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Article

Sustainable Extraction of Bioactive Compounds from Cocoa Shells Waste and Brewer’s Spent Grain Using a Novel Two-Stage System Integrating Ohmic-Accelerated Steam Distillation (OASD) and Supercritical CO2 Extraction (SSCO2)

by
Hao-Yu Ivory Chu
,
Xinyu Zhang
,
Yuxin Wang
,
Taghi Miri
* and
Helen Onyeaka
School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
*
Author to whom correspondence should be addressed.
Sustainability 2025, 17(16), 7373; https://doi.org/10.3390/su17167373
Submission received: 12 June 2025 / Revised: 4 August 2025 / Accepted: 12 August 2025 / Published: 14 August 2025
(This article belongs to the Section Waste and Recycling)
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Abstract

This study introduces a novel, two-stage extraction system that combines Ohmic-Accelerated Steam Distillation (OASD) with Supercritical CO2 Extraction (SSCO2) to efficiently recover bioactive compounds from plant-based wastes with varying cell wall complexities. Brewer’s spent grain (BSG) and cocoa shell were selected as representative models for soft and rigid cell wall structures, respectively. The optimized extraction process demonstrated significantly enhanced efficiency compared to traditional methods, achieving recovery rates in BSG of 89% for antioxidants, 91% for phenolic acids, and 90% for polyphenolic compounds. Notably, high yields of p-coumaric acid (95%), gallic acid (94%), ferulic acid (82%), quercetin (87%), and resveratrol (82%) were obtained with minimal cellular structural damage. For cocoa shells, despite their lignin-rich, rigid cell walls, recovery rates reached 73% for antioxidants, 79% for phenolic acids, and 74% for polyphenolic compounds, including chlorogenic acid (94%), catechin (83%), vanillin (81%), and gallic acid (94%). Overall, this hybrid technique significantly improved extraction efficiency by approximately 60% for BSG and 50% for cocoa shell relative to conventional approaches, highlighting its novelty, scalability, and potential for broad application in the sustainable valorization of diverse plant-based waste streams. This research presents a green and efficient platform suitable for valorizing agri-food by-products, supporting circular economy goals. Further studies may explore scale-up strategies and economic feasibility for industrial adoption.

1. Introduction

The global cocoa industry generates approximately 1 million tons of cocoa shell waste annually [1], while the brewing industry produced up to 39 million tons of Brewers’ spent grain (BSG) [2]. Although bioactive compounds are present only in small quantities, their antioxidant and phenolic components are highly valuable according to their potent biological activities [3,4]. Natural antioxidants and phenolics effectively delay food oxidation, extend shelf life, and preserve sensory attributes such as color and flavor [5,6]. Additionally, these compounds offer significant health benefits, including reduced risks of chronic diseases and enhanced nutritional and functional properties in food products [7].
However, traditional extraction techniques for these bioactive compounds face several limitations, including low extraction efficiency, high energy consumption, extensive use of organic solvents, and inconsistent product purity. These limitations have hindered the commercial viability of such processes [8,9]. Moreover, significant structural differences in the cell walls of various agricultural by-products make it difficult for conventional single-method extractions to be universally applicable [10]. For instance, cocoa shells exhibit a high cell wall hardness of approximately 19.2 MPa [11], primarily due to their elevated insoluble fiber content, which ranges from 50.1% to 64.1% [12]. This structural rigidity presents challenges in cell disruption and limits the efficient recovery of intracellular compounds [12,13]. To address this, methods such as supercritical CO2 extraction (SSCO2), microwave-assisted extraction (MAE), and ultrasound-assisted extraction (UAE) have been widely applied. These techniques leverage high pressure, thermal energy, or ultrasonic waves to break down or expand rigid cell walls [14,15,16,17,18]. However, they also introduce drawbacks, such as over-disruption of cellular structures, leading to decreased compound purity and high operating temperatures that degrade thermolabile bioactives, ultimately affecting final product quality [19,20,21].
In contrast, BSG has softer cell walls, with a hardness of approximately 2.62 MPa [22], attributed to its lower insoluble fiber content (15–20%) [23]. For such materials, techniques including hydrothermal extraction, acid or enzymatic hydrolysis, and subcritical water extraction (SWE) have proven effective in degrading cell structures and facilitating compound release [24,25]. Thus, selecting an appropriate extraction strategy tailored to the specific cell wall structure is crucial for maximizing recovery efficiency [26,27]. For hard cell wall materials, SSCO2 and UAE remain commonly used [28]. Despite their effectiveness in disrupting rigid cell structures, these techniques still face critical limitations, such as excessive structural breakdown that compromises selectivity and purity and thermal degradation of sensitive compounds [29].
Most importantly, due to the structural diversity of agricultural residues existing extraction methods struggle to accommodate both rigid and soft cell wall types simultaneously, posing a major challenge for industrial scale applications [26,30,31]. To address this, the present study proposes a novel two-stage extraction system designed to optimize the recovery of bioactive compounds from materials with differing cell wall structures. In the first stage, ohmic-accelerated steam distillation (OASD) [32,33] is employed as a pre-treatment. OASD provides rapid, uniform internal heating via electric currents, which softens cell walls and facilitates the release of intracellular contents. Simultaneously, it integrated steam distillation to recover heat-sensitive volatile and semi-volatile bioactive compounds. In the second stage, supercritical CO2 extraction (SSCO2) [34] is applied to further extract non-volatile and less heat-sensitive components. A graphical abstract summarizing the approach is presented in Figure 1.

