Article

Modulating Coffee Fermentation Quality Using Microbial

Inoculums from Coffee By-Products for Sustainable Practices in
Smallholder Coffee Production
Luisa-Fernanda Duque-Buitrago 1 , Karen-Dayana Calderón-Gaviria 1 , Laura-Sofia Torres-Valenzuela 1, * ,
Martha-Isabel Sánchez-Tamayo 2 and José-Luis Plaza-Dorado 1, *

1 GIPAB Group (Agrifood and Biotechnological Processes Research Group), School of Food Engineering,
University of Valle, Cali 760042, Colombia; [email protected] (L.-F.D.-B.);
[email protected] (K.-D.C.-G.)
2 Faculty of Agronomic Engineering, University of Tolima, Ibagué 730006, Colombia; [email protected]
* Correspondence: [email protected] (L.-S.T.-V.); [email protected] (J.-L.P.-D.)

Abstract: This study developed an inoculum culture for semi-controlled coffee fermen-
tation using lactic acid bacteria (LAB) and yeast, with coffee production by-products as
carbon sources. The viability of the inoculum was optimized by using a mixture design
to vary the proportions of coffee pulp (CP) and wastewater (CWW) in 0.25 increments; as
a process variable, fermentation time ranged from 36 to 48 h for LAB and 12 to 36 h for
yeast. Soluble solids (SS), pH, and titratable acidity (TA) were monitored, and the response
variable was the variation in microbial viability. The optimized inoculums were used for
coffee fermentation alone and in combination, and fermentation parameters and sensory
evaluation were measured. The optimal by-product combination for LAB inoculum was
100% CP, with a 48 h fermentation, reaching a maximum of 7.8 × 107 CFU/mL. The opti-
mal formulation for yeast was 100% CWW for 36 h, achieving a maximum concentration
of 8.3 × 108 CFU/mL. Experimental results for both inoculums were fit to a quadratic
statistical model with R2 of 0.84 and 0.88 and Adj-R2 of 0.77 and 0.83 for LAB and yeast, re-
spectively. The optimized inoculums produced high sensory scores, particularly in balance,
Academic Editor: Philip Soladoye
fragrance, and acidity. Using mixed inoculums, we achieved the highest fragrance/aroma
Received: 28 October 2024
score (8.25) and an improved balance, attributed to higher TA and lower pH, which are
Revised: 2 December 2024
Accepted: 3 December 2024
linked to enhanced flavor complexity. This demonstrates that by-product-based inoculums
Published: 20 February 2025 can not only increase microbial viability but also improve the sensory quality of coffee,
Citation: Duque-Buitrago, L.-F.;
supporting sustainable practices in coffee processing.
Calderón-Gaviria, K.-D.; Torres-
Valenzuela, L.-S.; Sánchez-Tamayo, Keywords: coffee pulp; coffee wastewater; waste valorization; lactic acid bacteria; yeast
M.-I.; Plaza-Dorado, J.-L. Modulating fermentation; rural development; specialty coffee
Coffee Fermentation Quality Using
Microbial Inoculums from Coffee
By-Products for Sustainable Practices
in Smallholder Coffee Production.
1. Introduction
Sustainability 2025, 17, 1781. https://
doi.org/10.3390/su17051781 Coffee, a globally consumed commodity, faces multifaceted challenges that impact
its economic, social, and environmental sustainability, ranging from socioeconomic fac-
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
tors to environmental concerns and market volatility. In response, different strategies
This article is an open access article have been used to reduce volatility, increase productivity, and adopt sustainable practices
distributed under the terms and to improve quality and prices. Producing nations have invested in differentiating their
conditions of the Creative Commons products as ‘specialty coffees’ through origin, processing techniques, and sustainability
Attribution (CC BY) license
practices. Colombian mild-washed coffee serves as an exemplary case, distinguished by its
(https://creativecommons.org/
unique characteristics that meet growing consumer demands for high-quality, sustainably
licenses/by/4.0/).

Sustainability 2025, 17, 1781 https://doi.org/10.3390/su17051781

Sustainability 2025, 17, 1781 2 of 21

produced coffee. At the producer level, diversifying postharvest processes, such as fermen-
tation reactions, is an essential strategy for enhancing coffee distinctiveness and shaping
sensory attributes and overall quality [1]. The link between postharvest processes and
sensory characteristics emphasizes the importance of exploring modifications to traditional
fermentation techniques. This topic has garnered significant attention from industry and
academic researchers in recent years [2].
In wet coffee processing, coffee cherries are de-pulped and fermented after harvesting
to remove residual mucilage, a process that significantly influences flavor through the
production of microbial metabolites [3]. This workflow involves key steps: de-pulping,
washing, fermentation lasting 12–18 h, and drying beans to achieve a final moisture content
of 10–12%. However, this process generates substantial environmental challenges. On
average, processing one ton of dried coffee beans generates approximately 3.6 tons of fresh
by-products [4]. Approximately 40–45 L of wastewater (CWW) are produced per kilogram
of processed coffee, with a chemical oxygen demand (COD) of 3000–7000 mg/L [5]. Un-
treated CWW and by-products often lead to water contamination, microbial proliferation,
and unpleasant odors in rural areas, where waste management infrastructure is limited.
Coffee pulp (CP), comprising 29–43% (w/w) of the fruit, and mucilage, which contains
84.2% water, 8.9% protein, and 4.1% sugar [6,7], are among the key by-products of wet
processing. These materials are rich in carbohydrates, providing valuable carbon sources
for microbial fermentation. Additionally, CWW is rich in sugars and bioactive compounds,
including caffeine and phenolics, and has potential applications in food, pharmaceutical,
and cosmetic industries [8].
Despite their environmental risks, these by-products present an opportunity for sus-
tainable reuse. Their composition makes them ideal substrates for microbial fermentation,
transforming waste into valuable resources and aligning with circular economic principles
in coffee production. Such innovations can mitigate environmental impacts while contribut-
ing to the sustainable development of the coffee industry. Fermentation is a critical stage
in coffee processing and shapes flavor and aroma profiles. Natural fermentation occurs
regardless of the post-harvest method used to obtain parchment coffee [9,10]. In wet or
submerged fermentation, the medium is excess water, while in dry or semi-dry methods, it
is the mucilage itself. Innovations in post-harvest operations include the introduction of
specific microorganisms, such as LAB and yeast, either alone or in combinations, sourced
externally or internally isolated from the same coffee process [11,12]. These microorganisms
are intentionally added to initiate fermentation, leading to shorter fermentation times and
desirable outcomes. These modifications aim to amplify desired flavor attributes while
mitigating undesirable ones, resulting in coffee with consistent flavors. These innovations
increase the marketability of specialty coffees, reinforcing the importance of fermentation
in the pursuit of coffee quality and differentiation, and are being studied in different steps
of the processes, as in the coffee processing per se [13,14], in the green coffee beans already
processed [15,16], and in the development of new beverages of brewed coffee [17–19].
LAB and yeast are significant components of the indigenous microbiota associated with
the submerged fermentation of de-pulped coffee cherries in wet processing [9,20]. During
mucilage fermentation, LAB produces organic acids, such as lactic and acetic acid, and
volatile compounds that significantly contribute to the flavor of the coffee profile [21]. These
organic acids enhance the acidity and overall complexity of the coffee. Additionally, LAB
contributes to developing desirable fruity and floral notes by producing esters and other
volatile compounds. LAB also plays a crucial role in reducing undesirable compounds that
may negatively affect coffee flavor by outcompeting spoilage microorganisms and lowering
the pH, thus creating a favorable environment for beneficial flavors [22,23]. Conversely,
yeast produces volatile metabolites, particularly aromatic esters, that can remain after
Sustainability 2025, 17, 1781 3 of 21

