From Waste to Taste: Coffee By-Products as Starter Cultures for Sustainable Fermentation and Improved Coffee Quality

Abstract

Utilizing coffee by-products in the fermentation process of coffee offers a sustainable strategy by repurposing agricultural waste and enhancing product quality. This study evaluates the effect of applying a starter culture, derived from coffee residues, on the dynamics of reducing and total sugars during coffee fermentation, as well as the composition of aromatic compounds, organic acids, and the sensory profile of coffee inoculated with yeast (Saccharomyces cerevisiae) and lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus), in comparison to a spontaneously fermented sample. Volatile compounds were identified and quantified using dynamic headspace gas chromatography-mass spectrometry (HS/GC-MS), with predominant detection of 2-furancarboxaldehyde, 5-methyl; 2-furanmethanol; and furfural—compounds associated with caramel, nut, and sweet aromas from the roasting process. A reduction in sugars (glucose, fructose, and sucrose) occurred over the 36 h fermentation period. Lactic acid (2.79 g/L) was the predominant organic acid, followed by acetic acid (0.69 g/L). The application of the inoculum improved the sensory quality of the coffee, achieving a score of 86.6 in evaluations by Q-graders, compared to 84 for the control sample. Additionally, descriptors such as red apple, honey, and citrus were prominent, contributing to a uniform and balanced flavor profile. These findings indicate that controlled fermentation with starter cultures derived from coffee by-products enhances sustainability in coffee production. It achieves this by supporting a circular economy, reducing reliance on chemical additives, and improving product quality. This approach aligns with sustainable development goals by promoting environmental stewardship, economic viability, and social well-being within the coffee industry.

1. Introduction

Coffee is a vital crop in many countries and is one of the most consumed beverages globally. In 2021/22, global coffee consumption reached an estimated 175.6 million bags, reflecting a 0.6% increase compared to the previous year [1]. The widespread interest in coffee stems largely from its diverse flavors and aromas. Specialty coffees, recognized for their unique sensory profiles, have gained prominence in recent years as appreciation for high-quality coffee has grown. These attributes depend on factors such as growing regions, coffee varieties, and post-harvest processes, including fermentation, drying, and roasting [2]. Fermentation plays a key role in producing high-quality beans. Microbial activity during this process decomposes the pulp surrounding the seed, generating metabolites that integrate into the beans [3]. Well-ripened coffee cherries are processed using wet, dry (natural), or semi-dry techniques [4].

The global coffee industry faces significant challenges in achieving environmental and social sustainability. Traditional production methods often consume large amounts of water, generate waste, and produce inconsistent product quality. These practices negatively affect the environment and local communities reliant on coffee cultivation. To address these challenges, stakeholders increasingly implement sustainable practices aimed at improving product quality while reducing environmental impacts. Controlled fermentation offers a pathway to improving sensory outcomes, optimizing resource use, and minimizing waste, thereby supporting a more sustainable and resilient coffee production system.

Coffee by-products, such as mucilage and pulp, contain sugars, including sucrose, glucose, and fructose, which serve as essential carbon sources for microbial metabolism during fermentation. These by-products also include significant quantities of proteins, organic acids, and minerals such as potassium, magnesium, and calcium, which collectively support microbial growth and activity [5]. Their high-water content facilitates enzymatic and microbial processes, making them suitable substrates for fermentation. By utilizing these by-products, producers transform agricultural waste into valuable resources, reduce environmental burdens, and enhance fermentation efficiency [6,7].

The adoption of sustainable practices aligns with the United Nations Sustainable Development Goals (SDGs), particularly SDG 12 (Responsible Consumption and Production) and SDG 15 (Life on Land). In many countries, producers rely on traditional fermentation with autochthonous microorganisms associated with coffee cherries. However, greater consistency can result from controlled fermentation with starter cultures. Researchers have demonstrated that specific microorganisms, such as yeasts, significantly influence the final quality of coffee. For example, [2] showed that P. fermentans YC5.2 promoted the synthesis of fumaric acid and volatile aromatic compounds, such as ethanol, acetaldehyde, ethyl acetate, and isoamyl acetate, during wet processing. This approach reduced lactic acid formation. Sensory analysis classified these samples as high-quality coffees with distinctive vanilla flavors and floral aromas. Similarly, Lactobacillus plantarum LPBR01 enhanced the acidity and fruity, floral sensory notes in coffee [2], while Lactobacillus rhamnosus HN001 produced profiles characterized by caramel and roasted attributes [8]. Fermentation with Leuconostoc mesenteroides CCMA 1105 and Lactobacillus plantarum CCMA 1065 developed volatile compounds, such as isovaleric acid, 2,3-butanediol, phenethyl alcohol, linalool, ethyl linoleate, and ethyl 2-hydroxypropanoate, which enhance sensory quality [9].

Starter cultures in controlled fermentation offer a valuable approach for achieving sustainable production goals in the coffee industry. These cultures improve consistency in sensory outcomes, reduce the reliance on chemical additives, and shorten processing times, contributing to environmentally responsible practices. Furthermore, controlled fermentation incorporates circular economy principles by converting coffee by-products, such as pulp and mucilage, into substrates for microbial growth. This process decreases waste generation, optimizes resource efficiency, and provides socio-economic benefits in regions where coffee farming serves as a primary livelihood, supporting poverty reduction and economic development (SDG 1 and SDG 8). Additionally, the microbial interactions involved in this method create distinct flavor profiles, which enhance the sensory and aromatic attributes of coffee while lowering the environmental impact of its production, aligning with sustainable product development and eco-design frameworks.