2. Materials and Method

2.1. Material and Chemicals

Brewers’ spent grain (BSG) was collected fresh (80% moisture) from ATTICBREW CO. (Birmingham, UK), and cocoa shells (40% moisture) were sourced from Cadbury (Birmingham, UK). Both samples underwent the same preparation procedure: freeze-drying at −40 °C for 24 h until reaching approximately 2% moisture, followed by grinding (IKA-Werke GmbH & Co., Staufen im Breisgau, Germany) into fine powder and sieving through a 0.5 mm mesh to reduce particle size variation. The processed powders, characterized by irregularly shaped particles, were stored at 4 °C in airtight containers until use. Sodium chloride (NaCl) and all other chemicals used in the extraction processes were supplied by Merck Chemicals Ltd. (Southampton, UK).

2.2. Two-Stage Extraction System

A novel two-stage extraction process was applied, noted as “Two-stage (OASD + SSCO2)”, consisting of ohmic-accelerated steam distillation (OASD) followed by supercritical CO2 extraction (SSCO2). For OASD, 10 g of biomass was placed in a 100 mL three-necked glass reactor fitted with two stainless steel electrodes spaced 4.25 cm apart and separated by a PTFE cover. The setup (Figure 2) [35] was operated at 30 V, with the temperature maintained about 30–35 °C. Steam distillation was integrated using standard glassware (Sigma-Aldrich, Darmstadt, Germany) and a condenser [36]. A mild alkaline solution (0.01 M NaOH) was used to enhance extraction efficiency [37].
For the second stage, supercritical CO2 extraction was carried out using a pilot-scale SSCO2 unit (Figure 3) equipped with CO2 cylinders and a high-pressure pump (VERITY 3011, Gilson, UK). Each experiment used 10 g of pre-treated biomass, packed in the extraction vessel between a stainless-steel mesh and a 90 mm paper filter to avoid sediment displacement. The system was pre-cooled to 2 °C, and ethylene glycol was added to prevent freezing. CO2 was liquefied at −20 °C and delivered at 240 bar [34]. Extraction was performed at 45 °C [38] with a CO2 flow rate of 1–3 mL/min [39], following protocols adapted from previous studies [40,41].

2.3. Measurement and Quantification

2.3.1. HPLC

Bioactive compounds including antioxidants, fatty acids and alkaloids were identified and quantified using reverse-phase HPLC (Agilent 1260 Infinity II HPLC (Agilent Technologies, Santa Clara, CA, USA)). Samples were dissolved in HPLC-grade methanol (1–5 g/mL for antioxidants, [42] 0.5–1 mg/L for fatty acids [43], 0.1–100 µg/mL for alkaloids [44,45]). Specific reversed-phase C18 columns (250 mm × 4.6 mm, 5 µm) were used under tailored gradient elution programs, with flow rates of 1.0–1.2 mL/min and UV detection at 210, 254, 280, and 360 nm depending on the compound class. Mobile phases included mixtures of water, methanol, acetonitrile, sulfuric acid, and formic acid, optimized per compound type.