roasting and contribute to the fruity and floral attributes of the roasted coffee [2,20–25]. The
metabolic activities of yeast significantly influence the modulation of soluble carbohydrates,
organic acids, alcohols, volatile compounds, and other substances in the coffee pulp and
mucilage. This metabolic exchange alters flavor-related constituents in green coffee beans,
modifying the final roasted coffee flavor [2].
Previous studies on coffee fermentation were conducted with monocultures of vari-
ous commercial microorganisms, including fungi, yeasts, and LAB [12,26]. The favorable
biomodifications of coffee aroma by the tested microorganisms during green coffee bean
fermentation were mainly achieved through the direct production of fruity volatile com-
pounds by yeasts [25] and the acidification of green coffee beans by sugar-supplemented
LAB fermentation, which indirectly elevated the production of caramel-smelling furan
derivatives during roasting [16,22,27]. Also, not only have individual microbial applica-
tions taken place, but the synergistic interactions between yeast and LAB such as Pichia
fermentans and Pediococcus acidilactici have been reported to impact aroma complexity
significantly [23].
However, some methodologies used in these studies are not feasible at the farm scale,
as they often require sterilization of coffee or rely on advanced technology and specialized
knowledge. While scientifically valid for understanding the effects of each inoculum,
this approach is impractical for smallholder coffee growers due to their particular char-
acteristics [28]. Additionally, the preparation of inoculums using specific microbiological
media presents a significant drawback. These media often demand controlled laboratory
environments and precise formulations, which are not easily replicable outside specialized
facilities. This further limits the applicability of these methods for farmers with limited
resources or access to advanced equipment.
Despite operating on a small scale, smallholder coffee farmers are vital to the indus-
try. They represent 95% of coffee farms, with 84% managing less than two hectares of
land [28,29]. Developing accessible, low-cost, and sustainable fermentation methods that
utilize coffee by-products can contribute to sustainability by empowering smallholder
farmers, promoting rural development, ensuring equitable access to innovation, and en-
couraging environmentally conscious coffee production. This study aimed to optimize the
use of coffee by-products in developing inoculum for semi-controlled fermentation with
commercial LAB and yeast at a smallholder scale. By focusing on accessible and replicable
methods, the study seeks to advance sustainable practices in coffee processing, reducing
waste, improving resource efficiency, and enhancing the livelihoods of smallholder farmers.
To achieve this, a three-step procedure was followed: (1) designing and optimizing mixture
proportions of CP and CWW for microbial viability variation, (2) applying these optimized
inoculums to coffee fermentation, and (3) evaluating the fermentation parameters and
sensory qualities of the final product.

2. Materials and Methods

A research roadmap was designed to ensure a replicable approach toward advancing
sustainable coffee processing practices. This structured approach is illustrated in Figure 1,
providing a clear overview of the study’s methodology.
 2. Materials and Methods
A research roadmap was designed to ensure a replicable approach toward advanc
Sustainability 2025, 17, 1781 4 of 21
sustainable coffee processing practices. This structured approach is illustrated in Fig
1, providing a clear overview of the study’s methodology.

Figure
Figure 1. Research 1. Research roadmap.
roadmap.

2.1. Coffee Beans

Freshly harvested coffee cherries (Coffea arabica var Colombia) were obtained at 1557 m
above sea level in Morales, Cauca, Colombia. During harvest, cherries were selected based
on health and maturity criteria; any green, overripe, or damaged cherries were discarded.

2.2. Coffee Pulp

The coffee pulp (CP) was collected immediately from the de-pulping machine during
a traditional wet process. It was washed to remove debris, stored in plastic bags, and frozen
until needed. The pulp was thawed, blended in a commercial blender with water at a 1:2
pulp-to-water ratio, and boiled for 5 min to stop spontaneous fermentation.

2.3. Coffee Wastewater

The coffee wastewater (CWW) was produced during the traditional wet fermentation
process, in which de-pulped coffee cherries were submerged in water at a 1:1 ratio and
allowed to ferment spontaneously for 24 h. Following fermentation, the coffee mass was
removed, and the CWW was immediately boiled for 5 min, cooled, and then frozen.

2.4. Microorganisms
Fresh commercial yeast Saccharomyces cerevisiae (Levapan, Cali, Colombia) and lac-
tic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus
commercial yogurt (Colacteos, Pasto, Colombia) were used for fermentation.

2.5. Analytical Methods

2.5.1. Microorganism Viability
Viable aerobic bacterial counts were obtained by enumerating LAB on de Man Rogosa
Sharpe agar and culturing yeast on potato dextrose agar (Scharlab, Barcelona, Spain). For
microbiological analysis, 1 mL samples from each CWW were diluted in a 10-fold series
with peptone water (Scharlab, Barcelona, Spain), ranging from 101 to 106 . Microbial counts
were determined using the standard spread plate method, with agar plates incubated at
37 ◦ C for 48 h for bacteria and 24 h for yeasts. Total viable bacterial counts were obtained
by counting the colony-forming units (CFU). The reported population data represents the
means of triplicate analyses.

2.5.2. pH
The pH of the inoculum and the coffee fermentation liquid was monitored by taking
samples from each at every sampling point according to AOAC method 981.12 [30]. The
Sustainability 2025, 17, 1781 5 of 21

pH of the brewed coffee was determined using a technique described by Mazzafera (1999),
where 2.25 g of ground coffee was mixed with 50 mL of deionized water heated to 80 ◦ C,
then cooled to room temperature before measuring the pH. The reported data represents
the mean values from triplicate analyses [31].

2.5.3. Soluble Solids

The total soluble solids (SS) of the inoculum and the coffee fermentation liquid were
monitored by taking samples from each at every sampling point using a digital and portable
refractometer (Atago, Tokyo, Japan), according to AOAC Official Method 932.12 [30].

2.5.4. Titratable Acidity

Titratable acidity (TA) was determined by mixing 1 mL of liquid media from the
inoculum, fermentation, and brewed coffee with distilled water. Acidity was titrated using
0.1 N sodium hydroxide with phenolphthalein as the indicator. The results reported are the
means of triplicate analyses with standard deviation, according to AOAC Official Method
942.15 [30], using lactic acid as the standard acid with a specified equivalence of 0.009.

2.6. Coffee Cup Quality Evaluation

Cup quality was evaluated by an external coffee taster certified as a Q-Arabica grader.
The evaluation was performed in triplicate as a blind assessment, with each sample assessed
three times by the same certified grader to ensure consistency. The coffee samples were
prepared following Specialty Coffee Association (SCA) protocols [32], roasted at 115.6 ◦ C
for 9–10 min until reaching a roast degree of 48–63 Agtron, and ground according to
SCA cupping protocols. For brewing, 150 mL of hot water (92–94 ◦ C) was poured over
8.25 ± 0.25 g of ground coffee, and the mixture steeped for 4 min. Sensory assessments
commenced at 65 ◦ C for olfactory evaluation and 43 ◦ C for gustatory evaluation. Attributes
assessed included aroma, taste, acidity, body, balance, aftertaste, and overall quality, each
rated on a scale from 0 to 10 in 0.25 increments. A descriptive analysis and a total score, with
a maximum of 100 points, were assigned, covering fragrance/aroma, flavor, acidity, body,
uniformity, balance, sweetness, clean cup, and overall quality. To ensure an anonymous
review, samples were masked, and the grader was not informed of the experimental
treatments of each sample.