The application of starter cultures derived from coffee by-products in coffee fermentation remains underexplored. Therefore, the objective of this research was to investigate the use of starter cultures (a medium based on coffee byproducts with yeast Saccharomyces cerevisiae and lactic acid bacteria Lactobacillus delbrukei subsp. bulgaricus and Streptococcus thermophilus) and apply them in a semi-wet fermentation process of Castillo variety coffee. The study evaluates the behavior of reducing and total sugars, organic acids, aromatic profile, and sensory attributes in the cup, compared to a spontaneously fermented sample. This research introduces biotechnological innovations to traditional processes, contributing to the creation of sustainable food products while promoting economic, environmental, and social sustainability within the coffee industry.

2. Materials and Methods

The experimental methodology of this study is organized into five sections. Section 2.1 describes the preparation of the starter inoculum, including the formulation and cultivation of microorganisms using coffee by-products. Section 2.2 details the fermentation setup, covering the treatments applied, bioreactor configuration, and sampling schedule. Section 2.3 explains the post-fermentation processes, including drying, threshing, and roasting of the coffee samples. Section 2.4, Section 2.5 and Section 2.6 present the analytical procedures used to analyze sugars, organic acids, volatile compounds, and sensory attributes of the coffee samples. Finally, Section 2.7 integrates statistical methods to evaluate the data and ensure reliable results.

2.1. Preparation of the Starter Inoculum

In previous research [10], fermentation of pulped Castillo variety coffee was conducted using coffee by-products, specifically mucilage broth and coffee pulp, as substrates in a formulation optimized to enhance microbial activity and fermentation efficiency. The optimal composition determined included 74.54% coffee pulp, 18.23% mucilage broth, 2.81% yogurt, and 4.42% yeast. This sustainable approach illustrates a circular economy model by repurposing coffee waste into valuable resources for fermentation, thereby reducing environmental impact and enhancing the socio-economic value of coffee production. This formulation was applied in the current study, where bioreactors were configured in two setups: one with 10% optimized inoculum containing the starter cultures Saccharomyces cerevisiae (Fleischmann brand), Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus (Tapioka commercial yogurt, Cooperativa de Productos Lácteos de Nariño, Ltd.a., Nariño, Colombia), and another allowing spontaneous fermentation with endogenous microbiota over 36 h (time established as optimal in a previous research [10]). After fermentation, samples were dried at 40 °C for 48 h, threshed using a Lab Coffee Huller C-200, and roasted with an Aillio Bullet R1V2 roaster.

During fermentation, samples were collected at different times (0, 18, 24, and 36 h) for analysis of organic acids and total and reducing sugars by HPLC. The roasted samples were analyzed for aromatic profile using gas chromatography-mass spectrometry and cup profile by Q-graders.

2.2. Analysis of Carbohydrate Variation

Monomeric sugars were quantified using High-Performance Liquid Chromatography (HPLC). The Shimadzu UFLC Prominence chromatograph equipped with a RID-20A refractive index detector (Shimadzu, Kyoto, Japan) was employed for this analysis. Quantification was performed using the BioRad Aminex7 HPX87H column. The flow rate was set at 0.6 mL/min, and the oven temperature was maintained at 45 °C. Elution was carried out isocratically with a 0.005 mol/L sulfuric acid solution, following the protocol established by [11], which facilitates the separation and detection of sugars by refractive index.

2.3. Analysis of Organic Acid Variation by HPLC

The analyses were conducted using the methodology established by [12] with some modifications. High-Performance Liquid Chromatography (HPLC) on an Agilent 1100 UV/VIS system was used. Separation was achieved with the SUPERCOGEL H column. The mobile phase comprised a 0.1% HPO₄ solution. The flow rate was set to 0.5 mL/min, and the injection volume was 20 μL. The injector temperature was maintained at 25 °C, and the column temperature was set to 50 °C. Samples were injected directly into the chromatograph.

2.4. Roasting Process

This process was realized using the methodology reported by [13] with some modifications. Two hundred grams of sample was roasted using a roaster (Aillio Bullet R1V2, Copenhagen, Denmark) preheated to 190 °C. The first crack occurred at 8 min (160 °C), and the total roasting time was 10 min.

