B for 0.1–2 min, 25–100% B for 2–9 min, and 100%
B for 9–13 min, and 100–0% B for 13–13.1 min, then 0% B for 13.1–16 min. Mass spectra
were carried out in an electrospray ionization (ESI) source at –2800 V in negative mode and
3500 V in positive mode and scanned in the range of 70–1050 m/z. Data filtering, peak detec-
tion, alignment, and calculations were performed using Analyst 1.6.3 software (AB Sciex,
Framingham, MA, USA). To facilitate the identification/annotation of metabolites, accurate
m/z ratios were obtained for each precursor ion. Total ion chromatograms and extracted
ion chromatograms of QC samples were exported to give an overview of the metabolite
profiles of all samples. Metabolites were characterized by searching internal and public
databases (MassBank, KNApSAcK, HMDB, MoTo DB, and METLIN) and comparing their
m/z values, retention times, and fragmentation patterns with those of the standards and
comparing their m/z values, retention times and fragmentation patterns with those of the
standards. The chromatographic peak area of each was calculated. Positive and negative
data were combined to obtain a combined data set.

2.4. Analysis of Volatile Compounds by GC–MS

According to the result of the cupping analysis, volatile compounds (VCs) of W24
and W36 were evaluated using GC–MS. Firstly, the extract volatile compounds to 50 mg
of roasted coffee powder sample, 0.5 mL of 80% methanol was added. The mixture was
ground at –20 ◦ C and added 0.2 mL trichloromethane to ultrasonic extraction for 30 min.
Then the extract was quiescence at –20 ◦ C and centrifugated at 13,000× g for 15 min at 4 ◦ C
to obtain the supernatant. 80 µL 15 mg/mL methamphetamine hydrochloride pyridine solu-
tion was added to the supernation for reaction for 90 min at 37 ◦ C. 80 µL Bis (trimethylsilyl)
trifluoroacetamide (1% chlorotrimethylsilane) was added and reacted for 60 min at 70 ◦ C fol-
lowing a shocking for 2 min. The GC–MS analysis was carried out referencing Shen et al. [6],
using a model 8890B GC instrument with a DB–5MS (40 m × 0.25 mm × 0.25 µm) capillary
column and 5977B mass spectrometer (Agilent, Santa Clara, CA, USA). In brief, the carrier
Fermentation 2024, 10, 560 4 of 13

gas was 99.999% helium at a 1.0 mL/min column flow. The column temperature program
was set to 60 ◦ C held for 0.5 min and then raised to 310 ◦ C at an 8 ◦ C/min rate. Mass
spectra were recorded in electron impact (EI) ionization mode at 70 eV and scanned in the
range m/z 50–500. Chroma TOF 4.3X software (LECO Corporation, St. Joseph, MI, USA)
and the LECO-Fiehn Rtx5 database were utilized for raw peak extraction, baseline filtering
and calibration, peak alignment, deconvolution analysis, peak identification, and peak area
integration. Metabolite identification considered both mass spectrum and retention index
matches. Peaks detected in fewer than 50% of QC samples or with an relative standard
deviation greater than 30% in QC samples were removed. Following normalization of the
original peak area data to the total peak area, further analysis was conducted.

2.5. Statistical Analysis

Statistical analyses were performed using IBM SPSS Statistics v28.0.1.1 (SPSS Inc.,
Chicago, IL, USA). The data were expressed as mean ± standard deviation. LSD analysis
of variance was used for statistical analysis, and p < 0.05 was considered significant. The
analysis method for differentially changed compounds was as follows. Firstly, the response
intensity of the sample mass spectrum peaks was normalized using the sum normalization
method, and variables with a relative standard deviation (RSD) > 30% of QC samples (equal
volumes of all samples) were removed, followed by log10 calculations. Then, variable
importance in projection (VIP) analysis ranked the overall contribution of each variable
to the orthogonal partial least square discriminant analysis (OPLS-DA) model, and the
variables with VIP > 1.0, p < 0.05, fold change (FC) > 1.5 or FC < 0.67 were classified as
differentially changed non-volatile compounds (DCnVCs), and VIP > 1.0, p < 0.05, FC > 1.1
or FC < 0.9 were classified as differentially changed volatile compounds (DCVCs) [19].

