Special Issue

Published as part of ACS Sustainable Chemistry & Engineering special issue “Advancing a Circular Economy”.

Synopsis

Sustainable cocoa shell valorization using stingless bee honey as an edible solvent integrates biodiversity conservation with green chemistry principles.

1. Introduction

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Sustainable alternatives for industrial processes are crossing various sectors, including food, cosmetics, and chemicals. Aligned with this goal, the use of agro-industrial residues, which are often discarded or undervalued, has gained attention to recover valuable compounds. Valorizing agricultural byproducts is in accordance with the principles of the circular economy and biorefinery, which aim to maximize the use of raw materials, reduce waste, and enhance the potential value of the final product(s). (1)

One notable example of an agricultural byproduct is the cocoa bean shell (CBS), a residue generated during cocoa processing. Although CBS contains valuable bioactive compounds, particularly methylxanthines and phenolic compounds, it is still frequently discarded as waste. (2) Among the methylxanthines, theobromine and caffeine are notably relevant due to their widely known health benefits, including stimulant activity and potential cardiovascular protection. (3,4) However, the recovery of bioactive compounds from CBS often involves extraction procedures combined with elevated temperatures and prolonged processing times. (5) For instance, subcritical water extraction (SWE) efficiently extracts bioactive compounds from CBS. (6,7) Nevertheless, such processes may also lead to the degradation of thermolabile compounds, increased energy consumption, and the need for additional steps, such as drying or purification processes. (8)

Moreover, other alternative methods employing green solvents, such as biobased solvents and natural deep eutectic solvents (NADES), have also been proposed for the valorization of CBS. (9) While these options are more sustainable than traditional problematic organic solvents, they also present certain limitations. For instance, although effective, some NADES formulations or biobased solvents such as ethanol may not be suitable for direct consumption or can only be ingested in limited quantities.

Consequently, there has been growing interest in identifying edible solvents that simplify both extraction and formulation, enabling the production of ready-to-use extracts. (10) In this context, stingless bee honeys (SBHs) are particularly promising alternatives. Naturally rich in antibacterial and antioxidant properties, SBHs is already widely consumed as food. (11) Compared to Apis mellifera honey, they often exhibits lower viscosity, higher acidity, and greater water content, which potentially enhance the extraction of bioactive compounds. These characteristics lead to enhanced mass transfer and higher solubility, especially for compounds of medium to high polarity. The higher acidity can also help disrupt cellular structures to release target molecules, while potentially increasing their solubility by altering their ionization state.

Moreover, SBHs also offer a unique opportunity in biodiversity-rich regions such as Brazil, Malaysia, and Australia, where native honeys possess distinctive yet underexplored properties. (12) Their incorporation into innovative products promotes sustainable development. Traditional and native communities have long valued SBHs for their nutritional and medicinal properties. (13) Recognizing and integrating this knowledge not only advances scientific innovation but also strengthens socio-biodiversity and cultural heritage, supporting global efforts aligned with the United Nations Sustainable Development Goals, including SDG 3 (good health and well-being), SDG 10 (reduced inequalities), SDG 12 (responsible consumption and production), and SDG 15 (life on land). (14)

At the same time, there is a growing interest to intensify extraction processes in biorefinery contexts, making them faster, more efficient, and potentially more environmentally friendly. (15) Ultrasound-assisted extraction (UAE) is a valuable tool for process intensification, as it promotes the structural disruption of plant matrices, enhances solvent–solute interactions, and accelerates mass transfer. (16) In viscous media such as SBHs, UAE may be particularly effective in helping to overcome diffusion limitations and improve solvent penetration. Additionally, it offers specific advantages in honey processing, such as microbial inactivation, decrystallization, and the preservation of phenolic content and antimicrobial properties, as described by Pereira et al. (2023). (17)

This study presents a novel method for focused extraction of theobromine and caffeine from cocoa bean shells using high-intensity ultrasound combined with SBHs. This approach combines a natural and food-grade type of solvent with an intensified green extraction process. The result is a honey naturally enriched with bioactive compounds, ready to be applied in food, nutraceutical, or cosmetic formulations. Beyond its technical innovation, this strategy embraces key principles: (i) the valorization of an agro-industrial byproduct, (ii) the use of a sustainable and edible solvent, (iii) the intensification of extraction processes, and (iv) the support of local products from biodiversity and circular economy practices.

2. Material and Methods

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2.1. Chemicals and Raw Materials

Cocoa (Theobroma cacao L.) bean shells (CBS) were obtained as byproducts from the agro-industrial unit of CATI (Coordenadoria de Assistência Técnica Integral) (Rio Preto, São Paulo, Brazil). The shells, which were already dried to approximately 7% moisture, were milled and sieved to obtain particles ranging from 250 to 500 μm and stored at – 20 °C until their utilization. SBHs were employed as natural solvents and were supplied by Meliponário Guaiçara (Santos, São Paulo, Brazil). Honeys were collected from five species, with the scientific names followed by their respective traditional names in parentheses: Tetragona clavipes (Borá), Tetragonisca angustula (Jataí), Melipona quadrifasciata (Mandaçaia), Scaptotrigona postica (Mandaguari), and Frieseomelitta varia (Moça-branca). All honey samples were stored under refrigeration at 4 °C until used to preserve their physicochemical properties. All honey samples were stored under refrigeration at 4 °C until they were used to preserve their physicochemical properties. Prior to extraction, SBHs were brought to room temperature. The CBS and SBHs have been registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SISGEN) under registration number AB5A8F0.