2.5. Analysis of Volatiles Compounds in Roasted Coffee

This analysis was realized following the protocol established by [14] roasted coffee sample was ground to a powder using a Kitchen Aid BCG111ER grinder. One gram of coffee powder was weighed into a 20 mL vial, which was sealed and preheated to 40 °C for 6 min. A solid phase microextraction (SPME) fiber (50/30 μm DVB/CAR/PDMS, SUPELCO, PA, Sigma-Aldrich, St. Louis, MI, USA) was then employed to extract volatile compounds for 20 min. The fiber was desorbed in the injection port of the gas chromatograph (Agilent 5973N) at 250 °C for 4 min. Separation of the desorbed volatile compounds was conducted using gas chromatography-mass spectrometry (Agilent 6890N GC-7000 Mass Triple Quad, Santa Clara, CA, USA) equipped with a capillary column (DB-WAX, 60 m × 0.25 mm × 0.25 μm, J&W Scientific, Folsom, CA, USA) and a quadrupole mass detector [15]. The injector operated in split mode with a 5:1 split ratio, utilizing helium as the carrier gas at a constant flow rate of 0.8 mL/min. The GC oven temperature was initially set to 32 °C for 10 min, then increased to 40 °C at a rate of 3 °C/min and held for 15 min. Subsequently, the temperature was raised to 160 °C at 3 °C/min, then to 230 °C at 4 °C/min, and maintained for 5 min. The mass spectrometer was configured in electron ionization mode, with the ion source temperature set to 230 °C and an ionization energy of 70 eV. Scanning mode was utilized with a mass range of 25 to 400 m/z. Data analysis was performed using Agilent Mass Hunter Qualitative Analysis B.04.00, and volatile compounds were identified by comparing mass spectra with NIST02.L, NIST5a.L, and NIST98.L libraries. The information was generated by the MSD ChemStation software, Version D.03.00. The LOD and LOQ were determined using a signal-to-noise ratio of 3:1 and 10:1, respectively.

2.6. Sensory Analysis with Certified Q-Grader Tasters

Sensory analysis was conducted to compare the sensory profiles of coffee produced through spontaneous and controlled fermentation. The coffee samples were prepared following protocols established by the Specialty Coffee Association [16]. Beans were roasted at 115 °C for 10 min, achieving a roast degree of approximately 48 to 63 on the Agtron scale, and then ground according to the SCA cupping protocol. Hot water (92–94 °C) was added to the ground coffee for 4 min. The total score for the coffee quality sensory test is out of 100 points, encompassing fragrance/aroma, flavor, acidity, body, uniformity, balance, sweetness, clean cup, and overall impression. A score above 80 classifies the coffee as excellent specialty coffee. Sensory analysis was performed by two certified Quality (Q)-Arabica graders from the Coffee Quality Institute (CQI) using five cups of each sample. The Q-graders identified relevant sensory attributes to describe the coffee, utilizing the SCA cupping form. The final score was calculated by summing the individual scores assigned to each attribute, representing the overall quality of the coffee. A higher final score indicates a better coffee quality classification, with scores exceeding 80.0 considered excellent specialty coffee.

2.7. Statistical Analysis

Results are presented as the mean ± standard deviation (SD) of independent triplicate analyses (n = 3). Statistical analysis was conducted using Minitab 19.0 Statistical Software (Minitab^®, Minitab Inc., State College, PA, USA). Significant differences between samples were evaluated using an independent two-sample t-test when necessary to compare the behavior of sugars and organic acids in two independent treatments (control and inoculum). Prior to the analysis, homogeneity of variances was verified at a significance level of p < 0.05.

3. Results and Discussion

The results are organized into four sections. Section 3.1 evaluates key fermentation parameters, including the changes in total and reducing sugars and organic acids, such as lactic and acetic acid, during the coffee fermentation process. The analysis compares treatments with and without starter cultures, demonstrating how the use of coffee by-products as fermentation substrates improves fermentation conditions and reduces dependency on chemical additives, contributing to sustainable production practices. Section 3.2 analyzes volatile compounds in roasted coffee, describing the methodology used to identify and compare the aromatic profiles of samples fermented with and without inoculum. Enhancing the aromatic profile through the use of by-products as fermentation substrates exemplifies the transformation of waste into high-value resources, producing coffee products that meet consumer demand for sustainable specialty goods. Section 3.3 presents the sensory evaluation of coffee samples, assessing attributes such as aroma, acidity, body, and overall flavor, and highlights the improvements in sensory characteristics linked to the application of starter cultures. Finally, Section 3.4 contextualizes these findings within global sustainability frameworks.

3.1. Monitoring of Fermentation Parameters

3.1.1. Analysis of Total and Reducing Sugars

Figure 1 shows an initial concentration of 0.3 g/100 g for fructose, which increases from 18 h to 24 h (15.8 g/100 g) and decreases again at 36 h (10.77 g/100 g). The initial increase likely results from sucrose hydrolysis [17], while the subsequent decrease can be attributed to the consumption of sucrose by microorganisms active during fermentation. Figure 1 and Figure 2 also show a decline in sucrose concentration throughout the fermentation period. This reduction reflects the metabolism of microorganisms involved in the process. These microbial transformations highlight a sustainable approach to processing, as naturally occurring sugars in coffee by-products serve as substrates, reducing reliance on external additives and aligning with circular economy principles. Similarly, this decrease coincides with an increase in lactic acid for both treatments (Figure 3 and Figure 4), which may indicate greater consumption of sucrose by lactic acid bacteria for growth on the substrate, leading to acid production. This behavior was also observed by [18] in coffee fermentation with the yeast Yarrowia lipolytica, who reported a significant decrease in the concentration of total sugars attributed mainly to the decrease in sucrose concentration which is a consequence of sucrose metabolism that is unique to the Y. lipolytica strain (NCYC 2904) used in that research.

In this research, a higher concentration of glucose was observed for both treatments (7.56 and 9.32 g/100 g) with a reduction (3.9 and 3.16 g/100 g) at the end of fermentation. Figure 2 illustrates a behavior similar to that reported by [19], where glucose was the most consumed sugar during the fermentation of pulped coffees, followed by a slower decrease in sucrose and fructose. The study indicates the presence of sugars even after 96 h of fermentation, with reductions of 72% in sucrose, 96% in glucose, and 95% in fructose when fermented with endogenous microorganisms. In this study, the controlled fermentation process lasted 36 h, resulting in a decrease in sugar concentrations comparable to other research, which indicates that approximately 60% of the sugars are consumed during the process [20].

