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Article

Bioprocessing of Spent Coffee Grounds as a Sustainable Alternative for the Production of Bioactive Compounds

by
Karla A. Luna
1,
Cristóbal N. Aguilar
1,
Nathiely Ramírez-Guzmán
2,
Héctor A. Ruiz
3,
José Luis Martínez
1 and
Mónica L. Chávez-González
1,*
1
Bioprocesses and Bioproducts Research Group, Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280, Coahuila, Mexico
2
School of Biological Sciences, Universidad Autónoma de Coahuila, Unidad Torreón, Torreón 27276, Coahuila, Mexico
3
Biorefinery Group, Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280, Coahuila, Mexico
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(7), 366; https://doi.org/10.3390/fermentation11070366
Submission received: 25 April 2025 / Revised: 13 June 2025 / Accepted: 23 June 2025 / Published: 26 June 2025
(This article belongs to the Special Issue Valorization of Food Waste Using Solid-State Fermentation Technology)
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Abstract

Spent coffee grounds are the most abundant waste generated during the preparation of coffee beverages, amounting to 60 million tons per year worldwide. Excessive food waste production has become a global issue, emphasizing the need for waste valorization through the bioprocess of solid-state fermentation (SSF) for high added-value compounds. This work aims to identify the operational conditions for optimizing the solid-state fermentation process of spent coffee grounds to recover bioactive compounds (as polyphenols). An SSF process was performed using two filamentous fungi (Trichoderma harzianum and Rhizopus oryzae). An exploratory design based on the Hunter & Hunter method was applied to analyze the effects of key parameters such as inoculum size (spores/mL), humidity (%), and temperature (°C). Subsequently, a Box–Behnken experimental design was carried out to recovery of total polyphenols. DPPH, ABTS, and FRAP assays evaluated antioxidant activity. The maximum concentration of polyphenols was observed in treatment T3 (0.279 ± 0.002 TPC mg/g SCG) using T. harzianum, and a similar result was obtained with R. oryzae in the same treatment (0.250 ± 0.011 TPC mg/g SCG). In the Box–Behnken design, the most efficient treatment for T. harzianum was T12 (0.511 ± 0.017 TPC mg/g SCG), and for R. oryzae, T9 (0.636 ± 0.003 TPC mg/g SCG). These extracts could have applications in the food industry to improve preservation and functionality.

Graphical Abstract

1. Introduction

The coffee industry is one of the most extensive in the world, driven by the daily consumption of the beverage by millions of people and its biological benefits, which have led to a great demand in the global economy. Coffee is a rich source of antioxidants, which can help increase energy and concentration. It also has a preventive effect on chronic diseases, such as cardiovascular diseases and those related to some types of cancer, as it reduces cell damage [1]. However, despite this beverage’s health and economic benefits, there is also a negative side related to industrial waste, specifically coffee waste, which is generated during production and consumption. The first type of waste is generated during the harvesting of the beans. After this step, the husk, mucilage, and parchment are eliminated to obtain the green beans. The second type of waste is generated during consumption and includes spent coffee grounds (SCGs). SCGs are a solid residue that remains after the coffee is prepared through filtration. In addition, the beverage preparation process generates other waste, such as disposable materials like filters and containers. Each year, more than two million tons of waste is generated from production to beverage consumption [2]. These residues contain organic compounds such as caffeine, chlorogenic acid, caffeic acid, and tannins, which could damage the environment if not properly managed [3]. SCGs also contain important components that make their recovery desirable, such as lipids, cellulose, polyphenols, and proteins, which could be an opportunity to address the waste issue as they can be exploited innovatively and sustainably [4]. There are various strategies for the valorization of spent coffee grounds, including several physicochemical and thermochemical techniques, such as solvent extraction, pyrolysis, and acid hydrolysis, which have been studied with the aim of recovering bioactive compounds or producing energy. However, these extraction methods present significant limitations, including the use of toxic reagents, high energy demands, and low yields under sustainable conditions [5]. As a result, bioprocesses, particularly SSF with filamentous fungi, have emerged as environmentally friendly, efficient, and low-cost alternatives for converting coffee grounds into value-added products [6]. Currently, various strategies have been proposed to incorporate SCGs into bioplastics, cosmetics, functional foods, and biofuel production. However, the results have been limited and may not be scalable level, highlighting the ongoing need for new valorization alternatives that prioritize sustainability, efficiency, and low cost [7].
In this context, bioprocesses are an alternative in reducing coffee residues, showing promising results in obtaining bioactive compounds through the SSF strategy. This process is carried out using microorganisms, particularly filamentous fungi such as T. harzianum and R. oryzae, which play an essential role in the biodegradation of the residues and the recovery of compounds of interest [8]. Rhizopus is efficient in both the degradation of organic compounds and the production of enzymes that release different nutrients [9]. The metabolic capabilities of both fungi are essential in bioprocessing, as they possess enzymes that degrade organic matter, improve the bioavailability of bioactive compounds, and facilitate the release of substances such as polyphenols from SCGs. Both strains are considered safe, with R. oryzae classified as Generally Recognized as Safe (GRAS). Furthermore, the use of these microorganisms supports a circular bioeconomy model by converting agro-industrial waste into value-added products [10,11].
Determining the operational parameters in bioprocesses is essential to optimize the production and recovery of bioactive compounds, improve efficiency, reduce costs, and increase the quality of the final product. A proper optimization design increases the compounds obtained during the process and reduces the environmental impact by responsibly utilizing them [12,13]. Once optimized through bioprocesses, these compounds can be converted into a wide range of value-added products such as bioethanol, biofertilizers, and food supplements [14,15,16]. Therefore, this work aims to optimize the bioprocess of SSF SCGs, using filamentous fungi and promoting bioprocesses to transform agro-industrial waste into bioactive compounds. It is not only favorable from an economic and environmental point of view but also contributes to the circular bioeconomy by transforming waste efficiently for the innovation of new products with great potential in the food, pharmaceutical, and agricultural industries.