2.3.2. Quantification of Bioactive Materials

Quantification Assays
Phenolic compounds were quantified using a commercial Phenolic Compounds Assay Kit (Sigma, Livonia, MI, USA) with a catechin-based standard curve (0–10 nmol/well), measuring absorbance at 480 nm. Total antioxidant capacity (TAC) was determined using the Sigma MAK334 kit (Livonia, MI, USA) with Trolox standards (0–1000 µM), measuring absorbance at 570 nm. Both assays were conducted in 96-well plates, and results were expressed as catechin or Trolox equivalents.

2.3.3. Confocal Laser Scanning Microscopy (CLSM)

CLSM DM5000B (Leica, Wetzlar, Germany) was used for structural and compound-specific imaging, analyzed with Fiji/ImageJ. Samples were stained with specific dyes: Safranin O/Toluidine blue for fibers [46], fluorescent ROS probes for antioxidants [47], ferric chloride/potassium cyanide and Fastgreen for phenolics [48], and Coomassie blue R-250 for polyphenols [49]. Excitation/emission wavelengths ranged from 405/633 nm to 488/210 nm.

2.3.4. Extraction Rate

Post-extraction, antioxidant contents were quantified by HPLC and the extracts were freeze-dried (SP Scientific, Warminster, PA, USA) at −40 °C. The extraction rate (%) was calculated using the following equation:
Extraction Rate   ( % ) = ( E x t r a c t e d   C o n c . ( m g L ) O r i g i n a l   C o n c . ( m g L ) ) × 100
where Extracted Conc. represents the antioxidant concentration measured in the extract solution, and Original Conc. corresponds to the antioxidant concentration determined from the raw BSG and cocoa shell samples prior to extraction, both expressed on a dry weight basis. All extraction procedures, separation processes, and analytical measurements were performed in triplicate, and results are presented as mean ± standard deviation to ensure reproducibility and statistical reliability.

3. Result and Discussion

3.1. Demonstration of Comprehensive Applicability: Analysis of Hard and Soft Cell Walls

The extraction efficiency of the proposed two-stage system (OASD + SSCO2) was demonstrated through the recovery rates of antioxidant and Phenolic compounds from two representative substrates. Figure 4 presents a comparison of extraction rates obtained from BSGs and cocoa shells. Error bars represent standard deviations from triplicate experiments and suggest good reproducibility across measurements.
The extracted antioxidant content was slightly higher in BSG (82.5%) than in cocoa shell (79.8%). Similarly, phenolic compounds were more efficiently extracted from BSGs (88.2%) compared to cocoa shells (83.6%). In both cases, extraction rates exceeded 75%, indicating effective compound recovery for both substrates using the integrated system. This yield is considerably higher than those typically reported for single-stage extraction methods, such as pressurized liquid extraction [50], which often achieve less than 30% recovery [2] while also requiring longer processing times and greater solvent consumption.
The extraction efficiencies for phenolic compounds were consistently higher than those for antioxidants in both materials. This outcome is expected, as phenolic compounds are generally more polar and more uniformly distributed within the plant matrix, making them more easily released under the applied extraction conditions [51]. Similar trends have been reported in previous studies [52] using both conventional and emerging extraction techniques, confirming the consistency and comparability of our findings.