3. Results
3.1. The Results of Cupping Analysis and Sensory Characteristics of Different Fermentation Durations
The cupping test is a very important evaluation method for coffee flavor and charac-
teristics. Each coffee sample was evaluated by three certified professionals with expertise
in cupping analysis based on the SCAA cupping protocol. If the total score of the cup-
ping analysis is in the range of 70.00–79.00, it is considered premium [20]. C. arabica is
the main cultivated species of coffee in Yunnan province, China, which is a well-known
cultivation base of C. arabica in the world [21]. All coffee samples from different fermenta-
tion durations were premium with total scores of 75.25 ± 0.14 (UC), 75.75 ± 0.25 (W12),
76.50 ± 0.14 (W24), 77.25 ± 0.25 (W36), and 76.00 ± 0.14 (W48), respectively. The scores
of detailed attributes were evaluated as shown in Figure 1. According to the score, the
score of fragrance/aroma in different coffee samples was 6.75, which was not influenced
by the fermentation duration. The score of 10 for uniformity, sweetness, and clean cup
means these coffee samples had an absence of defect. The scores of UC and W12 were
significantly lower than others (p < 0.05) and had a weak roast nut aroma, short aftertaste,
and low tea body. This indicates that a short fermentation duration was not beneficial for
the forming of coffee flavor. Body, sweetness, and balance were promoted by lengthening
fermentation duration. Meanwhile, the scores of W24 and W36 were significantly higher
than other fermentation duration samples (p < 0.05), and W36 had the highest sensory score
and had obvious roast cereal and nut aromas, orange acid, medium tea body, and low cof-
fee sweet. Roasted nut flavors often relate to pyrazine, such as 2,5-dimethylpyrazine,
2,6-dimethylpyrazine,2,3-diethyl-5-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, and
67-dihydro-5-methyl-5H-cyclopentapyrazine, while carbohydrates are related to the sweet-
ness of coffee, such as sucrose, glucose, fructose [22].
 to pyrazine, such as 2,5-dimethylpyrazine, 2,6-dimethylpyrazine,2,3-diethyl-5-methylpyra-
zine, 2-ethyl-3,5-dimethylpyrazine, and 67-dihydro-5-methyl-5H-cyclopentapyrazine,
Fermentation 2024, 10, 560 while carbohydrates are related to the sweetness of coffee, such as sucrose, glucose, fructose
5 of 13
[22].

76.00 ± 0.14 c

77.25 ± 0.25 a

76.50 ± 0.14 b

75.75 ± 0.25 c

75.25 ± 0.14 d

Figure 1. The scores of the coffee cupping test from fermentation duration of C. arabica in Yunnan
Figure 1. The scores of the coffee cupping test from fermentation duration of C. arabica in Yunnan Province.
Province. Different lowercase superscripts indicate significant differences between comparisons (p
Different lowercase superscripts indicate significant differences between comparisons (p < 0.05).
< 0.05).
3.2. The Result of Non-Volatile Compound Analysis by UHPLC–MS/MS
3.2. TheNon-volatile
Result of Non-Volatile
compounds Compound Analysis
in roasted coffee by UHPLC–MS/MS
beans are very important to coffee flavor.
TheNon-volatile
non-volatile compounds
compounds in inroasted
roastedcoffee
coffee beanssuch
beans, are as
very important
trigonelline, to coffee flavor.
chlorogenic acids,
Thecarboxylic acids,
non-volatile carbohydrates,
compounds polymeric
in roasted coffee polysaccharides,
beans, such as lipids, protein,
trigonelline, melanoidins,
chlorogenic ac-
and minerals, can contribute to the overall aroma, astringency, bitterness,
ids, carboxylic acids, carbohydrates, polymeric polysaccharides, lipids, protein, mela- sweetness, and
other sensory
noidins, properties
and minerals, of coffee beverages
can contribute [22].aroma,
to the overall Based astringency,
on the resultbitterness,
of cupping test,
sweet-
W36
ness, hadother
and the highest
sensoryscore following
properties with W24.
of coffee W36 showed
beverages [22]. Based significant differences
on the result from
of cupping
other fermentation duration samples with a value of p low than
test, W36 had the highest score following with W24. W36 showed significant differences0.05. Among them, the
value of p between W36 with W24 was 0.023, which was the highest
from other fermentation duration samples with a value of p low than 0.05. Among them, in all groups. A total
theofvalue
2567 non-volatile
of p betweencompounds
W36 with W24 (nVCs)waswere detected
0.023, which in wasW24theand W36 in
highest using UHPLC–
all groups. A
MS/MS (Table S1), which were classified as 17 super-classes, shown in Figure 2. They
total of 2567 non-volatile compounds (nVCs) were detected in W24 and W36 using
included 502 lipids and lipid-like molecules (comprising 19.56% of the total number of
UHPLC–MS/MS (Table S1), which were classified as 17 super-classes, shown in Figure 2.
nVCs), 487 organoheterocyclic compounds (18.97%), 461 organic acids and derivatives
They included 502 lipids and lipid-like molecules (comprising 19.56% of the total number
(17.96%), 353 organic oxygen compounds (13.75%), 301 phenylpropanoids and polyketides
of (11.73%),
nVCs), 487 255organoheterocyclic
benzenoids (9.93%),compounds
72 nucleosides,(18.97%), 461 organic
nucleotides, acids and
and analogues derivatives
(2.80%), 36 al-
(17.96%), 353 derivatives
kaloids and organic oxygen
(1.40%),compounds (13.75%),
31 organic nitrogen 301 phenylpropanoids
compounds and polyke-
(1.21%), nine hydrocarbons
tides (11.73%),
(0.35%), 255 benzenoids
four lignans, neolignans, and(9.93%),
related72compounds
nucleosides, nucleotides,
(0.16%), and analogues
four hydrocarbon deriva-
(2.80%), 36 alkaloids and derivatives (1.40%), 31 organic nitrogen
tives (0.16%), two homogeneous non-metal compounds (0.08%), two organic 1,3-dipolar compounds (1.21%),
nine hydrocarbons
compounds (0.35%),
(0.08%), four lignans, neolignans,
one organohalogen compoundand related
(0.04%), compounds
one (0.16%), and
acetylide (0.04%), four
hydrocarbon derivatives (0.16%), two homogeneous non-metal compounds (0.08%), two
46 others (1.79%).
organic 1,3-dipolar compounds (0.08%), one organohalogen compound (0.04%), one acet-
ylide (0.04%), and 46 others (1.79%).
Fermentation 2024,
Fermentation 10, 560
2024, 10, x FOR PEER REVIEW 6 of613
of 13