Ultrapure water was obtained using a Purelab Flex 3 purification system (Elga Veolia, High Wycombe, UK). Analytical standards (≥99% purity) of caffeine, theobromine, and 5-hydroxymethylfurfural (5-HMF) were purchased from Sigma-Aldrich (St. Louis, MO, USA), as well as sodium carbonate, Folin-Cicalteau reagent, 2,2-diphenol-1 picrylhydrazyl (DPPH), Trolox, 2,2 ′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and acetic acid (99.9%). Galic acid was purchased at Nuclear (Maringá, PR, Brazil). All other reagents were of analytical or HPLC grade.

2.2. Characterization of Stingless Bee Honey

The characterization of the SBHs used in this study was based on determining key parameters that could influence the extraction process and other relevant compositional aspects. The methodologies described hereafter included the determination of viscosity, water content, ash content, acidity, pH, and sugar composition. Each measurement was made at least in duplicate.

2.2.1. Viscosity

The viscosity of the pure SBHs was measured using a Brookfield LVDV1 viscometer (Brookfield Engineering Laboratories, USA) equipped with an SC4–34 cone spindle and a UL adapter. (18) Measurements were performed at 25 °C under a constant rotational speed of 6 rpm, applying an oscillatory shear. The system was pre-equilibrated to ensure temperature stability throughout the measurement. Viscosity values were recorded in centipoise (cP) after stabilization.

2.2.2. Moisture Content

Moisture content was determined using a digital refractometer (Hanna Instruments, HI96801, Romania), following AOAC Method 969.38. (19) Readings were conducted at 20 °C, and moisture content was determined using standard calculations and reference tables for honey, in accordance with the analytical procedures described by the International Honey Commission. (20)

2.2.3. Ash

Ash content was determined by incineration according to AOAC Official Method 920.181. (19) Approximately 2 g of honey were accurately weighed into a previously ignited and tared crucible. The samples were then placed in a muffle furnace (SOLAB, SL-100, Brazil) at 550 ± 25 °C until a constant white or gray ash was obtained, which required approximately 2 h. The crucibles were then cooled in a desiccator and reweighed. Ash content was calculated as the percentage (%) of the dry residue relative to the initial sample mass.

2.2.4. Acidity and pH

Titratable acidity was determined by volumetric titration and expressed in milliequivalents of acid per kilogram of honey (meq/kg), using formic acid as reference, following AOAC Official Method 962.19. (19) Approximately 10 g of honey were dissolved in 75 mL of distilled water and titrated with 0.089 mol L–1 standardized sodium hydroxide (NaOH) until reaching pH 8.50, using a calibrated pH meter (Marte, One Sense pH 2506, Brazil). Results were calculated based on the volume of NaOH consumed and the sample mass. The pH was measured separately by diluting 10 g of honey in 75 mL of distilled water, followed by manual stirring until complete homogenization. Measurements were performed at room temperature using a calibrated pH meter.

2.2.5. Sugars

Sugars were analyzed by an Acquity UPLC H-Class system (Waters, USA) with a refractive index detector (RID). Separation was performed using a Rezex column (Phenomenex, model ROA-Organic Acid H+ (8%), 8 μm, 300 × 7.8 mm, Torrance, CA, USA), adopting an isocratic flow of 0.6 mL min–1 of H2SO4 (0.005 mol L–1) at 60 °C. The RID was maintained at 40 °C. For the analysis, the hydrolysates were diluted, centrifuged, and filtered through a 0.22 μm nylon membrane prior to injection. Then, 10 μL of hydrolysate was injected and the run was established for 48 min, according to Barroso et al. (2022). (21) The concentration (mg/g) of glucose and fructose were calculated from calibration curves of each standard.

2.3. Ultrasound-Assisted Extraction (UAE)

The extraction of bioactive compounds from CBS was carried out using a probe-type high-intense ultrasound-assisted extraction (UAEprobe) system equipped with a 20 kHz ultrasonic probe with a 13 mm diameter tip, providing a maximum nominal power of 400 W at a frequency of 20 kHz (Ultronique, Brazil). For each assay, the extraction was performed in a 50 mL Falcon tube using a fixed amount of 500 mg of CBS and the corresponding amount of SBH, according to the defined solvent-to-feed (S/F) ratio. The extracts were manually homogenized and immediately subjected to UAEprobe. After sonication, the extracts were centrifuged at 10,600 × g for 10 min at room temperature (Bench centrifuge, model K241R, Centurion Scientific West Ashling, UK), and the supernatants were collected and diluted prior to chromatographic analysis.