2. Materials and Methods

2.1. Chemical Reagents

Potato dextrose agar (PDA) BD Bioxon, Tween 80%, distilled water, reagent, Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power assay (FRAP) were all acquired from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Raw Material

SCGs were obtained from a local cafeteria in Saltillo, Coahuila, México. The variety of the SCG species used was Arabica coffee. The material was collected and transported in airtight bags to the Laboratory of Bioprocesses & Bioproducts at the Food Research Department at the Universidad Autónoma de Coahuila. The material was dried at 60 °C and packed in hermetic bags until later use.

2.2.1. Characterization of the Raw Material

Spent coffee grounds, both untreated and fermented, were characterized in terms of composition. Determinations of protein content, ash, and crude fiber were performed in triplicate, following the methods established by the AOAC [17]. Lipid extraction was carried out using the Soxhlet method, with hexane as the solvent. Protein content was quantified using the Kjeldahl method, applying a nitrogen-to-protein conversion factor of 6.25. Ash content was determined by weight difference after incineration in a muffle furnace at 600 °C. Crude fiber was measured using acid and alkaline digestion methods.

2.2.2. Determination of Cellulose, Hemicellulose, and Lignin Content

The quantification of cellulose, hemicellulose, and lignin in coffee grounds was performed through quantitative acid hydrolysis using 72% (w/w) H2SO4. A volume of 5 mL of the acid was added to the samples, which were then subjected to continuous stirring for 1 h, following the method described by Shiva et al. [18]. After hydrolysis, the liquid phase was separated and analyzed using high-performance liquid chromatography (HPLC). The analysis of monomeric sugars (glucose, xylose, and arabinose), as well as acetic acid, was carried out using an Agilent 1260 Infinity II HPLC system equipped with a refractive index detector. A MetaCarb 87 H column (300 mm × 7.8 mm, Agilent, Santa Clara, CA, USA) was used at 60 °C, with a mobile phase of 0.005 mol/L H2SO4 at a flow rate of 0.7 mL/min. Compound concentrations were determined using calibration curves prepared with pure standards. All analyses were conducted in duplicate for each substrate. The solid was determined as Lignin by gravimetry.

2.3. Solid-State Fermentation (SSF) Process

2.3.1. Microorganisms

In this study, the fungal strains Trichoderma harzianum (TH04) and Rhizopus oryzae (MUCL 28168) from the Food Research Department’s collection at the School of Chemistry of the Universidad Autónoma de Coahuila, Mexico, were evaluated.

2.3.2. Reactivation of Fungal Strains

To reactivate filamentous fungi, T. harzianum and R. oryzae were inoculated into 250 mL Erlenmeyer flasks containing PDA (BD Bioxon) and incubated at 30 °C for 7 days. Spores were harvested using 30 mL of sterilized Tween 80 solution (0.01%, v/v). The spore suspension was counted using a Neubauer chamber to ensure a known concentration prior to incubation in fermented media.

2.3.3. Solid-State Fermentation (SSF) Strategy

The fermentation process was carried out in 250 mL Erlenmeyer flasks using 3 g of SCGs as the substrate. The temperature (°C), inoculum size (spores/mL), and humidity (%) conditions are detailed in Section 2.3.4. For inoculum preparation, a spore count was performed using a Neubauer chamber. Fermentation was carried out for 48 h. Mechanical extraction was performed to obtain the fermentation extracts deposited in conical Falcon tubes. The liquid phase was separated by centrifugation (Hermle Labortechnik Z326 K, DEU, Wehingen, Germany) at 500 rpm for 10 min at 10 °C. The extracts were filtered through 0.45 µm pore size membrane filters and subsequently stored frozen for later use.

2.3.4. Preliminary Selection of Variables Using the Hunter & Hunter Method

An exploratory Hunter & Hunter design was carried out to identify the operational conditions for exploring variables for releasing total polyphenols, evaluating the factors at a minimum and maximum level for inoculum size, temperature, and humidity variables for 48 h (Table 1). The total polyphenol content (mg/g of substrate) was established as the dependent variable. The results were analyzed using STATISTICA 7 and mean comparison tests. A significance level of α = 0.05 was used for all statistical analyses. The total polyphenol content was evaluated in the exploratory design, as detailed in the following section.