In the treatment without inoculum, sucrose was predominantly hydrolyzed for 36 h, resulting in a 75.8% reduction, followed by a 48.4% decrease in glucose. Fructose was not completely consumed, and an increase was observed, primarily attributable to the activity of enzymes that break down complex compounds into simple sugars, such as the hydrolysis of sucrose into glucose and fructose. The ability of starter cultures to influence sugar breakdown and organic acid production highlights their role in creating a stable, high-quality product while aligning with sustainable food processing practices. In the inoculum treatment, a 66.1% decrease in glucose and a 16% reduction in sucrose were observed, along with an increase in fructose. This behavior coincides with that reported by [19], who investigated the impact of different fermentation methods on the quality of coffee beverages, specifically using the Catiguá MG2 variety, and also observed a decrease in sugars during a 96 h fermentation process. Studies [21,22] note that residual fructose at the end of fermentation is commonly observed. Low concentrations of sugars in coffee after fermentation may contribute to the sweetness of the beverage [21]. Likewise, the results obtained coincide with those reported by [22] who mentioned a decrease in glucose and sucrose during the fermentation time employed, with an average sucrose reduction of 51.67% at hour 32 of the process. The reduction in sucrose and glucose was also reported by Carvalho Ferreira [23], who emphasized that sucrose decreased significantly from the beginning of fermentation until 48 h in all treatments; in addition, the glucose content decreased significantly at 24 h. Fructose increased at 24 h in all fermentations, suggesting the modulation of microbiological activity by the starters; this behavior in fructose coincides with that observed in our treatments.

A relationship was established between the final value and the initial value to evaluate statistically significant differences in sugars during fermentation, as the initial sugar concentrations were not equal for both samples. A homogeneity of variances test was performed and then a t-test for two samples (control and inoculum). For sucrose, glucose and fructose at t = 36 h there were statistically significant differences (p = 0.003, p = 0.011 and p = 0.012, respectively). These findings demonstrate the potential of coffee by-products as functional substrates in controlled fermentation, supporting the goals of sustainable food science by reducing waste and enhancing the value of agricultural by-products. The mucilage surrounding the coffee beans is rich in sugars, mainly sucrose, glucose, and fructose. Microorganisms, including bacteria and yeasts, metabolize sugars into acids, alcohols, and other compounds, which are essential for developing the flavor profile of the coffee. The microbial diversity involved in the fermentation process is influenced by factors such as the variety of coffee and its natural microbiota, soil and climate conditions of the region, and post-harvest processing methods, among others [21,24]. For this reason, at the end of fermentation there was a greater presence of glucose and fructose (3.9 and 10.77 g/100 g) in the control sample compared to the sample with inoculum (3.16 and 2.51 g/100 g) due to the greater presence of lactic acid bacteria and yeasts responsible for the metabolic process.

3.1.2. Analysis of Organic Acids

Among the acids, citric and malic acids are naturally present in the coffee fruit, while others are more frequent after fermentation [21]. Figure 3 and Figure 4 show an increase in the concentration of the evaluated organic acids for both treatments (control and inoculum). This trend is primarily due to microorganisms decomposing simple sugars (glucose, fructose, sucrose) present in the mucilage surrounding the coffee beans during fermentation. For example, lactic acid bacteria ferment sugars to produce lactic acid as the final product. Other bacteria, such as acetic acid bacteria, oxidize ethanol (produced by yeasts) to form acetic acid [25].

Figure 3. Behavior of organic acids during the fermentation without inoculum (control).

Figure 3. Behavior of organic acids during the fermentation without inoculum (control).

Figure 4. Behavior of organic acids during the fermentation with inoculum.

Figure 4. Behavior of organic acids during the fermentation with inoculum.

This behavior was also observed by [26], who demonstrated that fermentations conducted in the presence of yeast increase the concentrations of alcohols, esters, aldehydes, glycerol, and organic acids such as acetic acid. Similarly, research by [27] showed that during fermentation, lactic acid was the predominant acid, reaching a maximum concentration of 2.33 g/kg. This was attributed to the presence of lactic acid bacteria of the genera Weissella, Leuconostoc, and Lactobacillus identified in the fermentation process. In this study, the concentration of lactic acid at 36 h of fermentation with inoculum was 2.79 g/L and without inoculum 5.78 g/L.

Lactic acid, which was the predominant acid in both treatments, can provide a milder, rounder flavor profile, often associated with milky or yogurt-like notes, potentially imparting creaminess to coffee [28]. This characteristic is desirable in sustainable coffee production, as it contributes to a high-quality sensory profile without relying on external flavoring agents. Its high production can be attributed to the high concentrations of sugar available in the fermentation processes, which are generated by lactic acid bacteria that metabolize hexoses and pentoses [29]. Lactic acid is a desirable component in coffee, and several studies report the use of lactic acid bacteria as inoculums in fermentation to improve product quality [30]. This acid is known to be one of the main and most abundant organic acids generated during coffee fermentation. The results obtained are similar to those reported by [3,22,31].

The analysis of organic acids during fermentation included a calculation of the ratio between the final and initial values for each sample, accounting for differences in initial acid concentrations. A homogeneity of variances test was performed and then a t-test for two samples (control and inoculum).