The preliminary screening of variables influencing the UAEprobe of methylxanthines, i.e., (y1) theobromine and (y2) caffeine, from CBS was conducted using a Plackett–Burman experimental design (Table S1). The experiments were performed using the SBH from S. postica as the extraction solvent. The honey from such species was selected at this stage as it presented intermediate viscosity and water content (section 3.1) and hence considered a representative matrix. The following five independent variables were evaluated: (x1) ultrasound nominal power (160–400 W), (x2) extraction time (1–5 min), (x3) S/F ratio (20–50 g/g), (x4) water content (20–50%, w/w), and (x5) lactic acid content (0–10%, w/w).

Based on the results of the Plackett–Burman design, a face-centered design (FCD) was employed to optimize the most significant factors affecting UAE efficiency (Table S2). In this second step, the ultrasound power (x4) was fixed at 400 W (maximum supported by the equipment), and the S/F ratio (x5) at 20 g/g, resulting in a more concentrated extract. The three remaining variables were studied: (x1) extraction time, (x2) water content in honey, and (x3) lactic acid content. The levels were kept unchanged as explained in section 3.2.

Subsequently, a validation step was conducted under the identified optimal UAEprobe condition, comparing it with the use of pure honey by applying a Student’s t test. Additionally, the temperature profile of each extraction medium, i.e., pure honeys and water, was monitored over 5 min, with measurements taken every 30 s.

2.4. Comparative Extraction Technique

The optimal extraction condition using the UAEprobe system was compared with a modified version of the protocol proposed by Yusof et al. (2019), employing a 37 kHz ultrasonic bath (Elmasonic, Schmidbauer GmbH, Singen, Germany) operating at 150 W (UAEbath). (22) Briefly, the S/F optimized for the UAEprobe was maintained, while temperature and time were set at 55 °C and 45 min. Pure water was used as the reference solvent for its green, nontoxic profile, wide acceptance in the food industry, and greater comparability to SBH. (23) The UAEbath from CBS using each SBH and water as solvents were performed in at least duplicate. Theobromine and caffeine contents were measured as response variables.

2.5. Quantification of Caffeine, Theobromine, and 5-Hydroxymethylfurfural by UPLC-PDA

CBS extracts and pure honeys were diluted 5-fold with ultrapure water prior to chromatographic analysis. The diluted extracts were then syringe-filtered using 0.22 μm nylon filters and analyzed using an Acquity UPLC H-Class system (Waters, USA). Separations were carried out on a Waters Acquity C18 column (2.1 × 50 mm, 1.7 μm) maintained at 40 °C. The mobile phases consisted of water (A) and acetonitrile (B), both containing 0.1% (v/v) acetic acid, under the following gradient: 5% B (0–0.5 min), 5–30% B (0.5–5.5 min), 30–100% B (5.5–6 min), and 100% B (6–7 min), followed by a 2.5 min column equilibration. The flow rate was 0.5 mL/min, and the injection volume was 3 μL. UV absorbance was monitored from 210 to 400 nm, with quantification performed at 272 nm for caffeine and theobromine, and 284 nm for 5-hydroxymethylfurfural (5-HMF). The limit of quantification (LOQ) was below 0.02 mg/g, in line with values previously reported for SBH samples. (24)

2.6. Evaluation of Total Phenolic Content and Antioxidant Capacity

The total phenolic content (TPC) and antioxidant capacities, determined by ABTS•+, DPPH, and FRAP assays, were evaluated using UV–Vis spectrophotometry (Q798U, Quimis, Diadema, Brazil). The methods employed were based on the study of Müller et al. (2010) and Sanches et al. (2024). (25,26) All assays were performed in triplicate.

2.6.1. Total Phenolic Content (TPC)

The total phenolic content (TPC) was measured using the Folin–Ciocalteu colorimetric method. In brief, 500 μL of the sample (or its dilution) was mixed with 2.5 mL of a 10% Folin–Ciocalteu reagent and 2.0 mL of a sodium carbonate solution (7.5 g/100 mL). The mixture was then incubated in the dark at 45 °C for 6 min. The absorbance was measured at 760 nm. A calibration curve was created using gallic acid, and the results were expressed as milligrams of gallic acid equivalents per gram of crude honey, honey extract, or solvent extract (mg GAE/g).

2.6.2. ABTS**•+** Radical Scavenging Activity

The antioxidant capacity of the extracts was assessed using the ABTS assay, based on the decolorization of the ABTS•+ radical. In brief, 200 μL of the extract (or its dilution) was mixed with 1.0 mL of ABTS solution. After 6 min of incubation in the dark, the absorbance was read at 734 nm. Antioxidant activity was quantified using a Trolox standard curve, and the results were expressed as micrograms of Trolox equivalents per gram of crude honey, honey extract, or solvent extract (μg TE/g).

2.6.3. DPPH Radical Scavenging Activity

The DPPH assay was performed by adding 200 μL of the extract (or its dilution) to 1.0 mL of DPPH solution. The mixture was incubated in the dark for 30 min, and the absorbance was measured at 517 nm. A Trolox calibration curve was used to calculate the antioxidant capacity, expressed as micrograms of Trolox equivalents per gram of of crude honey, honey extract, or solvent extract (μg TE/g).