502
1

7
Super-class

48
1
Acetylides
2

Alkaloids and derivatives

2
Benzenoids
Homogeneous non-metal compounds
4
461 Hydrocarbon derivatives
Hydrocarbons
4 Lignans, neolignans and related compounds
2,567 Lipids and lipid-like molecules
2567
Nucleosides, nucleotides, and analogues
9
Organic 1,3-dipolar compounds
353 Organic acids and derivatives
Organic nitrogen compounds
31
Organic oxygen compounds
Organohalogen compounds
Organoheterocyclic compounds
36

30 Others
1
Phenylpropanoids and polyketides
46

72

255

Figure 2. Super-classes of non-volatile compounds (nVCs) from the fermentation duration of C. ara-
Figure 2. Super-classes of non-volatile compounds (nVCs) from the fermentation duration of C. arabica
bica in Yunnan province. The circles represent super-classes of nVC, different colors represent dif-
in ferent
Yunnan province. The
super-classes, and circles
differentrepresent super-classes
sizes represent of nVC,
the different different
numbers colors represent
of non-volatile different
compounds.
super-classes, and different sizes represent the different numbers of non-volatile compounds.
These nVCs were further grouped into 162 classes. These classes mainly included 386
These nVCs
carboxylic acids were further grouped
and derivatives into 162
(comprising classes.352
15.04%), These classes mainly
organooxygen included
compounds
386 carboxylic acids and derivatives (comprising 15.04%), 352 organooxygen
(13.71%), 198 fatty acyls (7.71%), 148 prenol lipids (5.77%), 136 benzenes and substituted compounds
(13.71%), 198(5.30%),
derivatives fatty acyls (7.71%), 148
89 flavonoids prenol79lipids
(3.47%), (5.77%),
steroids 136 benzenes
and steroid derivativesand(3.08%),
substituted
64
derivatives (5.30%), 89 flavonoids (3.47%), 79 steroids and steroid
coumarins and derivatives (2.49%), 64 glycerophospholipids (2.49%), 62 phenols (2.42%), derivatives (3.08%),
6460coumarins
indoles and and derivatives
derivatives (2.49%),
(2.34%), 64 glycerophospholipids
52 pyridines (2.49%),51
and derivatives (2.03%), 62cinnamic
phenols (2.42%),
acids
60and
indoles and derivatives
derivatives (1.99%), 38 (2.34%), 52 pyridines
benzopyrans (1.48%),and derivatives (2.03%),
31 organonitrogen 51 cinnamic
compounds acids
(1.21%),
and
28 derivatives
keto acids and (1.99%), 38 benzopyrans
derivatives (1.48%), 31 organonitrogen
(1.09%), 28 imidazopyrimidines (1.09%),compounds
26 quinolines(1.21%),
and
28derivatives
keto acids (1.01%),
and derivatives
23 lactones (1.09%),
(0.90%),28 23imidazopyrimidines
isoflavonoids (0.90%), (1.09%), 26 quinolines
22 purine nucleosides and
derivatives
(0.86%), 22(1.01%), 23 lactones
naphthalenes (0.86%),(0.90%),
21 hydroxy 23 isoflavonoids (0.90%),(0.82%),
acids and derivatives 22 purine nucleosides
21 pyrimidine
(0.86%), 22 naphthalenes
nucleoside (0.86%), 21 hydroxy
(0.82%), 19 phenylpropanoic acidsacids and18derivatives
(0.74%), (0.82%),
diazines (0.70%), 1821 pyrimidine
dihydrofu-
nucleoside (0.82%),
rans (0.70%), 19 phenylpropanoic
17 pyrans acids (0.74%), 18
(0.66%), 13 peptidomimetics diazines
(0.51%), 13 (0.70%), 18 dihydrofurans
piperidines (0.51%), 12
(0.70%), 17 pyrans
azoles (0.47%), 12 (0.66%), 13 peptidomimetics
heteroaromatic compounds (0.47%), (0.51%),11 13 piperidines
pyrrolidines (0.51%),
(0.43%), 12 azoles
11 phenol
ethers 12
(0.47%), (0.43%), 11 macrolides
heteroaromatic and analogues
compounds (0.47%), 11 (0.43%), 10 pteridines
pyrrolidines (0.43%),and derivatives
11 phenol ethers
(0.39%),11and
(0.43%), 10 benzodioxoles
macrolides and analogues (0.39%). Quinic
(0.43%), acids, mono-caffeoylquinic
10 pteridines acids, di-
and derivatives (0.39%), and
10caffeoylquinic
benzodioxoles acids,
(0.39%).tri-caffeoylquinic acids, and feruloylquinic
Quinic acids, mono-caffeoylquinic acids, such acids,
acids, di-caffeoylquinic as
caffeoylquinic acid,
tri-caffeoylquinic isoquinoline,
acids, 4-O-p-coumarylquinic
and feruloylquinic acids, such asacid, 1-O-caffeoylquinic
caffeoylquinic acid, 5-
acid, isoquinoline,
caffeoylquinic acid, acid,
4-O-p-coumarylquinic 3-O-feruloyquinic
1-O-caffeoylquinic acid,acid,
4-O-caffeoyl-3-feruloylquinic acid, 1,3-
5-caffeoylquinic acid, 3-O-feruloyquinic
dicaffeoylquinic
acid, acid, 1,5-dicaffeoylquinic
4-O-caffeoyl-3-feruloylquinic acid, and 3-caffeoyl-4-feruloylquinic
acid, 1,3-dicaffeoylquinic acid, were
acid, 1,5-dicaffeoylquinic acid,
identified
and as common compounds
3-caffeoyl-4-feruloylquinic in coffee
acid, at differentasfermentation
were identified durations. in coffee at
common compounds
To fermentation
different compare the changedurations. in nVCs, the differentially changed non-volatile compounds
(DCnVCs)
To compare the change inFC
(VIP > 1.0, p < 0.05, > 1.5 or
nVCs, theFCdifferentially
< 0.67) between W36 and
changed W24 were compounds
non-volatile analyzed.
The result was shown in Figure 3, in which significantly increased
(DCnVCs) (VIP > 1.0, p < 0.05, FC > 1.5 or FC < 0.67) between W36 and W24 were analyzed. DCnVCs were repre-
sented by red circles and significantly decreased DCnVCs were represented
The result was shown in Figure 3, in which significantly increased DCnVCs were represented by blue cir-
cles. A total of 43 DCnVCs were detected in W36 vs. W24. They were
by red circles and significantly decreased DCnVCs were represented by blue circles. A total related to 16 lipids
of 43 DCnVCs were detected in W36 vs. W24. They were related to 16 lipids and lipid-like
molecules, six organic oxygen compounds, three organoheterocyclic compounds, three ben-
zenoids, three organic acids and derivatives, two nucleosides, nucleotides, and analogues,
 Fermentation 2024, 10, x FOR PEER REVIEW 7 of 13