For lactic acid and malonic acid at t = 36 h there were statistically significant differences (p = 0.0005 and p = 0.004). On the other hand, for citric acid, tartaric acid and acetic acid there were no statistically significant differences (p = 0.126, p = 0.053 and p = 0.054).

Other acids such as acetic, tartaric, malonic and citric acids were detected in both treatments, although in much lower amounts compared to lactic acid. Acetic acid levels increased during fermentation time for the control sample from 0.1 to 1.22 g/L and for the inoculated sample from 0.1 to 0.69 g/L. Acetic acid is a natural metabolic compound that serves as an intermediate in plant metabolism, can be produced by ethanol oxidation under aerobic conditions or by acetogenic bacteria in anaerobic environments. Although excessive formation of acetic acid can be undesirable in certain products due to its sharp taste and vinegar-like aroma, in coffee, it can enhance sweetness in the flavor profile [32,33,34]. Regarding tartaric acid, a similar trend was reported by [23], who observed concentrations of 0.90 and 0.96 g/kg at 24 h and 32 h of coffee fermentation, respectively. In our study, the control fermentation, tartaric acid reached 0.46 g/L at 36 h and with inoculum 0.16 g/L. Tartaric acid produced during coffee fermentation can influence the flavor profile by contributing acidic and fruity notes, characteristics associated with grape or wine flavors [35]. Tartaric acid is a naturally derived acid from coffee fruits, and as presented in the study of Martinez [36], with inoculation during fermentation, its production increases and improves the flavor of the fruit. As was evidenced in the research, since it was higher in the inoculated sample with respect to the control.

Malic acid, a natural component of fruit, is associated with apple or green fruit flavors, enhancing the complexity of the coffee profile with fruity overtones. Bastian [37] points out that malic acid is a precursor of other acids, such as citric acid, which is consistent with what was observed in this study. The presence of citric acid, for example, is valued in coffee for imparting an acidic profile with notes reminiscent of citrus fruits such as lemon and orange. Citric acid is one of the most prominent acids in green coffee [35]. This compound showed a decrease in the control sample from 0.12 to 0.09 g/L while it remained constant at 0.25 g/L for the inoculum sample. Similarly, Bressani [38] observed a decrease in citric acid concentrations after 40 h of fermentation of Bourbon Amarelo coffee variety, the reduction can be attributed to the fact that citric acid can serve as a precursor of other acid decomposition products, such as glutaric, fumaric and maleic acids. Additionally, the microbiota present in coffee beans during natural fermentation can increase citric acid levels, resulting in coffee with elevated flavor scores, as evaluated by certified Q-graders using the international coffee quality standardization method [39].

3.2. Analysis of Volatile Compounds in Roasted Coffee

3.2.1. Roasting Process

Toasting experts recommend a duration of nine to twelve minutes for roasters with capacities ranging from 100 g to 12 kg. In this investigation, a light roast was performed using a roasting time of ten minutes, which falls within this recommended interval [40,41]. Figure 5 and Figure 6 show that during the initial phase, the loading temperatures were 140 °C and 145 °C, respectively. These lower temperatures prolong the roasting process, facilitating sugar caramelization, which enhances fruity flavors and increases the body of the coffee [40,42]. The final drying stage, observable in the graphs after five minutes, is marked by the color change in coffee beans from bluish green to yellow consistent with expert recommendations of four and a half to six minutes. At this stage, the emergence of distinct flavors and aromas begins, occurring at 148 °C for both fermentation types. The steam pressure exerted internally leads to the fracture of the bean, which in this study occurred at 161.8 °C at 8.3 min and 158.8 °C at 8.5 min, respectively, at which point the beans exhibit the characteristic color of coffee [41,43]. The final roasting stage targeted a medium roast to avoid the development of bitterness. The beans were then rapidly cooled within a maximum of five minutes to a temperature below 30 °C, preventing overcooking of the coffee.

In this study, a mild roasting profile preserved natural flavors, minimized bitterness, and enhanced flavor complexity in both coffee samples fermented with starter inoculum and those subjected to spontaneous fermentation. This roasting method resulted in a delicate flavor profile that emphasizes sweetness and floral, fruity, and almond notes [44,45,46], as shown in Figure 7 and Figure 8. The acidity is less intense but provides a good body that allows floral aromas to be appreciated. Coffee brewed with beans fermented using starter inoculum exhibited superior flavor, aroma, acidity, body, and uniformity, with a noticeable fruity aroma after roasting compared to coffee brewed with beans subjected to spontaneous fermentation [47]. This was corroborated by the rating of the Certified Taster.

3.2.2. Analysis of Volatiles Compounds in Roasted Coffee

The aroma of coffee is determined by a diverse combination of volatile compounds from various classes. Chromatograms of the two roasted coffee samples revealed similar volatile profiles, though with variations in quantity [48]. Coffee with inoculum presented 62 aromatic compounds, whereas the control exhibited 57. All volatile compounds identified in both samples are listed in Tables S1 and S2 (Supplementary Materials).

For both samples, the main compounds identified (Figure 7 and Figure 8) were 2-Furanmethanol, 2-Furancarboxaldehyde, 5-methylfurfural, and furfural. This can be attributed to the Maillard reaction, which initially produces Amadori products that degrade into sugar fragmentation products, followed by dehydration, fragmentation, cyclization, and polymerization reactions [49].