2.6.4. Ferric Reducing Antioxidant Power (FRAP)

The FRAP reagent was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl, and 20 mM ferric chloride (FeCl3·6H2O) in a 10:1:1 ratio (v/v/v). In a cuvette, 2.7 mL of FRAP reagent was mixed with 90 μL of distilled water and 90 μL of the sample (or its dilution). After 30 min of incubation at 37 °C in the dark, the absorbance was measured at 593 nm. A standard curve of ferrous sulfate (FeSO4·7H2O) was used to express the results as micromoles of Fe (II) equivalents per gram of crude honey, honey extract, or solvent extract (μmol Fe2+/g).

2.7. Green Metric – Path2Green

The sustainability of the developed UAEprobe process, which use SBHs as natural solvents, was evaluated using the Path2Green metric. (1) This tool assesses compliance with the 12 principles of green extraction, covering key aspects of the extraction workflow: (1) Biomass, (2) Transport, (3) Pretreatment, (4) Solvents, (5) Scaling, (6) Purification, (7) Yield, (8) Post-treatment, (9) Energy, (10) Application, (11) Repurposing, and (12) Waste management. For the 12th principle, the waste was calculated by removing the residual biomass after extraction and centrifugation, washing it with water, and drying it to a constant weight. After this, the waste percentage was calculated based on the ratio between the final dry residue and the initial mass of the biomass used.

Each principle is evaluated on a scale from – 1.00 to +1.00, with higher scores denoting greater adherence to green chemistry principles. The overall score is adjusted based on the significance of environmental factors (weight 3), societal factors (weight 2), and economic factors (weight 1).

The Path2Green app assesses and summarizes process performance using pictograms. Each principle is represented by a color-coded icon, indicating strengths and improvement areas. The overall sustainability score can be shown as a carbon footprint (gCO2/g_biomass), calculated via an integrated regression model.

y(gCO2/gbiomass)=−1079.3Score(path2green)+721.3

(1)

2.8. Statistical Analysis

The statistical calculations in the optimization step, such as model fitting, coefficient significance (p < 0.05), and analysis of variance (ANOVA), were conducted using Protimiza Experimental Design software (Protimiza Experimental Design, Brazil). The extract recoveries were compared using ANOVA, followed by Tukey’s multiple comparison test or the Student’s t test, with a significance level set at 95% (p < 0.05). The analyses were conducted using GraphPad Prism 9 software (GraphPad Software, USA). A two-way ANOVA was also performed to compare the effects of both extraction method (UAEprobe vs UAEbath) and solvent type (different SBH and water). Interaction effects and multiple comparisons were evaluated, and Tukey’s posthoc test was applied when appropriate. The workflow proposed in this work is shown in Figure 1.

Figure 1

Figure 1. Workflow implemented to characterize and optimize the intensified recovery of bioactive compounds from cocoa bean shells using stingless bee honeys as solvents.

3. Results and Discussion

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3.1. Stingless Bee Honeys Characterization

The selected SBH samples demonstrated considerable variation in their physicochemical characteristics, reflecting the biological and ecological diversity of the bee species from which they were obtained. One relevant physicochemical aspect related to the subsequent extraction performance is viscosity, which ranged from 140 ± 0 cP for T. clavipes to 3161 ± 2 cP for F. varia, indicating substantial differences among samples. As expected, viscosity showed an inverse correlation with water content. Accordingly, F. varia exhibited the highest viscosity and the lowest moisture content (25.1 ± 0.9%), whereas T. clavipes presented the lowest viscosity and the highest moisture content (38.3 ± 2.4%). A notable and species-dependent variability in water content was also reported by Biluca et al. (2016) and Ávila et al. (2019). (27,28)

Following a similar variability tredn, acidity also differed notably among samples, while pH values ranged from 3.3 ± 0.1 to 4.2 ± 0.1. On the other hand, ash content was generally low across all samples, whereas glucose and fructose levels were relatively high, with fructose typically exceeding glucose content. The values observed for these properties are consistent with those previously reported for SBH. (29)

These results reinforce the importance of including a diverse selection of SBHs in this study, as their unique compositions may distinctly influence extraction performance and their functionality as edible solvents. Moreover, a relevant characteristic of honey related to its physicochemical properties is its potential to function as a NADES. (30,31) This is mainly due to its rich composition of sugars, e.g., glucose and fructose, alo ng with organic acids and other small molecules that form a structure similar to that of a NADES. (32) Therefore, honey may potentially enhance the extraction of bioactive compounds, as has already been demonstrated for a wide range of NADES. (10)

3.2. Optimization of UAEprobe

For the optimization of the UAE, the honey produced by S. postica was selected as the model solvent. This choice was based on its intermediate viscosity (410.50 ± 0.71 cP) among the five SBHs evaluated (Table 1), allowing for a balanced assessment of ultrasound propagation in a viscous medium without the extremes of low or high resistance to cavitation.