Fermentation 2024, 10, 560 7 of 13

and lipid-like molecules, six organic oxygen compounds, three organoheterocyclic com-
pounds, three benzenoids, three organic acids and derivatives, two nucleosides, nucleo-
tides, and analogues, two phenylpropanoids and polyketides, one organic nitrogen com-
two phenylpropanoids
pound, one hydrocarbon,andand
polyketides,
six others.one
Amongorganic
them,nitrogen compound,
the regulative levelsone hydrocarbon,
of 12 DCnVCs
and six decreased
were others. Among them, the
significantly (VIPregulative
> 1.0, p <levels of 12FCDCnVCs
0.05, and were decreased
< 0.67), including significantly
lipids and lipid-
(VIP
like> 1.0, p < 0.05, and
molecules FC <DCnVCs,
(six 0.67), including lipids and lipid-like molecules
PA(18:1(9Z)/18:2(9Z,12Z)), (six DCnVCs,
PA(18:1(9Z)/16:0),
PA(18:1(9Z)/18:2(9Z,12Z)),
PA(16:0/18:2(9Z,12Z)), PA(18:1(9Z)/16:0), PA(16:0/18:2(9Z,12Z)),
20-hydroxy-leukotriene E4, 20-hydroxy-leukotriene
deoxynivalenol, and
E4,DG(18:1(11Z)/18:3(9Z,12Z,15Z)/0:0)),
deoxynivalenol, and DG(18:1(11Z)/18:3(9Z,12Z,15Z)/0:0)), organic acids
organic acids and derivatives (two DCnVCs, imaza- and deriva-
tives
mox(two
and DCnVCs, imazamox
Thr-lue), organic oxygen andcompounds
Thr-lue), organic oxygen
(one DCnVC, compounds (one DCnVC,
4-O-beta-D-glucosyl-4-cou-
4-O-beta-D-glucosyl-4-coumaric
maric acid), organoheterocyclicacid), organoheterocyclic
compounds (one DCnVC,compounds Ⅰ), and
citrusinine (one DCnVC, citrusi-
phenylpro-
nine I), and
panoids andphenylpropanoids and polyketides
polyketides (two DCnVCs, (two DCnVCs,
dihydromethysticin dihydromethysticin and
and 3-demethylsimmondsin
2’’-(Z)-ferulate). Among
3-demethylsimmondsin these related
2”-(Z)-ferulate). Among decreased
these relatedDCnVCs, four DCnVCs,
decreased DCnVCs, four DCn-
PA(18:1(9Z)/18:2(9Z,12Z)),
VCs, PA(18:1(9Z)/18:2(9Z,12Z)), PA(18:1(9Z)/16:0),
PA(18:1(9Z)/16:0), imazamox,
imazamox, andandPA(16:0(9Z)/18:2(9Z,12Z)),
PA(16:0(9Z)/18:2(9Z,12Z)),
were
were significantly
significantly decreased
decreased witha avalue
with valueofofFC
FCless
lessthan
than0.2.
0.2.