Figure 7. Chromatogram fermentation with inoculum.

Figure 7. Chromatogram fermentation with inoculum.

Figure 8. Chromatogram of coffee sample without inoculum.

Figure 8. Chromatogram of coffee sample without inoculum.

The generation of these compounds not only enhances the sensory quality of coffee but also reduces the need for artificial flavor enhancers, supporting environmentally responsible practices. The generation of furfural arises from Amadori rearrangement products, particularly from deoxyosones when the sugar is a pentose. It can also be produced by the oxidation of furfuryl alcohol, a product of the reaction between (deoxy)ribose or sucrose and cysteine/methionine [50]. Similarly, furanones are an important group of volatiles in coffee aroma. Specifically, 3(2H)-Furanone, dihydro-2-methyl contributes sweet and caramel notes as well as providing a fruity flavor profile; 2-Furanmethanol contributes sweet and fruity notes and is also associated with a caramel aroma, enhancing the overall sweetness of coffee. 2-Furancarboxaldehyde, 5-methylfurfural generates caramel and nutty aromas, while furfural contributes a nutty and caramel aroma [51]. These flavor enhancements achieved through controlled fermentation and roasting processes support the creation of high-value, sustainable coffee products that meet consumer demands for quality and environmental responsibility. These notes were described by the tasters in the sensory profile of the coffee (Section 3.3). The descriptors identified by the Q-graders are related to the volatile compounds in the chromatogram, for example, compounds such as pyridine have been related to nutty, woody, and bready flavors; furans to cooked caramel nuances; furanones to creamy, waxy flavors with a citrusy fruity undertone; and finally, pyrazines to roasted, toasted almond, hazelnut, peanut, and peanut notes [52].

Pyrroles, pyranones, and furanones, identified in both samples, are generated from the sugar fragmentation of deoxyosones with the additional action of reductions. When other amino acids participate in the reaction, the Strecker reaction of aldehydes with aminoketones, followed by heterocyclization, produces a series of aroma-active volatile compounds, such as pyridines, pyrazines, thiazoles, and pyrroles [53].

Sulfur-containing volatiles are largely responsible for the roasted aroma of coffee [53]. In this study, coffee with inoculum contained the sulfur-containing compounds para-Methoxybenzenethiol and furfuryl isothiocyanate. Para-Methoxybenzenethiol contains a sulfur atom (S) in the thiol group (-SH), and furfuryl isothiocyanate contains a sulfur atom (S) in the isothiocyanate group (−N=C=S). In contrast, coffee without inoculum contained the sulfur compound 1H-Cyclopentacccthiophene, hexahydro-, cis. This compound belongs to the thiophene family, which includes sulfur in its structure (the “thio” group indicates the presence of a sulfur atom). Sulfur compounds have low flash points and are highly susceptible to oxidative degradation, and they are generally present in very small concentrations, often less than 0.01% of the total volatile profile of roasted coffee. Despite their minute quantities, these compounds significantly influence the perception of freshness and contribute to the overall sensory experience of coffee, playing a fundamental role in the aroma and flavor characteristics of the roasted product [54]. Other significant compounds, such as furans, pyridines, and pyrroles, are not considered strong odorants due to their high threshold values in air. Furans contribute underlying burnt and caramel-like notes, while pyridines and pyrroles are associated with smoky and burnt coffee aromas, respectively [50,55].

Carboxylic acids such as acetic acid, butanoic acid, 2-butenoic acid, and 3-methyl- contribute to the acidity of coffee [56] in both samples. Pyrazines are the second most abundant volatile compounds present in coffee samples and contribute a roasted aroma and earthy notes to roasted ground coffee [56]. Pyrazines identified in both samples include methylpyrazine, characterized by a nutty odor; acetylpyrazine, which generates nutty and toasted aromas with a sweeter profile; and pyrazinamide, which provides earthy notes and a slight sweet profile.

On the other hand, one of the differences in the results is that the coffee without inoculum presented two ester compounds (2-Propenoic acid, butyl ester and butanoic acid, butyl ester). Although esters can be present in low concentrations, their production can greatly influence the flavor of coffee, generating sweet and fruity flavors, although their specific contribution to the sensory profile of coffee depends on their concentration and interactions with other compounds [37]. In the brewing industry, this group represents the largest and most important group of active flavor compounds [57]. They are produced mainly by acid catalysis and a decrease in pH and are mostly derived from yeasts [58].

3.3. Sensory Evaluation of Coffee Samples

The sensory attributes of sweetness, sourness, and bitterness are detected by taste buds distributed on the tongue. These attributes belong to the five fundamental tastes (sweet, bitter, salty, sour, and umami) identifiable by the human taste system and play a key role in food characterization. Sweetness is associated with carbohydrates present in grains, while sourness is linked to organic acids, which are naturally present in grains and can also be generated during fermentation processes. Bitterness is related to compounds such as caffeine and chlorogenic acids [59].

According to [60], trigonelline contributes to the overall aroma perception in brewed coffee and generates key volatile compounds such as pyridines and pyrroles during the roasting process. The attributes of body and astringency are perceived as tactile sensations through the oral somatosensory system and are related to non-volatile components of the beverage, such as fatty acids. The aftertaste attribute evaluates the duration of sensations perceived in the mouth after consuming the beverage. All these sensory attributes must be balanced; they must interact and complement each other in terms of intensity and quality to determine the final score. Achieving this balance is crucial for developing high-quality coffee products that meet consumer expectations and promote social sustainability through enhanced consumer satisfaction and loyalty. This score represents the sensory quality of the coffee beverage.