Table 1. Physicochemical Properties of Selected Native Stingless Bee Honeys, i.e., Viscosity, Moisture Content, pH, Acidity, Ash Content, and Main Sugars, of Five Species: Tetragona clavipes, Tetragonisca angustula, Melipona quadrifasciata, Scaptotrigona postica, and Frieseomelitta variaa

sample	traditional name	viscosity (cP)	moisture content (%)	pH	acidity (meq/kg)	ash (%)	glucose (mg/g)	fructose (mg/g)
Tetragona clavipes	Borá	140 ± 0.0e	38.3 ± 2.4a	3.3 ± 0.1b	189.5 ± 5.6a	0.15 ± 0.01b	245.63 ± 1.23c	245.63 ± 1.23c
Tetragonisca angustula	Jataí	1071 ± 1b	28.5 ± 1.2bc	3.6 ± 0.1b	59.9 ± 2.0c	0.22 ± 0.01b	238.97 ± 1.48d	260.74 ± 0.13d
Melipona quadrifasciata	Mandaçaia	310 ± 0d	32.9 ± 1.1ab	3.4 ± 0.0b	43.7 ± 3.5d	N.D.	296.09 ± 1.12a	322.00 ± 1.52a
Scaptotrigona postica	Mandaguari	410 ± 1c	32.0 ± 1.7ab	3.3 ± 0.1b	111.6 ± 2.7b	0.01 ± 0.00c	216.02 ± 2.73e	272.04 ± 2.70c
Frieseomelitta varia	Moça-branca	3161 ± 2a	25.1 ± 0.9c	4.2 ± 0.1a	47.6 ± 3.8cd	0.36 ± 0.06a	268.58 ± 0.96b	299.57 ± 1.14b

a

Different lowercase letters indicate statistically significant differences between groups according to Tukey’s multiple comparison test (p < 0.05). N.D: Not detected.

The optimization of the UAE process began with an evaluation of key process variables using a Plackett–Burman design to identify the most influential factors affecting the extraction of theobromine and caffeine. Among the five variables tested, water content, lactic acid concentration, extraction time, and ultrasound power exhibited significant (p < 0.05) positive effects on the extraction efficiency of both compounds (Table S3). In contrast, the S/F ratio did not significantly influence the responses within the tested range (p = 0.49 for theobromine and p = 0.55 for caffeine). For the subsequent optimization step, the ultrasound nominal power was fixed at its maximum level (400 W), representing the highest output supported by the equipment. Meanwhile, the S/F ratio was fixed at the lowest tested level (20 g/g) to minimize honey consumption and obtain a more concentrated extract. Nevertheless, a lower S/F ratio was not considered, as the high viscosity of honey made it difficult to centrifuge larger quantities of CBS dispersed in the medium.

Based on the findings from the Plackett–Burman screening, a FCD was subsequently employed to optimize the three most significant variables: extraction time (x1), water content in honey (x2), and lactic acid content (x3) (Table S4). The range of each factor was maintained despite their positive effects, to avoid conditions that could compromise extraction integrity, i.e., excessive heating, dilution of the honey matrix, or acidification beyond higher values for potential food and nutraceutical applications. An increased time leads to a higher temperature as the thermal energy accumulates during ultrasound-assisted extraction, and consequently, could start to degrade phenolic compounds, as previously reported for (−)-epicatechin and procyanidin B2. These compounds undergo epimerization and oxidative transformations above 60 °C, forming dimers, trimers, and other degradation products that reduce their bioactivity and nutritional value. (33) In terms of water content, a higher water content in SBH could promote microbial fermentation, thereby reducing its quality and shelf life. (34) Similarly, excessive acid addition could negatively impact sensory characteristics, especially considering the potential for direct consumption.

As a result of the FCD, eqs 2 and 3 represent the predictive models for theobromine (y1) and caffeine (y2) extraction, respectively. Both models exhibited satisfactory fits, with coefficients of determination (R2) of 88.73% and 92.53% for theobromine and caffeine, respectively. For theobromine (eqs 2), extraction yield was significantly influenced by extraction time (x1), showing a positive linear effect and a negative quadratic effect, as well as by the interaction between time and lactic acid content (x1x3), and between water and lactic acid content (x2x3). In the case of caffeine (eqs 3), the model revealed a more complex response, with significant quadratic effects of extraction time (x12) and lactic acid (x32), as well as interactive effects similar for theobromine.

y1=9.85+1.19x1−2.18x12−0.65x1x3+0.54x2x3

(2)

y2=0.96+0.08x1−0.17x12−0.06x32−0.05x1x3+0.05x2x3

(3)

The predicted optimal condition for theobromine extraction was 10.78 mg/g, achieved at 3.84 min of extraction time, 20% water content in honey, and no lactic acid addition. The highest predicted yield for caffeine was 0.99 mg/g, obtained under slightly different conditions: 3.63 min, 20% water, and approximately 2.26% lactic acid. Therefore, the optimal condition was set at a slightly lower value to ensure precise application using the ultrasound equipment: 3.5 min of extraction time, 20% water content, and no lactic acid addition, with the S/F and ultrasound power previously fixed at 20 g/g and 400 W, respectively. Although the optimal values for water and acid content were at the lower end of the tested range, the response surfaces (Figure S1) show that higher concentrations also improved extraction under certain conditions, especially for caffeine, which exhibited a broader optimal region and notable x2x3 interaction. In contrast, theobromine extraction was more sensitive to elevated modifier levels. Extraction time (x1) consistently showed a positive linear effect, reinforcing the benefit of longer sonication for both compounds.