PA(16:0/18:2(9Z, 12Z))
Agmatine
3
PA(18:1(9Z)/16:0)
−Lg P

SM(d18:0/20:3(8Z, 11Z, 14Z)-2OH(5, 6))

2 PA(18:1(9Z)/18:2(9Z, 12Z))

Imazamox

−5 0 5
Log2FC(W36/W24)
Figure
Figure 3. 3.The
Thedifferentially
differentially changed
changed non-volatile
non-volatile compounds
compounds(DCnVCs)
(DCnVCs) between
betweenW36 andand
W36 W24.
W24.
Red circles represent significantly increased differentially changed non-volatile compounds, blue
Red circles represent significantly increased differentially changed non-volatile compounds, blue
circles represent significantly decreased differentially changed non-volatile changed compounds,
circles
gray represent significantly
circles represent decreasedchanged
non-significantly differentially changed
non-volatile non-volatile changed compounds,
compounds.
gray circles represent non-significantly changed non-volatile compounds.
Meanwhile, 31 DCnVCs were relatively increased significantly (VIP > 1.0, p < 0.05,
andMeanwhile, 31 DCnVCs
FC > 1.5), including lipidswere
and relatively increased(10
lipid-like molecules significantly (VIP > 1.0, p < 0.05,
DCnVCs, neoconvallatoxolo-
and FC > 1.5), including lipids and lipid-like molecules (10 DCnVCs,
side, PC(20:1(11Z)/14:0), PS(15:0/24:1(15Z)), tetranor 12-HETE, ganosporeric neoconvallatox-
acid A, por-
oloside,
rigeninPC(20:1(11Z)/14:0), PS(15:0/24:1(15Z)),
A, PI(16:0/18:2(9Z,12Z)), tetranor
sebacic acids, azelaic acid, 12-HETE, ganosporeric acid
and apo-12’-zeaxanthinal), or- A,
porrigenin A, PI(16:0/18:2(9Z,12Z)),
ganic oxygen compounds (five DCnVCs, sebacic acids, azelaic
gomphrenin acid, and apo-12’-zeaxanthinal),
II, CMP-2-aminoethylphosphonate,
organic oxygen compounds (five DCnVCs, gomphrenin
3-methylthiopropyl-desulfoglucosinolate, glucomannan, II, and
CMP-2-aminoethylphosphonate,
2,3-butanediol glucoside),
3-methylthiopropyl-desulfoglucosinolate,
benzenoids (three DCnVCs, tiapamil, neopine, glucomannan, and 2,3-butanediol
and salutaridinol), nucleosides,glucoside),
nucleo-
benzenoids
tides, and (three DCnVCs,
analogues tiapamil, 5’-methylthioadenosine,
(two DCnVCs, neopine, and salutaridinol),and nucleosides, nucleotides,
cytidine 5’-monophos-
and analogues (two DCnVCs, 5’-methylthioadenosine, and cytidine 5’-monophosphate-
N-acetylneuraminic acid), organoheterocyclic compounds (two DCnVCs, fenoldopam
and merbarone), organic acids and derivatives (one DCnVC, tyrosyl-tyrosine), hydrocar-
bons (one DCnVC, vinylcyclohexene), organic nitrogen compounds (one DCnVC, agma-
tine), and others (six DCnVCs, SM(d18:0/20:3(8Z,11Z,14Z)-2OH(5,6)), PG(6 keto-PGF1
alpha/i-17:0), PG(22:6(5Z,7Z,10Z,13Z,16Z,19Z)-OH(4)/20:1(11Z), PI(18:1(9Z)/18:1(12Z)-
 phate-N-acetylneuraminic acid), organoheterocyclic compounds (two DCnVCs, fenoldo-
pam and merbarone), organic acids and derivatives (one DCnVC, tyrosyl-tyrosine), hy-
drocarbons (one DCnVC, vinylcyclohexene), organic nitrogen compounds (one DCnVC,
Fermentation 2024, 10, 560 agmatine), and others (six DCnVCs, SM(d18:0/20:3(8Z,11Z,14Z)-2OH(5,6)), PG(6 keto- 8 of 13
PGF1 alpha/i-17:0), PG(22:6(5Z,7Z,10Z,13Z,16Z,19Z)-OH(4)/20:1(11Z),
PI(18:1(9Z)/18:1(12Z)-O(9S,10R)), 16alpha-hydroxy DHEA 3-sulfate, and PI(18:1(9Z)-
O(12,13)/18:1(11Z))). Among DHEA
O(9S,10R)), 16alpha-hydroxy these 3-sulfate,
related and
increased DCnVCs, two DCnVCs,
PI(18:1(9Z)-O(12,13)/18:1(11Z))). Among
SM(d18:0/20:3(8Z,11Z,14Z)-2OH(5,6)) and agmatine, were significantly
these related increased DCnVCs, two DCnVCs, SM(d18:0/20:3(8Z,11Z,14Z)-2OH(5,6)) increased with a and
value of FC over