2.7. Experimental Methods

2.7.1. Bacteria and Yeast Inoculums Optimized Production
The experiments utilized a mixture design, part of the response surface methodology
class. Mixtures were defined by varying the percentages of coffee pulp (CP) and coffee
wastewater (CWW) in experimental units of 100 mL with 5% (v/v) inoculation of LAB
and 1% (w/v) yeast. The optimization of the mixtures was based on the relative microbial
growth (RG) of CFU/mL from the initial inoculation to the maximum CFU/mL during
the 48 h fermentation, with measurements taken at 0, 12, 18, 24, 36, and 48 h. SS, TA,
and pH were also monitored during the 48 h inoculum fermentation. An analysis of
variance (ANOVA) was performed to determine the effect of these parameters on inoculum
generation during the process variable levels of each microorganism. Statistical analyses
were performed with MINITAB version 19.
Relative microbial growth (RG) was quantified as the maximum increase in microbial
viability and the time required to achieve it, as shown in Equation (1):

(CFUti − CFUt0 )
RG = (1)
CFUt0
Sustainability 2025, 17, 1781 6 of 21

where CFUti represents the highest viable count reached by each microorganism during
the experiment at time ti, and CFUt0 is the viable count at the initial time point.
The experiments were conducted in a randomized order to reduce systematic errors.
As indicated in Equation (2), the sum of the proportions equals one.

k
∑ Xi = 10 ≤ Xi ≤ 1 i = 1 . . . k (2)
i =1

where Xi stands for the proportions of the ith component of the mixture that represents the
total number of components.
This study employed a quadratic regression model, as shown in Equation (3), to
analyze the relationship between the dependent variable ŷ and the independent variables
Xi and Xj:
k k −1 k
ŷ = ∑ βiXi + ∑ ∑ βijXiXj (3)
i =1 i =1 j = i +1

where ŷ is the predicted dependent variable, Xi (CP) and Xj (CWW) represent independent
variables; βi and βij denote the linear and quadratic coefficients, respectively. The first
term captures the linear effects of the variables, while the second term accounts for the
interactions between variable pairs, capturing quadratic effects.
Table 1 presents the proportions of components, the experimental conditions of the
mixture design, the response, and the process variables. The experiments consisted of 18
runs, including cubic, central, and axial points. The proportions of the components were
evaluated for both yeast and LAB. Additionally, different growth times were assessed for
each microorganism: 36 and 48 h for LAB and 12 and 36 h for yeast.

Table 1. Component and factor levels used in the mixture design of the experiment.

Components (%) Process Variable Time (h)

Run Response Variable
CP CWW LAB Yeast
1 25 75 36 12
2 25 75 48 36
3 50 50 36 12
4 50 50 48 36
5 75 25 48 36
6 50 50 36 12
7 100 0 48 36
8 50 50 48 36
9 50 50 48 36
Relative Microbial Growth (RG)
10 50 50 36 12
11 0 100 48 36
12 0 100 36 12
13 75 25 36 12
14 100 0 36 12
15 50 50 48 36
16 50 50 36 12
17 50 50 48 36
18 50 50 36 12

An ANOVA was performed to assess the adequacy and significance of the model.
The statistical significance of the model and its equation terms were evaluated based on
the p-value (p < 0.05). Statistical analyses were carried out using MINITAB version 19.
The quality of the model was evaluated by computing the coefficient of determination
(R2 ) to determine the proportion of variance in the dependent variable explained by the
independent variables, as shown in Equation (4). The adjusted R2 to measure the variance
Sustainability 2025, 17, 1781 7 of 21

between model predictions (ŷ) and experimental data (y) as described in Equation (5).
Values close to one indicate strong agreement between experimental and simulated data.
The root mean square error (RMSE) was calculated to evaluate the predictive accuracy of the
model using Equation (6), where lower RMSE values suggest higher model precision [33].

2
∑in=1 (yi − ŷi )
R2 = 1 − 2
(4)
∑in=1 (yi − y)

1 − R2 ( n − 1)


2
Adjusted R = 1 − (5)
n−P−1
s
ˆ 2
∑in=1 yi − yi


RMSE = (6)
n
where yi represents the experimental data, yiˆ is the model prediction, y the experimental
data, n is the number of observations, and P is the number of predictors. SS, TA, and pH
were also monitored during the 48 h inoculum fermentation. An ANOVA was performed
to determine the effect of these parameters on inoculum generation during the process
variable levels of each microorganism. Statistical analyses were performed with MINITAB
version 19.

2.7.2. Optimization
A response optimizer was used to find the optimal mixture using the desirability
compound, aiming to maximize the RG as the response variable. All variables were equally
weighted. The selection was based on response variable models explaining over 70% of
behavior (R2 adj. > 0.7).

2.7.3. Experimental Validation

Validation experiments were performed by reproducing inoculum production under
optimal conditions. The observed responses were compared to the optimal predictions
from the optimization, using relative error (RE) as shown in Equation (7):

y − ŷ
RE = ∗ 100% (7)
y

The optimized procedure and control fermentation were performed three times, with
three samples per replicate.

2.8. Coffee Processing with Optimized Inoculums

The coffee processing was conducted using the wet method. Freshly harvested Coffea
arabica cherries were de-pulped using a DH-2 depulper (Penagos, Colombia), and the
resulting beans were mixed with potable water to ferment and remove the mucilage. The
laboratory-scale setup consisted of 1 L flasks containing 800 g of de-pulped beans and
400 mL of water.
LAB and yeast inoculums were prepared according to the optimized conditions ob-
tained in the Section 2.7.2. prior to coffee processing. The following six fermentation
treatments were applied with 6% (v/w) of the respective inoculum: (1) Direct LAB inoc-
ulation sourced from commercial yogurt without prior optimization; (2) Optimized LAB
inoculum prepared under optimized growth conditions; (3) Fresh yeast inoculation without
prior optimization; (4) Optimized yeast inoculum prepared under optimized growth con-
ditions; (5) Mixture of optimized LAB and yeast inoculum; (6) Control treatment without
microbial inoculation.
Sustainability 2025, 17, 1781 8 of 21

All coffee processing was conducted at room temperature without sterilization of

the materials to mimic real-world coffee farm conditions. The fermentation vessels were
left undisturbed for the process, allowing for the natural degradation of mucilage. After
fermentation, the coffee beans were removed from the vessels, washed with potable water,
and dried at 40 ◦ C for 24 h until the beans reached a moisture content of 10–12%.
A principal component analysis (PCA) was used to evaluate both physicochemical
and sensory attributes across the different coffee samples. The sensory data included
fragrance/aroma, body, flavor, uniformity, and overall score, while the physicochemical
parameters were assessed both at the initial (I) and end (E) stages.

3. Results
The results are structured into four sections. Section 3.1 presents the monitoring of key
fermentation parameters such as pH, SS, TA, and RG during the inoculum fermentation
process. Section 3.2 focuses on the statistical analysis of the development of the inoculum,
Sustainability 2024, 16, x FOR PEER REVIEW 8 of 21
providing results on RG, statistical models, and further analysis of the interactions between
coffee by-products and RG. Section 3.3 addresses the optimization of the results using the
desirability compound and the chosen model to maximize RG, along with the validation of
using the desirability compound and the chosen model to maximize RG, along with the
the optimized inoculum, comparing the predicted and observed outcomes to evaluate the
validation of the optimized inoculum, comparing the predicted and observed outcomes
accuracy of the optimization process. Finally, Section 3.4 evaluates the performance of the
to evaluate the accuracy of the optimization process. Finally, Section 3.4 evaluates the per-
optimizedofinoculum
formance during
the optimized coffee fermentation,
inoculum during coffeeemphasizing fermentation
fermentation, emphasizingparameters
fermenta-
and the
tion sensory evaluation
parameters of theevaluation
and the sensory final product.
of the final product.
3.1. Dynamic Monitoring of Inoculum Fermentation Parameters
3.1. Dynamic Monitoring of Inoculum Fermentation Parameters
The mixture
The mixture design
designof ofexperiments
experiments(DOE)
(DOE)forforboth
bothmicroorganisms
microorganisms included
includedeighteen
eight-
experimental runs in five formulations, each varying by 25%. The initial physicochemical
een experimental runs in five formulations, each varying by 25%. The initial physicochem-
parameters
ical parametersof CP andand
of CP CWW CWW used
usedasascarbon
carbonsources
sourcesininthe
theinoculum
inoculum were
were as follows:
follows:
pH 4.24 and 3.81±±0.06,
and 3.81 0.06,TA TA0.46
0.46 ± 0.02 and 0.65 ± 0.02, and SS 3.20 ± 0.33 ◦ Brix and
± 0.02 and 0.65 ± 0.02, and SS 3.20 ± 0.33 °Brix and 4.91 ±
0.33
4.91 ±°Brix, ◦ Brix, respectively.
0.33respectively. Figure 2Figure
shows2the growth
shows the of LAB and
growth yeastand
of LAB across theacross
yeast different
the
formulations. The initialThe
different formulations. microbial viability among
initial microbial formulations
viability varied between
among formulations variedvalues
betweenof
1.4 ± 0.1 × 10 6 CFU/mL for 6 LAB and 5.8 ± 0.4 × 10 7 CFU/mL for7yeast.
values of 1.4 ± 0.1 × 10 CFU/mL for LAB and 5.8 ± 0.4 × 10 CFU/mL for yeast.