Table 1. Cup analysis for fermented coffee without inoculum.

Attribute	Score
Fragrance/Aroma	8.00 ± 0.35
Flavor	7.75 ± 0.0
Aftertaste	7.75 ± 0.35
Acidity	7.63 ± 0.18
Body	7.75 ± 0.35
Uniformity	8.75 ± 1.77
Sweetness	10.00 ± 0.0
Clean cup	10.00 ± 0.0
Balance	8.75 ± 1.77
Cupper Score	7.63 ± 0.18
Total Score	84 ± 1
Descriptors of the cup analysis	cocoa nibs, caramel, hazelnut, orange, high residual, medium acidity, round body, panela sweetness, uniform and balanced.

and

Table 2. Cup analysis for fermented coffee with inoculum.

Attribute	Score
Fragrance/Aroma	8.40 ± 0.20
Flavor	8.20 ± 0.20
Aftertaste	8.00 ± 0.00
Acidity	8.20 ± 0.20
Body	8.00 ± 0.00
Uniformity	9.00 ± 1.40
Sweetness	10.0 ± 0.00
Clean cup	10.0 ± 0.00
Balance	9.00 ± 1.40
Cupper Score	8.00 ± 0.00
Total Score	86.70 ± 0.20
Descriptors of the cup analysis	red apple, honey, citrus, blueberry, aromatic, long residual, medium acidity, silky body, honey sweetness, even and balanced.

present the cup analysis for coffee fermented without inoculum and for coffee fermented with inoculum, respectively.

The fragrance/aroma attribute received the highest score of 8.4 in the inoculated fermented coffee. The aromatic diversity of coffee arises from the interaction between factors inherent to the green bean (such as variety and origin) and the chemical transformations triggered by roasting. Growing conditions and processing methods influence the concentration and type of volatile precursor compounds, leading to a wide range of aromatic profiles [61]. Different fermentation processes (inoculated and spontaneous) result in varying aroma complexities within the same coffee variety (Castillo).

The fragrance/aroma attribute received the highest score of 8.4 in the inoculated fermented coffee. The aromatic diversity of coffee results from the interaction between factors inherent to the green bean (variety, origin) and the chemical transformations induced by roasting. Growing conditions and processing methods influence the concentration and type of volatile precursor compounds, leading to a wide range of aromatic profiles [61]. Different fermentation processes (inoculated and spontaneous) result in varying aroma complexities within the same coffee variety (Castillo). The flavor profile of coffee is shaped by the chemical transformations that occur during roasting. Precursor compounds in coffee beans convert into volatile and non-volatile compounds, which contribute to the unique sensory characteristics of the beverage. This process is essential for developing high-quality coffee [62].

Mouthfeel, or body, is a fundamental characteristic in the sensory evaluation of coffee. This property, often measured in terms of total solids and sometimes associated with fat content, significantly influences the overall perception of the beverage. It is crucial for a coffee’s sensory profile to maintain a proper balance between body and other attributes such as acidity, bitterness, and astringency to ensure a satisfying drinking experience [63].

A two-sample t-test was conducted to identify statistically significant differences between treatments. The analysis revealed that only the acidity and flavor parameters showed statistically significant differences (p = 0.04). These differences are attributed to the starter inoculum, which included Saccharomyces cerevisiae yeast. This yeast is known to enhance the cup score of coffee through the production of phenylethyl alcohol, benzyl alcohol, malic acid, and methyl salicylate. These compounds contribute to slightly acidic, floral, menthol, sweet, and fruity notes, which may explain the higher flavor score observed for the inoculated beverage [17,37]. Furthermore, previous studies have demonstrated that co-inoculation of lactic acid bacteria with yeast can produce desirable aroma compounds, leading to the development of specialty coffees [31,54].

Similar to this research, the study by [36] demonstrates that bacteria and yeasts can function as a consortium during coffee fruit fermentation. Therefore, the sequential inoculation of bacteria and yeasts through mixed starter cultures has enabled the production of specialty coffees, even though the variety is not traditionally considered specialty.

3.4. Limitations

Differences between batches may occur even when using the same coffee variety due to variations in the initial microbial load. To mitigate this, it is recommended to implement thorough cleaning and disinfection protocols for all containers, equipment, and the coffee itself before fermentation. Additionally, factors such as climatic conditions, soil quality, and the use of fertilizers may affect bean quality, potentially leading to variations in behavior and perceived flavor profiles.

Future investigations could explore the feasibility of incorporating inoculum into coffee husk, another by-product of hulling, to assess its utility in controlled fermentations. Expanding the application of the inoculum to other coffee varieties, such as Caturra, Geisha, Bourbon, and Catuaí, could provide insights into sensory profile variations. Furthermore, mixture experiments combining different yeasts and lactic acid bacteria at varying concentrations may offer a deeper understanding of their influence on sugar metabolism, organic acid production, volatile compound formation, and sensory attributes.