agmatine, werefive. Non-volatile
significantly compounds
increased withina coffee
valueplay
of FCa key
overfunction in coffee fla- com-
five. Non-volatile
vor. For example, lipid compounds can contribute to the perceived texture
pounds in coffee play a key function in coffee flavor. For example, lipid compounds and mouthfeel can
ofcontribute
coffee beverages. Carbohydrates can impact the sweetness. Trigonelline,
to the perceived texture and mouthfeel of coffee beverages. Carbohydrates caffeine, and can
chlorogenic acids can impact the bitterness [22]. Although trigonelline,
impact the sweetness. Trigonelline, caffeine, and chlorogenic acids can impact the caffeine, andbitter-
chlorogenic acids did not show significant changes in these results, the function
ness [22]. Although trigonelline, caffeine, and chlorogenic acids did not show significant of these
DCnVCs
changeswill be worthy
in these of the
results, attention.
function of these DCnVCs will be worthy of attention.
3.3.
3.3.The
TheResult
Resultof of
Volatile Compound
Volatile Compound Analysis by GC–MS
Analysis by GC–MS
Aroma-volatile chemicals in roasted
Aroma-volatile chemicals in roasted coffeeare
coffee arethe
themost
mostimportant
importantquality
qualitydetermi-
determinant.
nant. Although more than 1000 volatile compounds including hydrocarbons,
Although more than 1000 volatile compounds including hydrocarbons, alcohols, alcohols, al-
aldehydes,
dehydes, ketones, carboxyclic acids, esters, pyrazines, pyrroles, and pyridines, have been
ketones, carboxyclic acids, esters, pyrazines, pyrroles, and pyridines, have been identified
identified in coffee, only a small number of them contribute to the coffee flavor and aroma
in coffee, only a small number of them contribute to the coffee flavor and aroma [22].
[22]. In total, 176 volatile compounds (VCs) from 19 classes were confirmed in W36 vs.
In total, 176 volatile compounds (VCs) from 19 classes were confirmed in W36 vs. W24
W24 (Table S2). The percentage of these 19 classes of VC were shown in Figure 4. The
(Table S2). The percentage of these 19 classes of VC were shown in Figure 4. The dominat-
dominating classes were acids (48 VCs, comprising 39.28 in W24 and 40.61% in W36, re-
ing classes were acids (48 VCs, comprising 39.28 in W24 and 40.61% in W36, respectively),
spectively), alcohols (28 VCs, comprising 21.46% and 19.91%, respectively), hydrocarbons
alcohols (28 VCs, comprising 21.46% and 19.91%, respectively), hydrocarbons (22 VCs, com-
(22 VCs, comprising 12.89% and 12.11%, respectively), aldehydes (six VCs, comprising
prising 12.89% and 12.11%, respectively), aldehydes (six VCs, comprising 6.22% and 614%,
6.22% and 614%, respectively), lactones (four VCs, 5.08% and 5.02%), ketones (20 VCs,
respectively),
comprising 2.89%lactones (four respectively),
and 2.57%, VCs, 5.08% and and5.02%), ketones
ethers (10 VCs, (20 VCs, comprising
comprising 0.64% and2.89%
and 2.57%,
0.59%, respectively),
respectively). and ethers
Acids, alcohols, and(10 VCs, comprising
hydrocarbons 0.64%
were the and 0.59%,
dominating respectively).
non-volatile
Acids, alcohols,
compounds. and hydrocarbons were the dominating non-volatile compounds.

100%

1 acid 2 alcohols

80% 3 hydrocarbons 4 ketones

5 esters 6 aldehydes

7 pyridines 8 benzenoids
60%

9 lactones 10 amides

11 amines 12 amino acids

40%
13 pyrimidines 14 piperidines

15 furanones 16 alkanes
20%
17 pyrazoles 18 others

19 ethers

0%
W24 W36

Figure 4. Percentage of volatile compounds from fermentation duration of C. arabica in Yunnan province.