Moreover, an important consideration when coupling SBH with UAEprobe is that, as neat honey contains elevated viscosity, it promotes localized heating during ultrasound propagation. A similar effect has also been reported for deep eutectic solvents (DES) coupled with UAE, as described by Siddiqui et al. (2025). (35) This temperature increase may have enhanced the diffusion and solubilization of the target compounds. On the other hand, at higher concentrations of water or lactic acid, the viscosity of the system was significantly reduced, resulting in a more fluid medium that improved ultrasound wave propagation, cavitation, and mass transfer. (36) Additionally, the acidifying effect of lactic acid may further enhance solubility and alter the structural properties of the matrix. (37) This effect contributes to more efficient mass transfer and improved extraction yields, especially in complex plant-based matrices such as CBS. Moreover, each condition, i.e., high viscosity with localized heating versus reduced viscosity with improved cavitation, may potentially offer specific extraction advantages.

In addition to the factorial combinations and optimal condition, a control experiment using neat honey, i.e., without added water or lactic acid, was also performed under the same optimal ultrasound conditions. The neat SBH extract yielded an average of 8.71 ± 0.10 mg/g of theobromine and 0.77 ± 0.01 mg/g of caffeine. In comparison, SBH with 20% added water yielded 11.12 ± 1.41 mg/g of theobromine and 0.84 ± 0.09 mg/g of caffeine. Although numerically higher, these differences were not statistically significant (p > 0.05). Using neat honey may also be advantageous, as it preserves the natural composition, stability, and sensory properties of the final extract. Moreover, SBHs naturally contains a considerable amount of water and has a reduced pH (Table 1), which plays a critical role in modulating its physicochemical behavior and thermodynamic advantages during extraction processes. On the other hand, adding cheaper components such as water and lactic acid can offer practical benefits, such as reducing production costs and potentially enhancing mass transfer and extraction efficiency.

Based on these results, neat SBHs was selected for the subsequent experimental steps. This allowed us to assess, under the same optimal UAEprobe conditions, whether the other four SBHs, characterized by lower viscosity and higher natural water content (Table 1), could exhibit similar or improved extraction performance.

3.3. Quantification of Bioactive Compounds and Antioxidant Properties

Figure 2 illustrates the phenolic compounds content and antioxidant properties of crude SBH and their respective CBS extracts obtained via UAEprobe. Across all measurements, i.e., (TPC; Figure 2A), ABTS (Figure 2B), DPPH (Figure 2C), and FRAP (Figure 2D), a clear enhancement in antioxidant activity was observed in the extracts compared to the crude honeys, confirming the effective transfer of bioactive compounds from CBS to the honey matrix through the extraction process.

Figure 2

Figure 2. Total phenolic content (TPC; A) and antioxidant capacities measured by ABTS (B), DPPH (C), and FRAP (D) assays of five stingless bee honeys and their respective cocoa bean shell extracts obtained by ultrasound-assisted extraction (UAEprobe): Tetragona clavipes, Tetragonisca angustula, Melipona quadrifasciata, Scaptotrigona postica, and Frieseomelitta varia. Water was included as a reference solvent. Crude (left) represents the properties of the pure honeys, while Extract (right) corresponds to the cocoa bean shell extracts obtained using each honey as the extraction solvent. Different lowercase letters indicate statistically significant differences between groups according to Tukey’s multiple comparison test (p < 0.05).

While water also demonstrated efficient extraction performance, honey-based extracts presented higher values in specific assays, particularly in terms of TPC and DPPH. This highlights the dual role of SBHs as both extraction media and sources of bioactive compounds that may act synergistically with those derived from CBS. Moreover, an interesting result was observed for the honey of M. quadrifasciata, which consistently exhibited the lowest antioxidant capacity in its crude state. Following extraction, it showed a marked increase across all parameters, indicating that this SBH possesses a notable capacity for extracting and enriching bioactive compounds. This characteristic is likely related to the solubility of this SBH for antioxidant compounds and its lower-intermediate viscosity (Table 1), which may have facilitated heat transfer and allowed it to reach a temperature similar to that of water during the extraction process (Figure S2A), in comparison to the other SBH samples.

Regarding the TPC of crude SBHs, our findings are consistent with those of Biluca et al. (2016), who reported a similar range in honeys from other stingless bee species, with values ranging from 0.103 to 0.980 mg GAE/g. (27) In our study, the TPC ranged from 0.460 ± 0.008 mg GAE/g for M. quadrifasciata to 1.286 ± 0.259 mg GAE/g for T. clavipes, respectively. In another study, the use of honey from A. mellifera, which is characterized by higher viscosity and lower water content, was tested as an extraction solvent. (38) Funari et al. (2019) employed honey from this species to extract compounds from propolis, achieving interesting results, although primarily for highly polar compounds. In line with this, the present study also focused on extracting methylxanthines from CBS, given their more polar nature compared to phenolic compounds.