2.4. Optimization of Solid-State Fermentation Strategy

Following the Hunter and Hunter exploratory design for the treatments, a Box–Behnken design was generated using STATISTICA 7 considering a significance level of α = 0.05. An experimental design was employed to optimize the solid-state fermentation process to maximize total polyphenols. For this experimental design, three levels were established: low, medium, and high. As a result of the optimization design, 15 treatments were obtained (see Table 2). The extracts obtained through solid-state fermentation with T. harzianum and R. oryzae were analyzed for total polyphenol content and antioxidant activity using the DPPH, ABTS, and FRAP methods.

2.5. Total Polyphenols

2.5.1. Condensed Tannin Content

The determination of condensed polyphenols was carried out according to the HCl-Butanol method [19]. A catechin solution was prepared for the 1000 ppm calibration curve in triplicate. Then, 500 μL of the sample, diluted 1:10, was taken, and 3 mL of HCl-Butanol (1:9) was added, followed by 0.1 mL of ferric reagent. The tubes were placed in a water bath at 100 °C for one hour. Finally, the sample was placed in a microplate and the absorbance was measured at 460 nm using an Epoch™ (BioTek Instruments, Winooski, VT, USA) Microplate UV–visible spectrophotometer.

2.5.2. Total Polyonehol Content

To determine total phenolic compound content using the Folin–Ciocalteu method, a stock solution of gallic acid was prepared for the calibration curve, ranging from 0 to 500 ppm. Then, 20 μL of each sample, diluted 1:10, was taken, and 20 μL of the Folin–Ciocalteu reagent was added and incubated for 5 min at room temperature. Subsequently, 20 μL of 0.01 M sodium carbonate was added, the sample was kept for 5 min at room temperature, and 125 μL of water was added. Each sample was transferred to a microplate to obtain an absorbance reading at 700 nm using an Epoch™ Microplate UV–visible spectrophotometer (BioTek Instruments, USA) [19].

2.6. Antioxidant Activity

2.6.1. Antioxidant Activity by DPPH

The methodology followed was based on the method proposed by Siller-Sánchez et al. [20]. The electron donor capacity of the samples was evaluated by preparing the DPPH reagent (1,1-diphenyl-2-picrylhydrazyl) using ethanol as the solvent (60 mM). Subsequently, 290 μL of DPPH radical solution was placed in the microplate for every 10 μL of the sample or standard curve (Trolox reagent) (Sigma-Aldrich, St. Louis, MO, USA). The reaction solution was incubated in the dark for 30 min, after which the absorbance of the samples was recorded at a wavelength of 517 nm (Epoch™ Microplate UV-Visible Spectrophotometer), and the results were expressed as grams of Trolox equivalents per liter of solution. All tests were performed in triplicate.

2.6.2. Antioxidant Activity Using the ABTS Method

The antioxidant capacity was evaluated using the ABTS•+ radical scavenging method, following the general procedure described by Van den Berge et al. [21], with slight modifications. To generate the ABTS•+ radical cation, a solution of ABTS (7 mM) was reacted with potassium persulfate (2.45 mM) and left to stand in the dark at room temperature for 12 h prior to use. The resulting ABTS•+ solution was then diluted with ethanol until an absorbance of 0.700 ± 0.002 at 734 nm was reached. For the assay, 50 μL of the fermentation extract at different concentrations were added to 950 μL of the ABTS•+ working solution. After allowing the mixture to react for 1 min, the absorbance was recorded at 734 nm.

2.6.3. Antioxidant Activity by FRAP

The ferric-reducing power was determined according to the method reported by Bautista-Hernández et al. [22] with some modifications. The FRAP reagent was prepared with 2.5 mL of a 10 mM 2,4,6-tripyridyl-s-triazine solution in 40 mM HCl plus 2.5 mL of FeCl3 (20 mM) and 2.5 mL of 0.3 M acetate buffer (pH 3.6). A total of 290 μL of FRAP reagent was mixed with 10 μL of sample or standard solution (Trolox). The reaction mixture was incubated in the dark for 15 min. Absorbance was determined using an Epoch™ Microplate UV–visible spectrophotometer (593 nm).

2.7. Fermentation Extract Identification Using HPLC-MS

Phenolic compound profiling was conducted using HPLC-MS, employing a Waters e2695 separation module connected to a photodiode array (PDA) detector (UV/Vis range 190–600 nm), following the procedure outlined by [23], with slight modifications. Separation was achieved on a Symmetry® C18 reversed-phase column (4.6 × 150 mm, 5 µm particle size, 100 Å pore size) coupled with a matching guard column (Waters, Milford, MA, USA). The mobile phases consisted of phase A—water, methanol (Panreac, Barcelona, Spain), and formic acid (Merck, Darmstadt, Germany) in a 92.5:5:2.5% (v/v/v) ratio; and phase B—methanol, water, and formic acid in a 92.5:5:2.5% (v/v/v) ratio. The gradient elution started with 100% of phase A, decreasing to 55% over 50 min. From minute 50 to 55, phase A returned to 100% and was maintained for 4 additional minutes (up to 59 min). The flow rate was set at 0.5 mL/min, and 20 µL of sample was injected for each run. Detection was carried out using the PDA detector, collecting spectral data from 200 to 600 nm at 2 nm intervals. Chromatograms were recorded at wavelengths of 280, 320, and 360 nm. Each sample was injected in triplicate. Compound identification was based on retention times and UV spectra compared against commercial standards.