Figure
Figure 2.
2. Lactic
Lactic acid
acid bacteria
bacteria (a)
(a) and
and yeast
yeast (b)
(b) growth
growth inin different formulations of
different formulations of inoculum.
inoculum. Labels
Labels
represent the percentage
represent the percentageofofeach
eachcomponent
componentininthe
thetotal
totalinoculum:
inoculum:CP CP(coffee
(coffeepulp)
pulp) and
and CWW
CWW (cof-
(coffee
fee wastewater). Data are reported as mean ± SD from measurement replicates, with experimental
wastewater). Data are reported as mean ± SD from measurement replicates, with experimental
replicates based on the DOE.
replicates based on the DOE.

The growth of each microorganism exhibited opposite trends. LAB viability was pri-
The growth of each microorganism exhibited opposite trends. LAB viability was pri-
marily influenced by the proportion of CP in the formulations, reaching 7.8 × 1077 CFU/mL
marily influenced by the proportion of CP in the formulations, reaching 7.8 × 10 CFU/mL
after 48 h of incubation in CP100CWW0. In contrast, yeast viability was driven by the
percentage of CWW, peaking at 8.3 × 108 CFU/mL after 36 h of incubation in CP0CWW100.
Figure 3 illustrates the fermentative parameters SS, pH, and TA for the different for-
mulations, highlighting the dynamic changes observed during fermentation for both in-
oculums. A consistent decline in SS was observed throughout the 48 h fermentation period
in all formulations. Initial SS values ranged from 2.2 to 4.6 °Brix, influenced by the pro-
Sustainability 2025, 17, 1781 9 of 21

after 48 h of incubation in CP100CWW0. In contrast, yeast viability was driven by the

percentage of CWW, peaking at 8.3 × 108 CFU/mL after 36 h of incubation in CP0CWW100.
Figure 3 illustrates the fermentative parameters SS, pH, and TA for the different
formulations, highlighting the dynamic changes observed during fermentation for both
inoculums. A consistent decline in SS was observed throughout the 48 h fermentation
period in all formulations. Initial SS values ranged from 2.2 to 4.6 ◦ Brix, influenced by the
proportions of CP and CWW in each formulation. As fermentation progressed, SS levels
gradually decreased, with the most significant drop observed in the yeast-inoculated
blends after 12 h, indicating sustained sugar consumption by the yeast formulations
CP0CWW100 with yeast and CP100CWW0 with LAB exhibited the sharpest declines,
suggesting higher sugar availability and consumption by the inoculated microorganisms
than initially measured.
The pH levels exhibited slight fluctuations during fermentation. In LAB-inoculated
formulations, particularly CP100CWW0 and CP75CWW25, pH values dropped after 24 h,
which is consistent with the production of organic acids as LAB metabolizes sugars and
growth. In contrast, yeast-inoculated formulations, such as CP0CWW100 and CP25CWW75,
Sustainability 2024, 16, x FOR PEER REVIEW 9 of 21
maintained stable pH levels or showed slight increases over time, indicating that yeast
metabolism results in less acid production compared to LAB.

% Total acidity

Figure 3. Fermentative parameters of lactic acid bacteria (a1–a3) and yeast (b1–b3) in different
Figure 3. Fermentative parameters of lactic acid bacteria (a1–a3) and yeast (b1–b3) in different inoc-
inoculum formulations: total soluble solids (SST) (1), pH (2), and titratable acidity (TA) (3). Labels
ulum formulations: total soluble solids (SST) (1), pH (2), and titratable acidity (TA) (3). Labels rep-
represent
resent thethe percentage
percentage of of each
each componentininthe
component thetotal
totalinoculum:
inoculum:CP
CP(coffee
(coffee pulp)
pulp) and
and CWW
CWW (coffee
(coffee
wastewater). Data are reported as mean ± SD from measurement replicates, with experimental
wastewater). Data are reported as mean ± SD from measurement replicates, with experimental rep-
replicates based
licates based on on
thethe DOE.
DOE.

TA
Theincreased
pH levelssignificantly in LAB-inoculated
exhibited slight formulations
fluctuations during over theIn
fermentation. 48LAB-inoculated
h fermentation
period, especially
formulations, in CP100CWW0
particularly CP100CWW0 and CP75CWW25.
and CP75CWW25,The sharp increase
pH values in TAafter
dropped between
24 h,
36 and 48 h aligns
which is consistent with active acid production by LAB, as seen in the corresponding
the production of organic acids as LAB metabolizes sugars and
growth. inInpH.
decrease contrast, yeast-inoculated
On the other formulations,
hand, yeast-inoculated such asexhibited
formulations CP0CWW100 minimaland
or
CP25CWW75,
stable changes maintained
in TA, with stable pH levelseven
CP0CWW100 or showed
showingslight increases
a slight over time,
reduction. This indicat-
further
ing that yeast
supports metabolism
the observation results
that yeastin less acid production
fermentation compared
produces lower to LAB.
amounts of acid compared
TA
to LAB. increased significantly in LAB-inoculated formulations over the 48 h fermenta-
tion period, especially in CP100CWW0 and CP75CWW25. The sharp increase in TA be-
tween 36 and 48 h aligns with active acid production by LAB, as seen in the corresponding
decrease in pH. On the other hand, yeast-inoculated formulations exhibited minimal or
stable changes in TA, with CP0CWW100 even showing a slight reduction. This further
supports the observation that yeast fermentation produces lower amounts of acid com-
pared to LAB.
Sustainability 2025, 17, 1781 10 of 21

3.2. Statistical Analysis

The independent and dependent variables were fitted to the various models proposed
in the experimental design. Table 2 summarizes the observed and predicted responses
for both microorganism inoculums based on the results of the mixture design experiment.
The linear predictive models for LAB and yeast are shown in Equations (8) and (9), respec-
tively, while the quadratic predictive models for these microorganisms are presented in
Equations (10) and (11).
The quadratic model demonstrated a better fit for the LAB and yeast inoculum, as
evidenced by statistical values. For the LAB inoculum, the quadratic model resulted in
an R2 of 0.84, an adjusted R2 of 0.77, and an RMSE of 4.71. Similarly, the quadratic model
yielded an R2 of 0.88, an adjusted R2 of 0.83, and an RMSE of 0.76 for the yeast inoculum.
These values indicate a reasonable fit between the predicted and observed data, especially
for the yeast inoculum.

Table 2. Experimental results of the maximum increment in microbial viability of lactic acid bacteria
(LAB) and yeast across different inoculum formulations, along with the model quality results for
both microorganisms.

Response Variable
Components Relative Growth (RG)

Exp. Process Process

Number Variable LAB Variable Yeast
(n) X1 X2 X3 X3
CP CWW Time Linear Quadratic Time Linear Quadratic
Observed Observed
(%) (%) (h) Model Model (h) Model Model
1 25 75 36 34.33 24.23 24.68 12 5.32 5.46 5.50
2 25 75 48 44.30 42.22 42.33 36 11.52 10.19 10.12
3 50 50 36 34.06 30.65 35.16 12 6.88 5.89 6.31
4 50 50 48 51.12 46.13 47.15 36 9.18 8.49 7.80
5 75 25 48 45.87 50.03 50.14 36 6.87 6.80 6.73
6 50 50 36 28.40 30.65 35.16 12 6.38 5.89 6.31
7 100 0 48 53.14 53.94 51.29 36 7.03 5.10 6.91
8 50 50 48 38.06 46.13 47.15 36 7.17 8.49 7.80
9 50 50 48 42.71 46.13 47.15 36 8.30 8.49 7.80
10 50 50 36 35.38 30.65 35.16 12 6.97 5.89 6.31
11 0 100 48 34.40 38.32 35.67 36 13.19 11.89 13.70
12 0 100 36 1.92 17.80 6.06 12 3.85 5.03 3.93
13 75 25 36 33.34 37.07 37.52 12 5.16 6.32 6.36
14 100 0 36 34.53 43.49 31.76 12 6.07 6.74 5.64
15 50 50 48 53.09 46.13 47.15 36 6.44 8.49 7.80
16 50 50 36 37.80 30.65 35.16 12 6.71 5.89 6.31
17 50 50 48 52.48 46.13 47.15 36 6.75 8.49 7.80
18 50 50 36 36.07 30.65 35.16 12 6.05 5.89 6.31
R2 0.6736 0.8364 0.7321 0.8797
Adj-R2 0.6036 0.7682 0.6747 0.8295
RMSE 6.6516 4.7093 1.1311 0.7598
R2 : Coefficient of determination; Adj-R2 : adjusted R2 to measure the variance between model predictions and
experimental data; RMSE: root mean square error.