Thereafter, the contents of theobromine and caffeine in CBS extracts obtained using five SBHs and water, under two UAE methods, i.e., the optimized UAEprobe condition and the reference UAEbath, are shown in Figure 3. Overall, UAEprobe demonstrated significantly higher extraction efficiency even in a short time (3.5 min) for both methylxanthines compared to UAEbath with employed 45 min, confirming the advantages of high-intensity ultrasound in promoting effective cell disruption, cavitation, and solute-matrix mass transfer. This trend was particularly evident in the context of viscous extraction media such as honey, where direct energy delivery is essential to overcome diffusional limitations. Moreover, UAE has already proven to be an efficient technique for extracting bioactive compounds from cocoa byproducts, using both low-viscosity solvents such as hydroethanolic mixtures and more viscous green solvents such DES. (39,40)

Figure 3

Figure 3. Theobromine (A) and caffeine (B) contents in cocoa bean shell extracts obtained using five stingless bee honeys and water as extraction solvents, comparing two ultrasound-assisted extraction methods: probe (UAEprobe) and bath (UAEbath). The honeys used were from Tetragona clavipes, Tetragonisca angustula, Melipona quadrifasciata, Scaptotrigona postica, and Frieseomelitta varia. Different lowercase letters indicate statistically significant differences between groups according to Tukey’s multiple comparison test (p < 0.05).

Considering the solvent systems, SBH samples with higher water content and lower viscosity generally exhibited higher average extraction yields, although not all differences were statistically significant. Among the tested matrices, the reference solvent water, recognized as a green, food-grade medium with high solubility for methylxanthines, showed elevated recoveries for both theobromine and caffeine under optimized UAEprobe conditions. For instance, at 25 °C, theobromine exhibits higher solubility in water than in methanol, ethanol, 1-propanol, ethyl acetate, or acetone. (41) Meanwhile, caffeine exhibits higher solubility in water than in ethanol at the same temperature. The highest theobromine recoveries were observed for T. clavipes (9.78 ± 0.48 mg/g) and M. quadrifasciata (9.80 ± 0.24 mg/g), which were comparable to water (10.40 ± 0.01 mg/g) (Figure 3A). For caffeine, T. clavipes (0.98 ± 0.01 mg/g), M. quadrifasciata (0.96 ± 0.02 mg/g), and T. angustula (0.84 ± 0.07 mg/g) exhibited the highest recoveries among the honeys, also comparable to water (1.00 ± 0.01 mg/g) (Figure 3B). In this case, the superior performance of these SBHs compared to the others may be attributed to their physicochemical properties, particularly their lower viscosity (Table 1) and the lower temperatures reached during extraction (Figure S2A), which more closely resemble the behavior of water. This behavior highlights the impact of lower viscosity on the extraction efficiency of theobromine and caffeine. Furthermore, these findings indicate that specific SBH types can closely match the performance of water for methylxanthine recovery when combined with high-intensity ultrasound, supporting their potential as effective and edible extraction solvents. Nevertheless, beyond technical performance, the use of SBHs also contributes additional functional properties to the extracts, such as antioxidant, antimicrobial, and prebiotic potential, while aligning with sustainability goals through the valorization of native biodiversity and traditional knowledge. (42,43)

Figure S2illustrates two other critical aspects of the UAE process employing honeys as solvents: the temperature evolution over time (A) and the formation of 5-hydroxymethylfurfural (5-HMF) (B), a key indicator of thermal degradation. Honeys with higher viscosity, such as F. varia, exhibited the fastest and highest temperature increase, reaching around 79 °C at 3.5 min, which was considerably higher compared to water, which reached approximately 56 °C at the same time. These differences reflect the role of viscosity in ultrasound propagation, as already discussed in section 3.2. Moreover, the neat honeys exhibited a higher temperature increase, as observed during the DoE optimization steps, with values mostly ranging between 40–60 °C. Although higher viscosity can lead to increased temperatures and potentially enhance extraction, it also results in reduced mass transfer. In this context, and as observed in our study, honeys with intermediate to low viscosity such as those from S. postica and M. quadrifasciata resulted in higher extraction yields.

On the other hand, higher temperatures can also increase the risk of thermal degradation, leading to the formation of 5-HMF, a compound derived from sugar degradation. (44) Nevertheless, 5-HMF remained undetectable in honeys such as T. angustula and M. quadrifasciata, but increased significantly in S. postica (nondetectable levels before and 0.230 ± 0.014 after UAEprobe) and F. varia (0.192 ± 0.001 mg/g before and 0.275 ± 0.007 mg/g after UAEprobe). These results indicate that high-temperature profiles can compromise product quality, particularly in food and nutraceutical applications where 5-HMF is a regulated contaminant. (45) Even though 5-HMF has been detected in SBHs, the levels generally do not exceed 0.060 mg/g, with most samples presenting values below 0.040 mg/g or even nondetectable. (24) In our study, the highest 5-HMF content in neat SBH was observed in F. varia, which may be attributed to factors such as its sugar concentration, potential spontaneous fermentation, and water activity. Moreover, the subsequent increase in 5-HMF content was associated with the higher temperature reached during extraction.