2.8. Statistical Analysis

The software used for data analysis and experimental design (exploratory design by Hunter and Hunter and Box–Behnken design) was Statistica 7 (Statsoft, Tulsa, OK, USA). All analyses were performed in triplicate (n = 3), and the values were expressed as mean ± standard deviation. The statistical significance of the differences between the extracts after SSF was determined using Tukey’s test (p ≤ 0.05).

3. Results

3.1. Characterization of the Raw Material

Table 3 presents the results of the proximate analysis, which revealed variations in the nutritional components of unfermented coffee grounds and treatments subjected to fermentation with T. harzianum and R. oryzae. Regarding protein content, unfermented spent coffee grounds had lower values compared to the fermented treatments. The highest protein content was observed in the R. oryzae-treated sample (26.14 ± 0.09% w/w), followed by T. harzianum-treated sample (24.06 ± 0.67% w/w). These results indicate a change in the initial composition of the substrate under SSF conditions. Regarding carbohydrate content, the highest level (41.31%) was recorded in coffee grounds fermented with T. harzianum, while the unfermented sample had a value of 39.66%. These findings reflect changes in the chemical profile of spent coffee grounds resulting from the metabolic activity of the filamentous fungi used during fermentation. Regarding crude fiber, higher levels were found in unfermented spent coffee grounds, which decreased after fermentation, particularly in the R. oryzae treatment (13.67 ± 0.29% w/w). These results demonstrate the biochemical transformation of the substrate during fermentation and suggest an improvement in the nutritional composition of spent coffee grounds, which could increase their applicability in various fields. Furthermore, as part of the initial substrate characterization, the lignocellulosic composition of the SCGs was determined before the SSF process. The cellulose content was 10.95 ± 0.06%, hemicellulose 27.13 ± 0.01%, and lignin 14.85 ± 0.17%. These values provide information on the original composition of the waste. It is important to note that no assessment was made of the biomass fermented with the microorganisms regarding these components; therefore, no data are available on their potential degradation or transformation during the process.

3.2. Hunter & Hunter Preliminary Method for Obtaining Polyphenols

Figure 1 shows the Hunter & Hunter exploratory design results for the solid-state fermentation of T. harzianum and R. oryzae for polyphenol release. This design allowed us to evaluate the impact of different variables on the polyphenol content released during the fermentation process: temperature, humidity, and inoculum size. The yield of total polyphenols showed differences among the treatments evaluated, with values ranging from 0.200 to 0.279 mg total polyphenol content (TPC) per gram of SCG. This suggests that fermentation conditions influenced the final yield. The best conditions for fermentation with T. harzianum achieved higher total polyphenol yield (0.279 ± 0.002 TPC mg/g SCG), indicating that conditions of 80% humidity, a temperature of 25 °C, and an inoculum size of 1 × 107 spores/mL were favorable for polyphenol recovery. Total polyphenol production during R. oryzae fermentation showed variability among treatments, ranging from 0.200 to 0.260 mg TPC per gram of SCG. Treatments three (T3) (0.250 ± 0.011 TPC mg/g SCG) and two (T2) (0.245 ± 0.017 TPC mg/g SCG) presented higher values of total polyphenols, with no significant difference between them, suggesting that the conditions favored the release of polyphenols. Treatment T2 was carried out at 30 °C and 70% humidity, while treatment T3 was carried out at 20 °C and 80% humidity, using an inoculum size of 1 × 107 spores/mL.

3.3. Pareto Analysis of Significant Factors Affecting Polyphenol Release Based on Hunter and Hunter Exploratory Design

A Pareto diagram was performed to identify which parameters evaluated had the most significant influence on the efficiency of solid-state fermentation using the two microorganisms. The results obtained are shown in Figure 2, where it is observed that the minimum inoculum size stands out as the main factor that affects the fermentation process with T. harzianum and R. oryzae. The vertical dashed line in (a) indicates a significant effect on the response variable, where the inoculum factor showed a significantly negative effect, while temperature and moisture did not reach the critical value for significance. In (b), the inoculum was again the only factor with a significant effect. These results indicate that the inoculum is the most influential factor on the studied variables under the evaluated conditions.