3.2. HPLC-Based Identification and Compound-Specific Recovery Analysis

The extracted bioactive compounds from BSG using the two-stage OASD + SSCO2 system were analyzed by HPLC. Representative chromatograms are shown in Figure 5, with peak identities listed in Table 1. For comparative purposes, Soxhlet extraction method, a well-established conventional technique, was independently performed and served as an internal benchmark for assessing the efficiency of the two-stage OASD + SSCO2 system.
The BSG chromatogram (Figure 5) revealed a wide range of peaks, corresponding to 27 identified bioactive compounds, including fatty acids, phenolic acids, flavonoids, and antioxidants. According to Table 1, the highest extraction rate which using the two-stage system (OASD + SSCO2) included: p-coumaric acid (95%), gallic acid (94.6%), quercetin (86.9%), and ferulic acid (82.6%). Other notable recoveries were syringic acid (72.6%), kaempferol (55.6%), and vanillic acid (9%). In contrast, lipid-based compounds like lecithin and phospholipids showed significantly lower recovery rates (3% and 2%, respectively), likely due to their poor solubility in CO2 under the applied conditions.
The total extracted antioxidant yield from BSG was 9.6 mg/g, surpassing the Soxhlet reference of 3.92 mg/g, indicating a more efficient and selective recovery of functional compounds via the two-stage (OASD + SSCO2) method.
As summarized in Table 2, the two-stage OASD + SSCO2 system achieved consistently high recovery rates of key phenolic compounds from soft cell wall BSG. For p-coumaric acid our method reached a recovery of 95%, which is remarkably higher than 21% reported by Baltacıoğlu et al. using high-pressure processing (HPP) at 500 MPa, 10 min, 20 °C, and 80% methanol + 1% hydrochloric acid as solvent [53]. This improvement can be attributed to two key factors: (i) the OASD pre-treatment, which enhances cell disruption through electric field induced swelling and localized heating, and (ii) the tunable solvent properties of supercritical CO2, which improve solubility and selectivity for phenolic compounds.
A similar trend was observed for gallic acid, where our recovery rate reached 94%, compared to the 68% obtained by Zhang et al. using microwave-assisted simultaneous hydrodistillation, hydrolysis, and extraction (MSHDHE) with natural acidic deep eutectic solvents (NADESs) from liquidambar formosana leaves [56]. For ferulic acid, our method yielded 82%, exceeding the 78% maximum reported by Pazo-Cepeda et al. using pressurized microwave water extraction (PMWE) and pressurized hot water extraction (PHWE) from wheat bran [58]. Quercetin recovery in our study was 87%, which is comparable to the 89% reported by Chandan et al. using microwave-assisted extraction combined with a three-enzyme system (pectinase, hemicellulose, and cellulase) from onion peel [60]. It is important to note that the comparison samples were selected not only for their recovery methods but also for their comparable insoluble fiber contents, as fiber levels strongly influence compound release. Collectively, these findings confirm that the two-stage OASD + SSCO2 system is highly effective for recovering bioactive compounds from soft cell wall materials such as BSG. The approach achieves high extraction efficiencies while preserving compound integrity, demonstrating its suitability for samples with moderate fiber content and weaker cell wall structures.
The outcome of the rigid cell wall requires analysis. Cocoa shells using the two-stage OASD + SSCO2 system were analyzed by HPLC in Figure 6 and Table 3.
Figure 6 and Table 3 show the compound profile from cocoa shell extracts. A total of 22 compounds were identified. Notably, the highest extraction yields from the two-stage system were for: gallic acid (93%), chlorogenic acid (93%), vanillin (81%), quercetin (74%), and catechin (83%). Lower recoveries were observed for caffeic acid (50%) and ferulic acid (62%). Similarly to BSG, lipid-based compounds such as oleic acid and linoleic acid showed limited extraction efficiency (1–3%).
The total antioxidant yields from cocoa shells was 33.6 mg/100 g, slightly lower than the Soxhlet benchmark of 37.46 mg/100 g, suggesting that for certain compounds, particularly polar and hydrophobic ones, Soxhlet may still outperform SSCO2 unless co-solvents are introduced.
The experimental results indicated that antioxidants had the highest extraction rate. The key components and their extraction rates were as follows: chlorogenic acid (93%), gallic acid (93%), catechin (83%) and vanillin (81%).
As shown in Table 4, the two-stage OASD + SSCO2 system achieved high recovery rates for several bioactive compounds from cocoa shells. For chlorogenic acid, the extraction rate reached 93%, slightly higher than the 92% reported by Gupta et al., who used tetrahydrofuran (THF) as a solvent and itaconic acid as a functional monomer to synthesize molecularly imprinted polymers (MIPs) for purification from spent coffee beans [61]. While the yields were comparable our method avoided toxic solvents and complex polymer synthesis, instead using ohmic heating to soften the cell wall followed by high-pressure CO2 extraction, providing a greener, simpler, and more energy-efficient approach.
The gallic acid extraction rate was also 93%, exceeding the 9% reported by Gavarić et al. who extracted gallic acid from rosehip seed waste using ultrasound-assisted extraction (UAE) with natural deep eutectic solvents (NADESs) following supercritical CO2 pretreatment [64]. Cocoa shells, with their relatively high insoluble fiber content, were specifically compared to materials with lower fiber levels, such as mango seeds and rosehip seeds, to highlight the advantage of our system in overcoming cell wall barriers.
For catechin, the recovery rate was 83%, substantially higher than the ~27% obtained by Nathalia et al. from Euterpe oleracea (açaí) using energized dispersive guided extraction (EDGE) [66]. Similarly, vanillin recovery in our study reached 81%, compared to the 43% achieved by Xu et al. using NADES-based extraction from vanilla pods under optimized conditions (33.9% water content, 64.6 °C, 32.3 min, and a solid–liquid ratio of 44.9 mg/mL) [68].
Following OASD treatment, the cell wall architecture became more porous, significantly enhancing the efficiency of subsequent extraction steps. In the second stage, the extraction technique was selected based on the specific cell wall characteristics of the treated sample.
For rigid cell wall materials pre-treated with OASD, SSCO2 was employed to further disrupt the structure and promote compound release [69]. Through this dynamic and adaptable two-stage system, the study achieved high recovery yields while improving product purity and reducing both energy and solvent consumption. The innovation of this research lay in the integration of OASD as a high-efficiency pre-treatment technique, followed by SSCO2 extraction.
This section of the work indicates the technique works for both hard and soft cell walls. It is important to investigate more in the next stage the extraction of cell walls and cell interiors to confirm if our approach can entirely remove the antioxidants within the cells.