3.4. Environmental, Social, and Economic Impacts of Extraction Process Based on Path2Green

The evaluation of the CBS extraction process using UAEprobe and SBHs as natural solvents through the Path2Green metric offers a comprehensive perspective on its sustainability, based on the 12 principles of green biomass valorization (1) (Table S5). Overall, the process presents several advantages aligned with green chemistry and circular bioeconomy principles, while also revealing areas that merit improvement to enhance its overall sustainability profile. The use of CBS as raw material strongly contributes to the sustainability of the process (Principle 1), as this agro-industrial byproduct is naturally sourced, low-cost, and typically discarded. This valorization aligns well with circular economy strategies and received the highest score in the Path2Green assessment.

In terms of solvent selection (Principle 4), the process achieved a full score (+1.00) by employing SBHs, edibles, biodegradables, and ready-to-use solvents, which eliminated the need for downstream purification and allowed the direct application of the enriched extract. This also positively influenced Principle 6 (Purification) and Principle 8 (Post-treatment), as no additional steps were required following extraction, significantly reducing energy and material inputs. (46) Moreover, the final product, rich in theobromine, caffeine, and phenolic compounds, is safe and versatile for food, nutraceutical, and cosmetic applications, thus fully addressing Principle 10 (Application).

Despite these strengths, the method exhibited limitations related to scalability (Principle 5), as the extractions were conducted in batch mode, which hinders reproducibility and continuous processing. (47) Similarly, while UAE is recognized as a high-efficiency technique, its energy demand (Principle 9) led to a moderate score (−0.50), even though renewable energy sources were used. The extraction yield (∼20%) also impacted Principle 7 (Yield), as a large proportion of the biomass remained unexploited. This contributed to additional challenges under Principle 12 (Waste Management), where the estimated 80% of residual biomass led to a score of – 0.60. (48)

Regarding biomass transport (Principle 2), the process incurred a modest penalty due to the 200 km distance between biomass source and processing site, which translated to a score of +0.12 according to the established logarithmic correlation. Finally, Principle 11 (Repurposing) received a neutral score (0.00), since the use of honey as an edible solvent precludes its recovery or reuse, although it does not generate hazardous waste.

The Path2Green score for the developed UAEprobe method for CBS extraction using SBHs was +0.118, as illustrated in the summary pictogram (Figure 4). The distribution of scores reflects a method that is innovative, food-safe, and aligned with several green extraction principles, but that also requires further development, particularly in terms of energy optimization, process scaling, and residue valorization, to fully maximize its sustainability potential.

Figure 4

Figure 4. Pictogram showing the final Path2Green score of +0.118, which represents how well the process aligns with the 12 principles of green extraction from biomass. Each principle is illustrated with a color-coded icon, where green indicates strong adherence, yellow reflects a neutral score, and red signals low adherence and areas for improvement.

4. Conclusions

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This study presents an innovative and intensified process for valorizing CBS by employing UAEprobe employing SBHs as a natural and edible solvent. Moreover, it generated a ready-to-use final product rich in methylxanthines and phenolic compounds, exhibiting high bioactivity. This is potentially of interest to the food and cosmetic sectors, while also aligning with biorefinery and circular economy principles by valorizing an agro-industrial byproduct. Additionally, the use of SBHs, a biodiversity-derived natural product, contributes to the promotion of socio-biodiversity and supports local socioeconomic development. The proposed methodology involved sequential optimization steps, achieving an optimal condition of 3.5 min of extraction time, using neat honey, at a S/F ratio of 20 g/g and an ultrasound power of 400 W. Through this process, it was possible to recover high contents of both methylxanthines, theobromine and caffeine, as well as to increase the levels of phenolic compounds and bioactivity in the honey used as the extraction solvent, compared to the crude samples.

Finally, and from a future perspective, the viability of potential applications will require additional experiments: (i) stability, (ii) degradation pathways, (iii) microbiological tests, and (iv) sensory analyses focused on organoleptic properties preferred by consumers. These will enhance and ensure product acceptance.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.5c04842.

(Figure S1) Response surface plots and contour graphs from the face-centered design showing effects of time, water content, and lactic acid on theobromine and caffeine extraction; (Figure S2) temperature profiles during UAE and 5-HMF quantification before and after extraction; (Table S1) Plackett–Burman design matrix and responses for theobromine and caffeine extraction; (Table S2) face-centered design matrix and responses; (Table S3) ANOVA results for Plackett–Burman design; (Table S4) ANOVA results for face-centered design; (Table S5) detailed scoring of the Path2Green metric based on the 12 principles of green extraction (PDF)

• sc5c04842_si_001.pdf (855.08 kb)

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Author Information

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Felipe Sanchez Bragagnolo - Laboratório Multidisciplinar de Alimentos e Saúde (LabMAS), Faculdade de Ciências Aplicadas (FCA), Universidade Estadual de Campinas (UNICAMP), RuaPedro Zaccaria, 1300, 13484-350, Limeira, São Paulo, Brasil; https://orcid.org/0000-0002-8017-9551; Email: [email protected]

Mauricio Ariel Rostagno - Laboratório Multidisciplinar de Alimentos e Saúde (LabMAS), Faculdade de Ciências Aplicadas (FCA), Universidade Estadual de Campinas (UNICAMP), RuaPedro Zaccaria, 1300, 13484-350, Limeira, São Paul