3.4. Box-Behnken Design for the Production of Polyphenols

In the analysis of optimizing solid-state fermentation with T. harzianum and R. oryzae, different treatments were evaluated under various temperature, humidity, and inoculum size conditions. The results obtained are detailed in Figure 3. In the case of fermentation with T. harzianum, the results showed a range of total polyphenol concentrations, from 0.261 to 0.511 mg/g SCG. Treatment T12 presented a higher release of total polyphenols during fermentation, reaching a concentration of 0.511 ± 0.003 TPC mg/g SCG. This treatment was carried out under the following parameters: temperature 25 °C, humidity 80%, and inoculum size 1 × 108 spores/mL. For fermentation with R. oryzae, a polyphenol concentration range of 0.350 to 0.646 mg/g SCG was obtained. Treatment T9 presented the highest concentration among all the treatments evaluated, with a value of 0.646 ± 0.017 mg/g SCG, representing a significant difference from the other treatments. The conditions for this treatment were as follows: temperature 25 °C, humidity 70%, and inoculum size 1 × 106 spores/mL. During SSF, microorganisms grow under optimal conditions.

3.5. Response Surface Analysis for Polyphenol Release in Solid-State Fermentation

Figure 4 shows the response surface plot of the SSF process with both microorganisms, describing the influence of temperature, humidity, and inoculum size on polyphenol release. The colors in the 3D surface plots represent the magnitude of the dependent variable, in this case, total polyphenols (mg/g). Red indicates high values of total polyphenols, yellow represents intermediate values and green corresponds to low values.
Table 4 shows the analysis of critical values to determine the optimal conditions under which this fungus released the maximum amount of polyphenol.
Fermentation with T. harzianum showed that the maximum release of total polyphenols occurs under intermediate conditions of temperature (25 °C), humidity (77%), and inoculum concentration (1 × 107 spores/mL). The interaction of inoculum size and temperature favored the maximum release of polyphenols. On the other hand, the interaction between humidity and inoculum is displayed, indicating that excessive humidity reduces polyphenol release.
Fermentation with R. oryzae showed that the maximum release of polyphenol content depends on the interaction between inoculum size and humidity. Intermediate to low humidity values combined with high inoculum concentrations were observed to favor maximum polyphenol release. The maximum polyphenol release was reached at a temperature of 28 °C, 76.76% humidity, and an inoculum concentration of 1 × 107 spores/mL. These results suggest that maintaining humidity at an optimal level is crucial to maximizing polyphenol release in solid-state fermentation.

3.6. Antioxidant Activity of the Selected Treatments

Figure 5 shows the antioxidant activity of the selected extracts treated with T. harzianum and R. oryzae, chosen according to the Box–Behnken design for polyphenol release. Both microorganisms obtained a higher antioxidant activity, measured using the DPPH method: 13 for T. harzianum treatment (0.173 ± 0.026 mg GAE/g) and 9 for R. oryzae treatment (0.186 ± 0.001 mg GAE/g).

3.7. Identification of Phenolic Compounds in the Fermented Extract Using HPLC-MS

The extracts obtained from the fermentation of SCGs with T. harzianum and R. oryzae and the unfermented extract were characterized using HPLC. The identified compounds are summarized in Table 5. Caffeic acid and caffeoylquinic acid were identified in the unfermented extract. In the extracts fermented with T. harzianum, four compounds of the hydroxycinnamic acid family were identified: caffeoylquinic acid, caffeic acid cynarin, and ferulic acid. The bioprocess considerably improved the release of these compounds compared to the initial sample, demonstrating its effectiveness in recovering bioactive compounds. On the other hand, in the extracts fermented with R. oryzae, the compounds caffeoylquinic acid, caffeic acid, and ferulic acid were identified.

4. Discussion

4.1. Material Characterization

The spent coffee ground (SCG) residue is considered a suitable substrate for solid-state fermentation (SSF) due to its nutritional content, fibrous structure, and wide availability. These characteristics support the growth of filamentous fungi and facilitate the transformation of the substrate’s composition during the fermentation process [24]. The results showed that SSF significantly modified the nutritional composition of the coffee residue. An increase in protein content was observed in the fermented treatments compared to the unfermented sample, which could be attributed to the development of fungal mycelium during fermentation and the incorporation of microbial biomass into the substrate [25]. In contrast, the reduction in crude fiber content may be related to the enzymatic activity of the fungi, which secrete hydrolytic enzymes capable of breaking down the structural components of the substrate, such as cellulose and hemicellulose [26].