Linear model,

RGLAB = 12.163 X1 − 43.744 X2 + 0.870 X3 X1 + 1.710 X3 X2 (8)

RGyeast = 7.563 X1 + 1.609 X2 − 0.068 X3 X1 + 0.285 X3 X2 (9)

Quadratic model
Sustainability 2025, 17, 1781 11 of 21

RGLAB = −26.848 X1 − 82.755 X2 + 216.060 X1 X2 + 1.628 X3 X1 + 2.467 X3 X2 − 4.196 X1 X2 X3 (10)

RGyeast = 5.002 X1 − 0.952 X2 + 14.185 X1 X2 + 0.053 X3 X1 + 0.407 X3 X2 − 0.672 X1 X2 X3 (11)

where RG represents relative microbial growth; X1 and X2 are independent variables CP

and CWW, respectively; and X3 is process variable.
The ANOVA of quadratic models revealed significant effects of the concentrations of
CWW, CP, and time on the viability of the microorganisms. Both CWW and CP concen-
trations, along with incubation time, were statistically significant components and factors
for both inoculums. LAB viability increased with higher time values and was significantly
influenced by the proportion of CP in the mixture (p = 0.025). Similarly, yeast viability
increased with higher time values but was primarily driven by the percentage of CWW
(p = 0.00) (Supplementary Materials Table S1).

3.3. Optimization and Validation

The response variable was optimized using the desirability function to maximize
microbial growth for both the LAB and yeast inoculums. According to the model, the
highest RG for the LAB inoculum was predicted with 100% CP (CP100CWW0) and a
fermentation time of 48 h, yielding a predicted RG of 51.3, equivalent to a theoretical count
of 7.1 × 107 CFU/mL. For the yeast inoculum, the highest RG was predicted with 100%
CWW (CP0CWW100) and a fermentation time of 36 h, resulting in an RG of 13.7 and a
theoretical count of 8.5 × 108 CFU/mL.
Validation experiments were performed in triplicate under these optimized con-
ditions. At the end of the fermentation period, the experimental RG values for LAB
were 45.98 ± 4.30 (equivalent to 6.4 ± 0.6 × 107 CFU/mL), while for yeast, they were
12.14 ± 1.16 (equivalent to 7.6 ± 0.7 × 108 CFU/mL). These results confirmed the accuracy
of the model, as the experimental values closely matched the theoretical predictions. The
RE was 10% for LAB and 11% for yeast, demonstrating the reliability of the optimization
process, as shown in Table 3.

Table 3. Validation model for LAB and yeast inoculum.

Parameter Experimental Value Predicted Value RE (%)

RGlab 45.98 ± 4.30 51.3 10.35
RGyeast 12.14 ± 1.16 13.70 11.33
RE: relative error.

3.4. Performance Evaluation of Optimized Inoculum

The viability of LAB and yeast in coffee fermentation was evaluated using both direct
inoculation from commercial sources and optimized inoculums. The initial LAB count from
direct inoculation using commercial yogurt (fermentation 1) was 2.9 ± 0.5 × 106 CFU/mL,
while the optimized LAB inoculum (fermentation 2 and 5) had an initial count of
7.1 ± 0.7 × 107 CFU/mL. For yeast, direct inoculation using fresh yeast (fermentation 3)
showed an initial count of 3.5 ± 0.4 × 109 CFU/mL, whereas the optimized yeast inoculum
(fermentation 4 and 5) started with 8.2 ± 0.8 × 108 CFU/mL. The SS, pH, and TA were also
measured during fermentation, as detailed in Table 4.
Sustainability 2025, 17, 1781 12 of 21

Table 4. Experimental results of lactic acid bacteria (LAB) fermentation parameters and yeast-
inoculated coffee.

Fermentation SS (◦ Brix) pH TA (%)

Initial End Initial End Initial End
1 5.3 5.9 5.04 3.84 13.21 65.76
2 5.1 3.1 4.88 3.78 12.31 54.95
3 2.3 3.3 4.58 4.77 31.83 33.03
4 4.3 3.1 4.48 4.20 20.72 33.03
5 4.4 3.0 4.61 3.90 16.81 46.24
6 5.0 6.1 5.38 3.86 7.51 62.43
SS: soluble solids; TA: titratable acidity. 1. Direct LAB inoculation sourced from commercial yogurt without
prior optimization. 2. Optimized LAB inoculum prepared based on optimized growth conditions. 3. Fresh
yeast inoculation without prior optimization. 4. Optimized yeast inoculum prepared based on optimized
growth conditions. 5. Mixture of optimized LAB and yeast inoculum. 6. Control treatment without any
microbial inoculation.

Regarding pH, LAB-inoculated coffee (fermentations 1 and 2) showed a decrease,

indicating increased acidity, which is typical of LAB-driven fermentations. This aligns
with the expected production of organic acids by lactic acid bacteria. In contrast, yeast-
fermented coffee (fermentations 3 and 4) maintained stable pH levels or exhibited only
slight fluctuations, indicating lower acid production compared to LAB-fermented coffee.
Regarding TA, coffee inoculated with LAB (fermentations 1 and 2) demonstrated a signif-
icant increase in acidity during fermentation, consistent with the observed pH decrease.
Conversely, yeast-fermented samples (fermentations 3 and 4) exhibited more stable TA
levels, indicating lower acid production. The control treatment (fermentation 6) followed a
similar TA trend to yeast-fermented coffee. LAB-inoculated coffee (fermentations 1 and
2) showed a substantial increase in TA, correlating with the pronounced pH decrease,
confirming that LAB fermentation was characterized by elevated acid production from
the breakdown of carbohydrates into organic acids, driving the overall acidification of the
coffee. In contrast, TA levels in yeast-fermented coffee (fermentations 3 and 4) showed a
modest increase, indicating that yeast fermentation resulted in lower acid production. This
aligns with the more stable pH values observed, suggesting that yeast activity primarily
influenced other metabolic pathways, such as the production of flavor compounds, rather
than significant acid generation.
The inoculation of LAB and yeast on the sensory properties is shown in Table 5.
Regarding the sensory evaluation, variations in fragrance/aroma, flavor, aftertaste, and
acidity scores were recorded across the fermentation setups. Treatment 5, which involved a
mixture of optimized LAB and yeast inoculum, consistently scored highest in key sensory
attributes, such as fragrance/aroma (8.25), flavor (8), and overall score (8.25), indicating
a direct relationship between fermentation parameters and sensory outcome as the ob-
served increase in titratable acidity in treatment 5, which showed the highest final TA and
lowest pH.
Sustainability 2025, 17, 1781 13 of 21

Table 5. Sensory profile of lactic acid bacteria (LAB) fermentation parameters and yeast-inoculated coffee.