3.5. Integration of Sustainability Aspects and Alignment with Sustainable Development Goals (SDGs)

The analysis indicates that controlled fermentation with starter cultures could play an important role in advancing sustainability within coffee production. Optimizing fermentation parameters, such as regulating total and reducing sugars and maintaining a balance in organic acid production, improves resource efficiency and minimizes waste generation. Utilizing coffee by-products, including mucilage broth and coffee pulp, as fermentation substrates illustrates a circular economy approach, which could transform agricultural waste into valuable resources. This practice reduces the environmental impact of coffee production while adding socio-economic value, potentially benefiting communities engaged in coffee farming [5,64,65,66].

Enhancing volatile compound profiles through controlled fermentation and mild roasting processes enhances the quality of specialty coffee products, specialty coffee products [67]. By increasing the concentration of desirable aromatic compounds and improving sensory attributes, this approach aligns with consumer preferences for premium and sustainably produced goods. These improvements could support economic sustainability by enhancing product marketability and competitiveness, potentially raising income for coffee producers [19,68].

The integration of starter cultures and coffee by-products into controlled fermentation processes not only aligns with SDG 12 (Responsible Consumption and Production) and SDG 15 (Life on Land) but also contributes significantly to SDG 8 (Decent Work and Economic Growth) and SDG 1 (No Poverty). By creating value-added products from agricultural waste, this approach can enhance the income potential of coffee-producing communities, promoting inclusive economic growth and stable employment opportunities [66,67]. Furthermore, the reduction in waste management costs and the increased marketability of specialty coffee products can alleviate economic pressures on smallholder farmers, contributing to poverty reduction (SDG 1). These practices support sustainable economic systems by fostering innovation, resource efficiency, and equitable wealth distribution within coffee-growing regions [68].

The methodological approach of this research incorporates key sustainability principles, including eco-design and life cycle assessment (LCA), aiming to align each stage of the production process with sustainability goals [66]. Reducing chemical additives and optimizing fermentation conditions could help preserve the natural qualities of coffee while promoting environmentally responsible practices. Such practices are essential for advancing the SDGs [67], particularly those related to Responsible Consumption and Production (SDG 12), Industry, Innovation, and Infrastructure (SDG 9), and Decent Work and Economic Growth (SDG 8).

The study aligns with SDG 12 by supporting Responsible Consumption and Production through efficient resource use and waste reduction. Innovative fermentation techniques could stimulate industry innovation and sustainable infrastructure within the coffee sector, consistent with SDG 9. Furthermore, socio-economic benefits derived from improving product quality and marketability may contribute to SDG 8, which promotes sustained, inclusive economic growth, productive employment, and decent work for all [68].

Integrating starter cultures and utilizing coffee by-products in the fermentation process presents a viable pathway toward sustainable coffee production. These practices hold the potential to enhance coffee quality and marketability while contributing to broader sustainability objectives, including resource efficiency, waste reduction, and economic and social well-being in coffee-producing regions [10,69].

4. Conclusions

Inoculation with yeast (Saccharomyces cerevisiae) and lactic acid bacteria (Lactobacillus delbrukei subsp. bulgaricus and Streptococcus thermophilus) directly impacted the sensory quality of coffee. Both samples showed a reduction in evaluated sugars (glucose, fructose, and sucrose); however, it was more significant in the inoculated sample (3.16, 2.51, and 1.45 g/100 g for glucose, fructose, and sucrose, respectively). The analysis identified lactic acid, tartaric acid, citric acid, malonic acid, and acetic acid, which contributed to the aromas and flavors perceived by tasters. Volatile compounds in the roasted samples were predominantly 2-furancarboxaldehyde, 5-methyl-2-furanmethanol, and furfural, which are associated with sweet and caramelized aromas, such as caramel, roasted sugar, nutty, roasted, dried fruit, or almond aromas. Finally, the inoculated sample achieved a higher score (86.70 ± 0.20) with notes of red apple, honey, citrus, and blueberry compared to the control sample (84 ± 1) with notes of cocoa nibs, caramel, hazelnut, and orange, demonstrating a clear difference in sensory descriptors. This research contributes to sustainable development in coffee production by emphasizing the role of controlled fermentation and starter cultures, which are based on the reuse of coffee by-products, in establishing a more efficient production system with a reduced environmental impact. The findings support the integration of scientific methods into sustainable agriculture, promoting practices that align product quality with the sustainability objectives of the coffee industry and contribute to the broader sustainability agenda.

The alternative proposed in this research is particularly suited for small producers, as the starter cultures used are both accessible and practical. Coffee hulls, a primary by-product generated during the pulping process, and mead (the water used to wash the beans) serve as the main substrates. Fresh yeast (Saccharomyces cerevisiae) is widely available and can be stored under refrigeration, while yogurt containing microorganisms such as L. delbrueckii subsp. bulgaricus and S. thermophilus is also readily obtainable in the market. The inoculum formulation, detailed in previous research referenced in Section 3, has been shown in this study to significantly enhance the sensory profile of the coffee. The results demonstrate improved sugar and organic acid behavior, a more diverse volatile compound profile, and unique sensory descriptors compared to spontaneous fermentation (control), supporting its efficacy in controlled fermentation.

Future research could expand on these findings by exploring the potential of other microbial consortia in enhancing the sensory and nutritional quality of coffee. Investigating the influence of different coffee varieties and regional climates on fermentation outcomes could further refine controlled fermentation techniques. Moreover, scaling up the use of coffee by-products for microbial inoculum development offers opportunities to strengthen circular economy practices in the coffee industry.