4.2. Exploratory Design: Hunter & Hunter Method

The results from the exploratory Hunter & Hunter study showed that both treatments achieved similar concentrations of polyphenol release, suggesting that, despite differences in temperature and humidity conditions, both processes promoted the metabolic activity of the microorganism. This may also explain the ability of fungi to adapt to different parameter conditions [27]. These results suggest that under the experimental conditions evaluated, both treatments had a similar optimal impact on polyphenol release. It is possible that each treatment achieved a similar maximum level of polyphenol release under the conditions evaluated, indicating that both fungal species responded similarly to the same environmental conditions. It has been demonstrated that in solid-state fermentation, agro-industrial residues support microorganisms, enabling greater interaction and facilitating the production and release of polyphenols, as well as other bioactive compounds [28]. The use of filamentous fungi in bioprocesses offers several advantages over other microorganisms, including higher enzyme yields and the ability to utilize agro-industrial waste for growth and metabolism [29]. The release of polyphenols during the fermentation process under optimal conditions has been reported to be due to the enzymatic action of microorganisms such as filamentous fungi (T. harzianum and R. oryzae). These fungi degrade the lignocellulosic matrix (lignin, cellulose, and hemicellulose) of SCGs, which contain approximately 20–25% lignin, 35–45% cellulose, and 15–20% hemicellulose [30]. Components that are difficult to decompose due to their complex and resistant structure include strong chemical bonds between lignin and cellulose polymers. However, these fungi can release ligninolytic and cellulolytic enzymes, thus facilitating the extraction of polyphenols in the cellular structure from the substrate [31,32]. The exploratory design of parameters for the solid-state fermentation bioprocess is a key tool for innovation, as it enables the exploration of new combinations of factors to obtain bioactive compounds. Parameter control achieves rapid monitoring, facilitating more efficient regulation and optimization, allowing process scalability and sustainability [33]. The results show that fermentation of spent coffee grounds with T. harzianum and R. oryzae significantly recovers total polyphenols, suggesting that this process can effectively generate value-added products. These results demonstrate the feasibility of utilizing coffee industry residues in fermentation processes, which could contribute to a circular economy model by converting these organic by-products into beneficial products.

Pareto Analysis—Hunter & Hunter Method

Regarding the Pareto chart of the exploratory design of Hunter & Hunter, previous studies, such as [34], have shown that a larger inoculum decreases the production of different enzymes. This is due to the high amount of inoculum that causes overcrowding of the spores, which refers to a high concentration of inoculum in a limited space. This can lead to competition for the nutrients needed for synthesis, affecting their metabolism and ability to produce enzymes during bioprocesses [35]. In this context, it is essential to highlight that enzyme production by these microorganisms is influenced by physical factors, such as temperature, pH, incubation time, and inoculum size, under conditions that ensure optimal growth and efficient enzyme production [36].
However, in the present study, only inoculum size significantly affected the process, minimizing the impact of temperature and humidity. This could be attributed to the fungus’s adaptability to the analyzed conditions, the potential interaction between variables, or the specific metabolic regulation involved in the release of polyphenols during bioprocessing. Studies have reported similar findings when analyzing microbial stability under different fermentation conditions [37,38,39]. On the other hand, humidity and temperature did not significantly impact the release of polyphenols during the fermentation process with either microorganism. It is possible that they were maintained at levels that allow for the development of the microorganism, as Trichoderma and R. oryzae grow in a temperature range of 20 °C to 30 °C and at humidity levels of 70% to 90% [40]. These conditions do not limit the metabolic activity of the fungi, although slight variations may influence the release or production of polyphenols.

4.3. Evaluation Using the Box–Behnken Design

Regarding the optimization design through Box-Behnken, as noted in the study by [32], these methods are widely recognized for their ability to produce cellulases, β-glucosidases, xylanases, and lipases, which break down substrates and release bioactive compounds. In this process, cellulases are responsible for the degradation of cellulose, one of the main components that contains polyphenols [41]. Ligninases act on lignin, another essential component of plant cell walls, while xylanases degrade hemicelluloses, facilitating the release of polyphenols. Additionally, the enzyme polyphenol oxidase is involved in the oxidation of polyphenols, which can result in modifications with new bioactive properties [42]. These enzymes are crucial for breaking down complex substrates into simpler ones. They break the chemical bonds of complex molecules, and some enzymes specifically break down carbohydrates into simple sugars [43]. This study demonstrates the fermentative potential for modifying the spent coffee ground matrix and improving its profile. Therefore, certain limitations should be considered for its large-scale implementation, such as heat transfer, homogeneous temperature control, moisture, and aeration within the system. Future research focused on scale-up, process control, and validation under pilot conditions is essential to ensure the reproducibility, efficiency, and economic feasibility of the bioprocess on an industrial scale [44].
This process holds promise for identifying and producing bioactive molecules to improve the efficiency and profitability of total polyphenol release, expanding its application in different industries and promoting innovation and sustainability.

4.3.1. Antioxidant Activity

Referring to the results of antioxidant activity in previous studies, [45] reported a 2.7-fold increase in the antioxidant capacity of extracts fermented with R. oryzae compared to unfermented material. This finding is consistent with [46], which reported increased antioxidant activity resulting from the release of total polyphenol compounds during fermentation with R. oryzae. The antioxidant activity of the optimized extracts from the fermentation process highlights a rich source of antioxidant compounds, which show a higher affinity for the free radical DPPH [45]. The compounds obtained during fermentation may have the ability to donate electrons or hydrogen atoms to free radicals, thereby neutralizing free radicals [47]. The ability to neutralize is related to preventing chronic diseases such as cancer and cardiovascular diseases, where oxidative damage plays a crucial role [47]. The growing interest in consumer health awareness and preference for natural additives has encouraged researchers to explore natural alternatives to synthetic antioxidants. According to the author, natural phenolic compounds exhibit antioxidant activity similar to that of commercial antioxidants, enabling the manufacture of oxidation-resistant products that improve product stability [48].
Therefore, polyphenols that have antioxidant capacity not only play an important role in the prevention of chronic diseases and protection against pathogens, but are also fundamental in the food industry [49], improving the quality and durability of food by driving the demand for these functional products and supplements, which are key to human health.