Fermentation
1 2 3 4 5 6
Characteristic Score
Fragrance/aroma 7.75 8 8 7.75 8.25 8
Flavor 8 8 8 7.75 8 8
Aftertaste 7.75 7.75 7.75 7.75 7.75 7.75
Acidity 7.75 7.75 7.75 8 7.75 7.75
Body 7.75 7.75 7.75 7.5 7.75 7.75
Uniformity 10 10 10 10 10 10
Sweetness 10 10 10 10 10 10
Clean cup 10 10 10 10 10 10
Balance 7.75 7.75 7.75 7.5 7.75 7.75
Overall 7.75 8 8 7.75 8 8
Final score 84.5 85 85 84 85.25 85
Fragrance of
tangerine with Fragrance of Fragrance of Fragrance of
walnut, aroma of Fragrance of honey with molasses and delicate mango
Fragrance of
almond and vanilla and maple hazelnut, aroma hazelnut, aroma with toasted
honey and
grapefruit, flavor syrup, aroma of of orange and of maple syrup hazelnut, aroma
molasses, aroma
of tangerine, red apple with maple syrup, with molasses, of orange with
of maple syrup
hazelnut, and honey, flavor of creamy flavor of flavor of honey, delicate
with anise, flavor
molasses, vanilla, butter, grapefruit, coriander seed, flavor of vanilla,
Sensory notes of lime, tangerine,
lingering and orange, tangerine, and celery, and molasses, and
and maple syrup,
honey-like molasses butter, honey molasses, herbal tropical fruits,
honey-like
aftertaste of aftertaste with aftertaste with and molasses caramel aftertaste
aftertaste, syrupy
sugarcane and tangerine, silky vanilla, creamy aftertaste, citrus with a subtle hint
body, juicy
walnut, creamy body, tangerine body, citrus honey-like acidity, of honey, silky
citrus acidity.
body, citrus citrus acidity. honey- light and body, bright
honey- like acidity. silky body. citrus acidity.
like acidity.
1. Direct LAB inoculation sourced from commercial yogurt without prior optimization. 2. Optimized LAB
inoculum prepared based on optimized growth conditions. 3. Fresh yeast inoculation without prior optimization.
4. Optimized yeast inoculum prepared based on optimized growth conditions. 5. Mixture of optimized LAB and
yeast inoculum. 6. Control treatment without any microbial inoculation.

Aftertaste showed little variation across treatments, suggesting that despite differences
in acidity, the aftertaste remained consistent. Similarly, body, or mouthfeel, exhibited slight
variation, with scores consistently between 7.5 and 7.75, indicating that while LAB and yeast
inoculum influenced acidity and flavor, their impact on body was minimal. Uniformity
maintained a perfect score of 10 across all setups, highlighting that the fermentation
processes were well-controlled and resulted in minimal sensory defects across samples,
regardless of physicochemical changes. Clean cup, which indicates the absence of off-
flavors or defects, remained constant at 10 for all fermentations. This stability implies
that the inoculum types did not introduce undesirable flavors, even in treatments with
higher acidity.
In contrast, balance and other key sensory characteristics such as fragrance/aroma and
flavor showed more variation, particularly in treatment 5. Balance scored 7.75 across most
treatments but increased to 8 in treatment 5, suggesting that the LAB and yeast inoculum
mixture in this treatment created a more harmonized sensory profile. This improvement
aligns with both physicochemical changes—especially higher acidity—and the enhanced
aroma and flavor scores, indicating a more cohesive sensory experience. The highest
fragrance/aroma score (8.25) was also observed in treatment 5, which had high TA and
a notably lower pH. This suggests that volatile organic acids and other by-products may
have contributed to enhanced aroma and flavor perception. Lower pH values in fermented
products are often linked to more complex profiles, as organic acids and other metabolites
contribute to sharper, more distinct tastes and aromas. The strong flavor profile of treatment
5 further supports this, indicating that the mixed inoculum enriched flavor complexity and
Sustainability 2025, 17, 1781 14 of 21

balance. Acidity also correlated with physicochemical changes; treatment 5 had a high
acidity score (7.75), corresponding to the higher TA (62.43%), suggesting that fermentation
increased both acid content and perceived acidity, enhancing the sensory experience.
Across all fermentations, certain sensory attributes appeared consistently. Notably,
citrus acidity with honey-like qualities was present in each sample. Fragrance and aroma
notes featured nutty undertones, such as hazelnut, walnut, and maple syrup. The body
of the coffee varied slightly from light and silky to syrupy, yet remained smooth across
treatments, suggesting minimal impact of inoculum variations on mouthfeel.
PCA was performed to visually assess the relationships between physicochemical
parameters and sensory attributes across the fermentation treatments (Figure 4). The
PCA biplot reduces data dimensionality, enabling the identification of14key
Sustainability 2024, 16, x FOR PEER REVIEW
contributors to
of 21
sensory variation and facilitating the interpretation of correlations between physicochemical
variables and sensory characteristics. The first two principal components (PC1 and PC2)
particularly evident
accounted for and
for 71.0% treatments
15.6%1,of2, the
3, 5,total
and 6, which cluster
variance, along PC1,effectively
respectively, showing capturing a
strong correlations with sensory attributes such as fragrance/aroma, body, flavor, uni-
significant portion of the variability in the data.
formity, and final score.

1.5
1
6
1.0
SS E
2
TA E
0.5
Flavor
Body
4
PC2 (15.6%)

Acidity Uniformity
0.0

pH E
-0.5
TA I Final score
Overall

-1.0 Fragance/aroma

-1.5
3
5
-2.0
-6 -5 -4 -3 -2 -1 0 1 2 3
PC1 (71.0%)

Figure 4. Biplot of the principal component analysis (PCA) of physicochemical and sensory attrib-
Figure 4. Biplot of the principal component analysis (PCA) of physicochemical and sensory attributes
utes across the different coffee samples. A total of 86.6% of the variance was described by the two
across the different coffee
principal components samples.
(PC). Dots A total
correspond of 86.6% of
to individual the variance
replicates was described
of 1. Direct by the two principal
LAB inoculation
sourced from commercial yogurt without prior optimization. 2. Optimized LAB inoculum prepared
based on optimized growth conditions. 3. Fresh yeast inoculation without prior optimization. 4.
components (PC). Dots correspond to individual replicates of 1. Direct LAB inoculation sourced
Optimized yeast inoculum prepared based on optimized growth conditions. 5. Mixture of opti-
from
mizedcommercial yogurt
LAB and yeast without
inoculum. priortreatment
6. Control optimization.
without 2.
anyOptimized LAB inoculum prepared based on
microbial inoculation.
optimized growth conditions. 3. Fresh yeast inoculation without prior optimization. 4. Optimized
4. Discussion
yeast inoculum prepared based on optimized growth conditions. 5. Mixture of optimized LAB and
yeastThis study focused
inoculum. on treatment
6. Control optimizingwithout
inoculumanyformulations by utilizing coffee pro-
microbial inoculation.
cessing by-products, specifically CP and CWW. Due to their large volumes, these by-prod-
The positioning
ucts present of the treatments
significant environmental in thefor
challenges PCA space
coffee farms.reveals distinctapprox-
CP constitutes profiles. PC1, which
imately 45% of the fresh coffee cherry, meaning that producing 1 ton of
aligns with variables such as final titratable acidity (TA(E)), soluble solids dried coffee beans (SS(E)), and
generates 3.64 tons of fresh CP. Additionally, wet processing methods produce 40–45 L of
overall
CWW per score, is positively
kilogram associated
of dried coffee, resultingwith higher
in around sensory scores.
40,000–45,000 L of CWWThisfor
trend
each is particularly
evident forcoffee
ton of dried treatments 1, 2,
beans [5]. 3, 5, and
Utilizing 6, by-products
these which cluster alongenvironmental
mitigates PC1, showing strong correlations
impact,
reduces
with waste, attributes
sensory and creates such
value-added products, therebybody,
as fragrance/aroma, improving theuniformity,
flavor, sustainabilityand final score.
and efficiency of the coffee production system.