In W24, the percentage of quininic acid was a maximum of 6.15% ± 1.13%, followed
by 3-[(tetrahydro-2H-pyran-2-yl)oxy-]-benzenamine and 2-ethylhexanal ethylene glycol
acetal with 6.06% ± 3.43% and 5.53% ± 0.67%, respectively, while 3-[(tetrahydro-2H-
pyran-2-yl)oxy-]-benzenamine was 7.54% ± 0.53% in W36, followed by quininic acid
(7.00% ± 0.21%) and 2-ethylhexanal ethylene glycol acetal (5.46% ± 0.26%). Nine VCs,
isochlorogenic acid, glycolic acid, 3-pyridinol, lactic acids, myo-inositol, galactinol, sucrose,
Fermentation 2024, 10, 560 9 of 13

D-(+)-trehalose, hexonic acid, and 3-deoxy-gamma-lactone were also significant VCs with
a percentage value over 3.00%.
Moreover, 22 significantly differentially changed volatile compounds (DCVCs) were
detected, as shown in Figure 5. These DCVCs came from seven alcohols (2,3-butanediol,
phloroglucinol, hexitol, butan-1-ol, pyrogallol, D-mannitol, and D-erythro-sphingosine),
five acids (pipecolic acid, 3-hydroxypropionic acid, nicotinic acid, 2-oxovaleric acid, and
lactic acid), three hydrocarbons (trehalose, ethylalpha-D-glucopyranoside, and 1,6-anhydro-
glucose), three ketones (1-(5-ethyl-2-hydroxy-4-methoxyphenyl)-2-(3,4-methylenedioxy-
phenyl)-ethanone, methylhydroquinone, and 1bata,12,12-trimethyl-7,11-dioxapentacylo
[15.3.0.0(4,16).0(5,10)]eicos-13-en-20-ol-8-one), two amides (2,3,6,7,8,8a-hexahydro-1,4-dioxo-
pyrrolo [1,2-s]prrazine-3-propanamide and benzalaniline), one pyrimidine (4-(2-hydroxy-5-
nitrophenyl)pyrimidine), and one aldehyde (2-fluoro-3-hydroxy-4-methoxy-benzaldehyde).
Based on DCVCs between W36 and W24, only three DCVCs including benzalaniline,
ethylalpha-D-glucopyranoside, and 2,3-butanediol were significantly reduced with pro-
longed fermentation duration; other DCVCs were significantly accumulated in roasted
coffee. Although the contribution of these DCVCs to coffee flavor is not confirmed in the
Fermentation 2024, 10, x FOR PEER REVIEW 10 of 13
current research report, their function is worthy of further study to find directly odor-
volatile compounds in coffee beverages.

Figure5.5.The
Figure Thevolatile
volatilecompounds
compounds(VCs)
(VCs)and
andtheir
theirdifferently
differentlychanged
changedvolatile
volatilecompounds
compounds(DCVCs)
(DCVCs)
between W36 and W24. “*” represents significantly differentially changed volatile compounds.
between W36 and W24. “*” represents significantly differentially changed volatile compounds. Red Red
represents that the content of VCs was higher, green represents that the content of VCs was
represents that the content of VCs was higher, green represents that the content of VCs was lower.lower.

4. Discussion
Coffee flavor is easily influenced by several factors, such as post-harvest primary pro-
cessing [5,9]. From the surface to the interior, coffee beans are surrounded by skin, pulp,
mucilage, parchment, and silver skin. To obtain green coffee beans, these five layers sur-
rounding coffee beans must be peeled by mechanical or other methods. The first step to
Fermentation 2024, 10, 560 10 of 13