4.3.2. HPLC-MS Analysis

The results obtained from the chemical characterization agree with the findings of Arancibia-Díaz [45], who demonstrated that solid-state fermentation of SCGs with different microorganisms resulted in a significant increase in bioactive compounds of the hydroxycinnamic acid family compared to unfermented SCGs. These compounds open up new scientific avenues, as they possess antioxidant capacity and present significant chlorogenic and caffeic acid values [50]. Previous studies [51] have demonstrated the presence of 5-caffeoylquinic in SCGs. This compound has shown high antioxidant activity in vitro and in different cellular models. These effects suggest that caffeoylquinic acid may be a beneficial compound for protecting cells against oxidative and inflammatory damage [52]. In addition, studies consider SCG extracts to be a valuable source of nutraceuticals that could be used to prevent neurodegeneration [52]. Moreover, SCG extracts have antimicrobial and antifungal properties against various pathogens [53,54].

5. Conclusions

The solid-state fermentation (SSF) bioprocess has proven to be a strategy for maximizing the recovery of total polyphenols, with control of variables such as temperature, humidity, and inoculum size being critical to the process. In the comparison between Trichoderma harzianum and Rhizopus oryzae fungi, evidence indicated that R. oryzae obtained higher efficiency in the release of polyphenol content, achieving the highest yield in treatment T9 (0.636 ± 0.017 mg/g SCG). These results highlight the ability of the approach to identify optimal conditions for polyphenol release during the solid-state fermentation process, thus improving the efficiency of biotechnological processes with biological activities. This optimization of SSF could significantly impact the food industry by enhancing product properties and health benefits. It also opens new possibilities in various sectors; polyphenols can be used to develop new food supplements and cosmetics that protect the skin. Therefore, this bioprocess presents a sustainable and integral solution for the recovery of compounds or nutrients, as well as the valorization of waste, such as spent coffee grounds, contributing to the preservation of the environment. Thus, the bioprocess can be the key to a circular bioeconomy, in which coffee waste is biotransformed into innovative industrial applications.

Author Contributions

K.A.L.: Conceptualization, formal analysis, investigation, methodology, writing—original draft, writing—review and editing. C.N.A.: Conceptualization, validation, writing—review and editing. N.R.-G.: Data curation, validation, writing—review and editing. H.A.R.: Validation, writing—review and editing. J.L.M.: Data curation, validation, writing—review and editing. M.L.C.-G.: Conceptualization, project administration, supervision, validation, writing—review and editing, resources, visualization, methodology, data curation. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We acknowledge, with many thanks, the Food Research Department for collaboration and support during the experimental work and CONAHCYT for scholarship support to K.A.L. (777738).