4.
4.1.Discussion
Impact of Coffee By-Products on LAB and Yeast Growth in Different Inoculum
Formulations
This study focused on optimizing inoculum formulations by utilizing coffee processing
The optimization
by-products, of inoculum
specifically CP and formulations
CWW. Due demonstrated
to their the critical
large role of coffee
volumes, these by-products
by-products, such as CP and CWW, in enhancing microbial viability. The results con-
present significant environmental challenges for coffee farms. CP constitutes approximately
firmed that LAB, by producing organic acids, lowered the pH of fermentation media, cre-
45% of the freshenvironment
ating a favorable coffee cherry, meaningspoilage
for inhibiting that producing 1 ton of
microorganisms and dried coffeede-
enhancing beans generates
sirable flavor attributes [10,19,31]. For example, the significant pH reduction observed in
CP-rich formulations correlates with the higher viability of LAB, aligning with its known
role in producing organic acids. Similarly, yeast-driven formulations, particularly those
enriched with CWW, showed increased production of aromatic esters and modulated
Sustainability 2025, 17, 1781 15 of 21

3.64 tons of fresh CP. Additionally, wet processing methods produce 40–45 L of CWW
per kilogram of dried coffee, resulting in around 40,000–45,000 L of CWW for each ton
of dried coffee beans [5]. Utilizing these by-products mitigates environmental impact,
reduces waste, and creates value-added products, thereby improving the sustainability and
efficiency of the coffee production system.

4.1. Impact of Coffee By-Products on LAB and Yeast Growth in Different Inoculum Formulations
The optimization of inoculum formulations demonstrated the critical role of coffee
by-products, such as CP and CWW, in enhancing microbial viability. The results confirmed
that LAB, by producing organic acids, lowered the pH of fermentation media, creating a
favorable environment for inhibiting spoilage microorganisms and enhancing desirable
flavor attributes [10,19,31]. For example, the significant pH reduction observed in CP-rich
formulations correlates with the higher viability of LAB, aligning with its known role in
producing organic acids. Similarly, yeast-driven formulations, particularly those enriched
with CWW, showed increased production of aromatic esters and modulated essential
substances, including soluble carbohydrates and organic acids, as reflected in the enhanced
sensory complexity of treatments involving yeast inoculum [34,35].
The predictive model that optimized inoculum formulations performed well in pre-
dicting microbial viability, though minor deviations were observed between theoretical
predictions and experimental outcomes. These discrepancies could be attributed to unmod-
eled environmental factors, such as variations in initial microbial load or slight temperature
fluctuations during fermentation. Nevertheless, the model proved robust, optimizing
conditions for maximizing LAB and yeast viability.
The composition of coffee by-products significantly influences microbial growth dur-
ing fermentation. A higher percentage of CP favored LAB viability, while higher concen-
trations of CWW enhanced yeast growth. Formulations with balanced ratios of CP and
CWW, such as CP50CWW50 and CP25CWW75, supported moderate growth rates for LAB
and yeast. However, after 36 h, yeast viability declined, which is associated with nutrient
depletion as the SS decreased or the accumulation of metabolic by-products [18].
The concentration of SS reflects the availability of fermentable sugars, a critical fac-
tor for microbial growth. Yeasts, particularly Saccharomyces cerevisiae and Pichia species,
thrive in CWW-rich environments due to their higher sugar content, leading to a more
rapid decrease in SS than CP-dominant formulations. The ability of the yeast to quickly
consume sugars supports the production of volatile compounds, which contribute to the
fruity and floral aromas of the coffee. LAB, however, metabolizes sugars more slowly,
particularly complex carbohydrates, resulting in a more gradual decline in SS. This dif-
ference in sugar utilization between yeast and LAB reflects their complementary roles in
fermentation dynamics.
Changes in pH, SS, and TA during coffee fermentation are closely tied to the metabolic
activity of LAB and yeast. The decision not to adjust initial physicochemical parameters,
such as pH, SS, or TA, was made to replicate the natural fermentation conditions typically
encountered on smallholder coffee farms, where such variations are not controlled. This
allowed us to evaluate the performance of LAB and yeast in realistic scenarios, ensuring
the findings are directly applicable to practical, field-scale operations. This approach is
particularly relevant as smallholder farmers often lack the resources or infrastructure to
precisely adjust these parameters during fermentation. The fermentative metabolism of
LAB and the respiratory metabolism of yeast, especially under aerobic conditions, lead to
distinct patterns in these parameters across different formulations.
Differences in SS between the formulations reflected variations in sugar content, with
CWW providing a more abundant carbon source for yeast due to its higher concentration
Sustainability 2025, 17, 1781 16 of 21

of soluble sugars [6,36]. Changes in pH levels across different formulations correlated

with carbohydrate availability. Microbial activity led to the hydrolysis of polysaccharides
into monosaccharides, producing organic acids, which caused a decrease in pH [3,6,37],
particularly in CWW-rich formulations [37]. After 36 h, pH levels in all formulations begin
to rise, signaling reduced microbial activity due to nutrient depletion or the accumulation
of inhibitory by-products. This shift indicates a decline in fermentation efficiency as LAB
and yeast become less metabolically active.
TA, an indicator of organic acid production, shows a marked increase during the
initial stages of fermentation, especially in yeast-inoculated formulations with higher CWW
content. The rapid fermentation of sugars by yeast results in the accumulation of organic
acids such as succinic and malic acid, contributing to the acidic profile of the coffee [38].
In LAB formulations with higher CP content, TA rises more gradually as LAB ferments
complex carbohydrates over a more extended period [26]. This prolonged acidification
enhances the sensory complexity, contributing to a well-rounded flavor and aroma.

4.2. Impact of LAB and Yeast Inoculum in Coffee Fermentation and Sensory Profile
Fermentation 1, involving direct LAB inoculation, increased SS instead of the expected
decrease. This suggests that microbial activity or enzymatic breakdown of coffee mucilage
components may have released more soluble solids into the fermentation leachate, con-
tributing to the higher ◦ Brix values. This result contrasts with typical fermentation patterns,
where microorganisms consume sugars. Fermentation 2, which used an optimized LAB
inoculum, showed a significant decrease in SS, indicating that this inoculum was more
efficient at metabolizing sugars. This sharp reduction is consistent with the production
of organic acids and fermentation by-products, which lead to a notable drop in soluble
solids. The pre-adaptation of the LAB in the inoculum facilitated an active metabolism of
the carbon sources typical of coffee fermentation.
Fermentation 3, using fresh yeast without prior optimization, showed an increase in
SS similar to that of direct LAB fermentation, suggesting that the yeast did not efficiently
consume sugars and released additional soluble solids from the coffee beans. In fermenta-
tion 4, the yeast inoculum resulted in a moderate decrease in SS, indicating active sugar
consumption, albeit slower than LAB fermentations. This slower utilization suggests that
yeast ferments sugar into alcohol and other compounds more gradually. In fermentation 6
(the control), an unexpected increase in SS was observed due to passive release from the cof-
fee beans in the absence of inoculated microorganisms. This implies that natural processes,
such as leaching or slight spontaneous fermentation by native microorganisms, contributed
to the accumulation of this soluble solid without a deliberate fermentation process.
The mixed inoculum fermentation (fermentation 5) balanced the two trends, showing
a moderate increase in TA and a slight decrease in pH. This suggests that the combination of
LAB and yeast resulted in more controlled acid production. The yeast’s metabolic activities
tempered LAB acidification, leading to a more balanced flavor profile. The significant SS
decrease and the pH and TA increase in treatments with combined inoculations indicated
an ecological interaction between these microbial groups. The interaction between LAB
and yeasts also involves yeast autolysis, which releases essential nutrients such as amino
acids and polysaccharides that support LAB growth. At the same time, LAB acidification
creates an environment conducive to yeast fermentation [23,26]. The control fermentation
(fermentation 6), which lacked microbial inoculation, exhibited minimal changes in pH and
TA, reflecting limited natural fermentation activity. Despite the lack of deliberate inocula-
tion, fermentation 6 demonstrated a significant increase in TA due to natural spontaneous
fermentation by indigenous microorganisms. The elevated TA alongside relatively stable
Sustainability 2025, 17, 1781 17 of 21