4. Discussion
Coffee flavor is easily influenced by several factors, such as post-harvest primary
processing [5,9]. From the surface to the interior, coffee beans are surrounded by skin,
pulp, mucilage, parchment, and silver skin. To obtain green coffee beans, these five layers
surrounding coffee beans must be peeled by mechanical or other methods. The first step
to obtaining green coffee beans is post-harvest treatment, which is a crucial step for the
chemical composition of coffee beans and the sensory quality of the coffee beverage [5]. Ac-
cording to previous studies, the post-harvest primary processing method can significantly
affect the chemical compounds of roast and green coffee beans [23,24]. In roasted coffee
beans, 1-O-caffeoylquinic acid, 5-hydroxymethylfurfural, and sugar alcohol can serve as
discriminant marker compounds for distinguishing different primary processing methods
for C. arabica in Yunnan [6].
In the wet processing, coffee cherries with the skin removed were submerged in
fermentation for 12–36 h to remove the pulp [5]. Fermentation is a multifactorial and
important process in coffee processing which has often been influenced by environmental
conditions [16]. Microorganisms (yeasts, bacteria, and filamentous fungi) are present in
coffee fermentation, which come from coffee cherries, soil, air, water, etc. [12]. During the
washed processing of C. arabica from Yunnan province, the chemical compounds of coffee
in fermentation were changed with the change in the structure of the microorganisms, and
some chemical compounds showed a strong positive or strong negative correlation with
the microorganisms [18]. For example, L-quinate showed a strong positive correlation with
Leuconostoc, Metschnikowia, and Apiotrichum. 3-deazaadenosine showed a strong negative
correlation with Tatumella and a positive correlation with Burkholderia, Dyella, and Candida.
In addition, microorganisms also showed interaction with other microorganisms. For
example, Metschnikowia and Apiotrichum fungi genera were extremely strongly positively
correlated with Leuconostoc [18]. Furthermore, these microorganisms can produce important
enzymes for degrading pectin [15]. Furthermore, coffee fermentation can produce a positive
or negative impact on coffee flavor and aroma [25]. Coffee beans themselves contain all
kinds of chemical compounds [26]; in some of these compounds are coffee flavor precursors
such as sugar, proteins, amino acids, and phenolic compounds [4,25]. These coffee flavor
precursors can form coffee aroma by Maillard reaction, Strecker degradation, carameliza-
tion reaction, fragmentation reaction, etc. [4]. During coffee fermentation, microbial activity,
the extent of fermentation, and microorganisms’ diverse metabolites can influence flavor
precursors, such as the concentrations of free sugars and free amino acids [27]. Therefore,
the selection of appropriate specific microorganisms for coffee fermentation has become a
popular method to improve the sensory profile of coffee beverages by producing extracel-
lular enzymes, volatile and non-volatile metabolites, and pH changes [23,28]. However,
controlling the coffee fermentation process is a major key factor and challenge for improv-
ing coffee flavor by microorganisms [3], because underfermentation, overfermentation, or
failing fermentation would develop spoilage microorganisms, producing adverse effects
on the coffee’s aroma and flavor. Moreover, fermentation time, temperature, pH conditions,
moisture content, and other fermentation conditions can influence coffee quality [29,30].
For example, overfermentation would produce undesirable chemical compounds, such
as notably propionic and butyric acids [3]. These compounds would feature an onion
taste [3]. Therefore, fermentation duration is one of the main useful fermentation condi-
tions that is important for coffee flavor. Coffee fermentation duration is often uncertain; a
complete coffee fermentation needs 12–36 h [5]. In addition, the study on the fermentation
method has become an effective way to improve coffee quality [31,32] and special microbial
fermentation also been used as a starter to improve coffee flavor [20,33].
Based on the study of the fermentation duration of C. arabica from Yunnan province,
China, the roasted coffee beans with a 36 h fermentation showed the highest cupping score
and the best sensory character, which was significantly different from other fermentation
coffee samples with a value of p lower than 0.05. Meanwhile, the results of differentially
changed non-volatile and volatile compound analysis found 43 DCnVCs and 22 DCVCs
Fermentation 2024, 10, 560 11 of 13

between fermentation at W36 and W24. Although all these compounds did not contribute
to the coffee flavor, the chemical compounds’ constitutions showed differences.

5. Conclusions
The nVCs, VCs, and coffee flavor of roasted C. arabica beans from Yunnan province at
different fermentation durations during the washed processing methods were compared in
this analysis. The fermentation duration of 36 h showed the highest total cupping score.
Furthermore, a total of 2567 nVCs from 17 super-classes and 176 VCs from 19 classes were
identified in W24 and W36, respectively. Among these lipids and lipid-like molecules,
organoheterocyclic compounds, organic acids and derivatives, organic oxygen compounds,
phenylpropanoids and polyketides, and benzenoids dominated the nVCs, comprising
91.90%. Acids, alcohols, hydrocarbons, aldehydes, and lactones dominated the VCs. More-
over, 43 nVCs were significantly differentially changed non-volatile compounds, and
22 were significantly differentially changed volatile compounds between W36 and W24.
Coffee fermentation is one of the key steps in coffee processing. These results imply that
fermentation duration can influence coffee flavor by the change in chemical compounds.
For the washed primary processing of C. arabica from Yunnan province, a fermentation
duration of 36 h may be the suitable fermentation duration, which presents potential value
as a reference for the washed processing of coffee in the food industry. This study will
provide the possibility for further study on coffee fermentation to improve coffee flavor
and coffee quality by changing coffee chemicals.

Supplementary Materials: The following supporting information can be downloaded at https://

www.mdpi.com/article/10.3390/fermentation10110560/s1, Table S1: The peak area of non-volatile
compounds in W24 and W36. Table S2: The peak area of volatile compounds in W24 and W36.
Author Contributions: Conceptualization, X.S. and K.L.; methodology, X.S., Q.W. and W.Y.; software,
W.Y.; formal analysis, X.S., Q.W., K.L. and W.Y.; resources, T.Z. and B.Y.; data curation, X.S., T.Z., B.Y.
and Z.Y.; writing—original draft preparation, X.S. and Q.W.; writing—review and editing, K.L. and
W.Y.; funding acquisition, X.S. and K.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Yunnan Fundamental Research Project (No. 202101BD070001-046),
the Reserve Talent Project of Young and Middle-aged Academic and Technical Leaders Yunnan
Province (No. 202405AC350064), the Innovation and Entrepreneurship Project of University Students
Yunnan Province (No. S202310676052, S202410676038), the Science and Technology Innovation Team
Project of Yibin Vocational and Technical College (No. ybzy21cxtd-03), and the Scientific Research
Project of Yibin Vocational and Technical College (No. ZRZD24-12).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The experimental data provided in this work are available in articles
and Supplementary Materials.
Conflicts of Interest: The authors declare no conflicts of interest.

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