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00366 g001
Figure 1. Total polyphenol content of extracts fermented by treatments (a) T. harzianum and (b) R. oryzae using the Hunter & Hunter exploratory design. Different letters show significant differences (α = 0.005).
Figure 1. Total polyphenol content of extracts fermented by treatments (a) T. harzianum and (b) R. oryzae using the Hunter & Hunter exploratory design. Different letters show significant differences (α = 0.005).
Fermentation 11 00366 g001
Fermentation 11 00366 g002
Figure 2. Pareto diagram of the significant factors influencing polyphenol release during solid-state fermentation with (a) T. harzianum and (b) R. oryzae.
Figure 2. Pareto diagram of the significant factors influencing polyphenol release during solid-state fermentation with (a) T. harzianum and (b) R. oryzae.
Fermentation 11 00366 g002
Fermentation 11 00366 g003
Figure 3. Total polyphenol content of SCGs fermented with (a) T. harzianum and (b) R. oryzae based on a Box–Behnken design. Different letters show significant differences (α = 0.005).
Figure 3. Total polyphenol content of SCGs fermented with (a) T. harzianum and (b) R. oryzae based on a Box–Behnken design. Different letters show significant differences (α = 0.005).
Fermentation 11 00366 g003
Fermentation 11 00366 g004
Figure 4. Response surface plot of the optimization of solid-state fermentation with (a) T. harzianum and (b) R. oryzae for total polyphenol content.
Figure 4. Response surface plot of the optimization of solid-state fermentation with (a) T. harzianum and (b) R. oryzae for total polyphenol content.
Fermentation 11 00366 g004
Fermentation 11 00366 g005
Figure 5. Antioxidant activity measured using the DPPH, ABTS, and FRAP methods for extracts fermented with (a) T. harzianum and (b) R. oryzae.
Figure 5. Antioxidant activity measured using the DPPH, ABTS, and FRAP methods for extracts fermented with (a) T. harzianum and (b) R. oryzae.
Fermentation 11 00366 g005
Table 1. Experimental matrix of the Hunter & Hunter exploratory design.
Table 1. Experimental matrix of the Hunter & Hunter exploratory design.
TreatmentTemperature (°C)Humidity (%)Inoculum (Spores/mL)
125701 × 107
230701 × 107
325801 × 107
430801 × 107
525701 × 108
630701 × 108
725801 × 108
830801 × 108
Table 2. Experimental matrix of the Box–Behnken design.
Table 2. Experimental matrix of the Box–Behnken design.
TreatmentTemperature (°C)Humidity (%)Inoculum (Spores/mL)
T120701 × 107
T230701 × 107
T320801 × 107
T430801 × 107
T520751 × 108
T630751 × 108
T720751 × 108
T830751 × 108
T925701 × 106
T1025751 × 106
T1125701 × 108
T1225801 × 108
T1325751 × 107
T1425751 × 107
T1525751 × 107
Table 3. Proximate analysis and lignocellulosic components (cellulose, hemicellulose, and lignin) of fermented and unfermented SCGs.
Table 3. Proximate analysis and lignocellulosic components (cellulose, hemicellulose, and lignin) of fermented and unfermented SCGs.
Content (% w/w)Unfermented SCGsSCGs Fermented with R. oryzae SCGs Fermented with T. harzianum
Protein24.06 ± 0.6726.14 ± 0.0925.85 ± 0.09
Carbohydrates39.6634.7241.31
Lipids16.94 ± 0.2625.31 ± 0.9719.17 ± 0.19
Fiber16.62 ± 0.5413.67 ± 0.2913.11 ± 0.03
Ash 2.72 ± 0.710.16 ± 0.710.54 ± 0.19
Cellulose10.95 ± 0.06N.D.N.D.
Hemicellulose27.13 ± 0.01N.D.N.D.
Lignin14.85 ± 0. 17N.D.N.D.
N.D.: No Determined.
Table 4. Critical parameters of temperature, humidity, and inoculum concentration affecting polyphenol release by T. harzianum and R. oryzae.
Table 4. Critical parameters of temperature, humidity, and inoculum concentration affecting polyphenol release by T. harzianum and R. oryzae.
FactorObserved
Minimum		Critical ValuesObserved
Maximum
T. harzianum
Temperature, °C202530
Inoculum,
spores/mL		1 × 1061 × 1071 × 108
Humidity, %707780
R. oryzae
Temperature, °C2028.630
Inoculum,
spores/mL		1 × 1061 × 1071 × 108
Humidity, %707680
Table 5. HPLC-MS characterization of spent coffee ground extracts: unfermented and those obtained from T. harzianum and R. oryzae fermentation.
Table 5. HPLC-MS characterization of spent coffee ground extracts: unfermented and those obtained from T. harzianum and R. oryzae fermentation.
Retention Time (min)Mass
(m/z −1)		CompoundFamily
Unfermented SCG Extract13.40353Caffeoylquinic acidHydroxycinnamic acids
15.58179Caffeic acidHydroxycinnamic acids
Extract
from T. harzianum Fermentation		13.01353Caffeoylquinic acidHydroxycinnamic acids
15.09179Caffeic acidHydroxycinnamic acids
19.41367Cynarin Hydroxycinnamic acids
20.75367Ferulic acidHydroxycinnamic acids
R. oryzae Fermentation Extract12.83353Caffeoylquinic acidHydroxycinnamic acids
19.22179Caffeic acidHydroxycinnamic acids
29.49193FeruloylquinicHydroxycinnamic acids
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Luna, K.A.; Aguilar, C.N.; Ramírez-Guzmán, N.; Ruiz, H.A.; Martínez, J.L.; Chávez-González, M.L. Bioprocessing of Spent Coffee Grounds as a Sustainable Alternative for the Production of Bioactive Compounds. Fermentation 2025, 11, 366. https://doi.org/10.3390/fermentation11070366

AMA Style

Luna KA, Aguilar CN, Ramírez-Guzmán N, Ruiz HA, Martínez JL, Chávez-González ML. Bioprocessing of Spent Coffee Grounds as a Sustainable Alternative for the Production of Bioactive Compounds. Fermentation. 2025; 11(7):366. https://doi.org/10.3390/fermentation11070366

Chicago/Turabian Style

Luna, Karla A., Cristóbal N. Aguilar, Nathiely Ramírez-Guzmán, Héctor A. Ruiz, José Luis Martínez, and Mónica L. Chávez-González. 2025. "Bioprocessing of Spent Coffee Grounds as a Sustainable Alternative for the Production of Bioactive Compounds" Fermentation 11, no. 7: 366. https://doi.org/10.3390/fermentation11070366

APA Style

Luna, K. A., Aguilar, C. N., Ramírez-Guzmán, N., Ruiz, H. A., Martínez, J. L., & Chávez-González, M. L. (2025). Bioprocessing of Spent Coffee Grounds as a Sustainable Alternative for the Production of Bioactive Compounds. Fermentation, 11(7), 366. https://doi.org/10.3390/fermentation11070366

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