pH indicates that acid production occurred, potentially driven by native lactic acid bacteria
or yeast in the environment.
LAB are known for enhancing acidity and flavor complexity in fermented foods,
including coffee by-products [39,40]. In our study, the behavior of LAB microbial growth re-
lated to pH reduction and increased TA, particularly when CP was used as a carbon source,
is similar to the findings of de Melo Pereira et al. (2015), where LAB fermentation also
resulted in similar pH shifts [40]. Additionally, the fermentation process led to considerable
sugar consumption, further supporting the active metabolism of LAB during the fermen-
tation of CP as a carbon source, which increased bacterial biomass. LAB species such as
Leuconostoc and Lactococcus produce volatile compounds such as diacetyl and acetoin, which
enhance flavor [21]. This aligns with previous work highlighting LAB’s role in producing
aroma-enhancing metabolites [41], which likely contributed to the sensory improvements
observed in our study. Although the metabolomic analysis of the resulting coffee was not
within the scope of this research, the relationship between specific metabolites produced by
LAB and the modulation of sensory profiles during coffee fermentation is consistent with
our results and other studies [21,39,41–43].
Yeasts, particularly Saccharomyces cerevisiae and Pichia species, also play a critical role
in the fermentation process by altering the carbohydrates, organic acid, and volatile com-
pound content of the coffee beans. They produce vital flavor-active compounds, such as
2,3-butanedione (buttery flavor), acetaldehyde (fruity flavor), and hexanal (green bean
flavor) [24,44]. Furthermore, S. cerevisiae contributes terpenes and esters derived from
amino acids, adding sweet, caramel, fruity, and floral characteristics to the final coffee
product [35,45]. Recent studies on mixed-culture fermentations involving LAB and yeasts
have demonstrated improved fermentation efficiency and enhanced volatile compound pro-
duction [23]. The combination of Pichia fermentans and Pediococcus acidilactici, for instance,
significantly increased lactic acid and ethanol production in ripe coffee beans, highlighting
the complementary roles of these microorganisms. Yeast activity, particularly in sugar
consumption, was more efficient with P. fermentans, especially in fructose utilization, com-
pared to LAB-only fermentations [23]. These observations are consistent with findings
from other fermentations, where LAB acidification and yeast activity jointly modulate
sensory outcomes. This ecological interaction leads to improved sensory attributes, such
as increased concentrations of ethanol (alcoholic notes), isoamyl alcohol (banana and pear
notes), and ethyl acetate (fruity notes), which were observed in spontaneous coffee fer-
mentations [26,46]. The balance of these microbial activities contributes to the overall
complexity and richness of the coffee’s flavor profile.
The sensory profile variations across different fermentation setups appear to be di-
rectly influenced by physicochemical changes occurring during fermentation. Specifically,
increased acidity and decreased pH in treatments, such as treatment 5. These findings sup-
port research showing that fermentation, especially with mixed LAB and yeast inoculum,
significantly alters the sensory profile of coffee by producing organic acids and metabolites
that enrich flavor complexity and aroma intensity [23,39,47]. Attributes such as body,
aftertaste, and clean cup were consistently maintained across treatments, indicating that
variations in fermentation conditions remained controlled and did not introduce negative
sensory defects. Treatment 5’s high TA and balanced acidity, paired with optimal aroma
scores, underscore how mixed inoculum fermentation can enhance sensory outcomes by
promoting beneficial by-products without compromising consistency [48,49].
Tailoring coffee flavor profiles through controlled fermentation offers substantial socio-
economic and environmental benefits. By leveraging microbial activity, smallholder produc-
ers can create stable cup profiles that meet commercial quality standards while appealing
to diverse consumer preferences. This adaptability supports long-term market viability
Sustainability 2025, 17, 1781 18 of 21

and provides a competitive advantage by enabling product differentiation. Furthermore,

this research integrates microbiology, food science, and socio-economic considerations [50],
contributing to a sustainable coffee production model that balances economic growth,
environmental stewardship, and social resilience.
In addition to socio-economic gains, controlled fermentation promotes resource effi-
ciency by utilizing local by-products such as CP and CWW. These practices reduce waste
and align with sustainable agricultural methods, allowing producers to enhance coffee
quality without compromising environmental responsibility. By refining fermentation tech-
niques, small-scale farmers can produce high-quality coffee tailored to market demands
while supporting environmental preservation and economic sustainability. This integration
of innovation and resourcefulness underscores the potential of microbial fermentation as a
transformative tool in coffee processing [51,52].
Although the overall final scores across treatments did not change significantly, this
consistency is a positive outcome, as it indicates that quality remains uncompromised
even as sensory profiles are modulated. The positive influence of LAB and yeast inoculum
on both physicochemical and sensory attributes is further evident in treatments 4 and 5,
which exhibited favorable parameters and scored highest in sensory qualities. These results
align with previous studies indicating that optimal fermentation conditions, particularly
through the synergistic effects of LAB and yeast, enhance desirable flavor and aroma
compounds [48].
Additionally, PCA results illustrate relationships between physicochemical and sensory
characteristics, showing how microbial activity shapes overall coffee quality [48,49,53,54].
Variables such as initial titratable acidity (TA(I)) and final pH (pH(E)) are more closely
aligned with sensory attributes related to acidity, indicating their influence on this spe-
cific sensory dimension. Treatments positioned in the lower-right quadrant of the biplot
(e.g., treatments 1, 2, and 6) exhibit higher overall sensory scores, suggesting a signifi-
cant influence of the final-stage physicochemical parameters (SS(E) and TA(E)) on sensory
outcomes. Interestingly, the contribution of PC2, while smaller, highlights variability not
fully explained by the evaluated physicochemical parameters. This suggests that achiev-
ing higher final scores and enhanced sensory attributes (e.g., fragrance/aroma) may also
depend on other variables not assessed in this study. For instance, treatments applying
inoculum developed with yeast or mixtures of LAB and yeast show strong associations
with improved sensory profiles, emphasizing the potential of these inoculums in enhancing
coffee quality.
These findings raise questions about additional physicochemical or biochemical factors
influencing sensory attributes, underscoring the need for further research to explore other
contributors to coffee quality beyond the parameters evaluated in this study. Carefully
controlled fermentation using LAB and yeast thus emerges as a promising method to
achieve specific sensory qualities, providing a foundation for future studies aimed at
optimizing fermentation to improve product development.

5. Conclusions
The optimized inoculum formulations enable the repurposing of coffee by-products,
such as coffee pulp (CP) and coffee wastewater (CWW), reducing waste while promoting
microbial production. By adjusting these formulations, microbial viability and sensory
enhancement were balanced—CWW concentrations boosted yeast growth, while CP con-
centrations supported LAB viability. These findings support large-scale applications in
coffee processing facilities, reinforcing sustainability, and delivering both environmental
and economic benefits. The optimized formulations balance the growth and viability of
LAB and yeast, offering a scalable solution that enhances process control while maintaining
Sustainability 2025, 17, 1781 19 of 21

the desired sensory quality of the final product. This research highlights the potential of
optimized microbial inoculums to enhance sensory attributes and promote sustainable
coffee fermentation practices, offering a scalable solution for smallholder farmers.

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

https://www.mdpi.com/article/10.3390/su17051781/s1.

Author Contributions: Conceptualization, L.-S.T.-V., M.-I.S.-T., and J.-L.P.-D.; methodology, L.-F.D.-B.

and K.-D.C.-G.; validation, L.-F.D.-B. and K.-D.C.-G.; formal analysis, L.-F.D.-B., K.-D.C.-G., and
J.-L.P.-D.; investigation, L.-F.D.-B. and K.-D.C.-G.; resources, L.-S.T.-V. and J.-L.P.-D.; writing—original
draft preparation, L.-F.D.-B. and K.-D.C.-G.; writing—review and editing, L.-S.T.-V. and J.-L.P.-D.;
visualization, L.-F.D.-B. and K.-D.C.-G.; supervision, L.-S.T.-V. and J.-L.P.-D.; project administration,
L.-F.D.-B.; funding acquisition, L.-S.T.-V., M.-I.S.-T., and J.-L.P.-D. All authors have read and agreed to
the published version of the manuscript.

Funding: This research was supported by the Ministerio de Ciencia, Tecnología e Innovación through
the Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación, Fondo Francisco
José de Caldas, and the Programa Orquídeas, Mujeres en la Ciencia: Agentes para la Paz (935-2023)
under funding contract 286-2023.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Materials; further inquiries can be directed to the corresponding author.

Conflicts of Interest: The authors declare no conflicts of interest.

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