3.3. Imaging Analysis Provides Includes Qualitative and Quantitative Assessment of Antioxidant Release from Cells

Confocal laser scanning microscopy (CLSM) was used to visualize the cell wall disruption caused by OASD pre-treatment and confirm the release and localization of antioxidant and polyphenolic compounds after HPLC and kit analyses.
For BSG (Figure 7), qualitative CLSM imaging (yellow: cell wall; green: antioxidants; blue: phenolic and polyphenolic acids) revealed significant structural changes before and after the two-stage (OASD + SSCO2) treatment. These qualitative observations were supported by the quantitative fluorescence area analysis (Table 1). Antioxidant fluorescence decreased from 7899.9 ± 5 to 854.2 ± 34.6, corresponding to an 89.19% extraction efficiency. Phenolic acids decreased from 6941.5 ± 5 to 612.4 ± 3, giving a 91.18% extraction efficiency, while polyphenolic acids declined from 4215.2 ± 3 to 421.0 ± 2, equivalent to 90.01% efficiency. Additionally, cell wall fluorescence decreased by 37.39% (from 4878.0 ± 2 to 3054.1 ± 3), indicating partial degradation that likely facilitated compound release. Compared with conventional extraction approaches such as ultrasound-assisted extraction (UAE) [70] and single-stage supercritical CO2 (SFE) [71], which often suffer from lower selectivity, longer extraction times, or higher solvent consumption, the two-stage system achieved superior efficiency while maintaining sustainability advantages.
For cocoa shells (Figure 8), fluorescence area analysis also confirmed effective extraction (Table 5). Antioxidants decreased from 3124.6 ± 1 to 842.5 ± 2 (73.03% recovery), phenolic acids from 3012.6 ± 5 to 624.5 ± 8 (79.27%), and polyphenols from 2243.5 ± 1 to 567.5 ± 2 (74.70%). Notably, cell wall fluorescence remained largely unchanged (4015.1 ± 2 before extraction vs. 3841.9 ± 34.2 after), signifying that the two-stage method preserved overall structural integrity while enabling efficient compound release. Compared with single-stage approaches such as supercritical fluid extraction (SFE) [72], which often require longer processing times and may lead to incomplete cell wall disruption, the two-stage (OASD + SSCO2) system achieved higher efficiency while maintaining sustainability advantages. Together, these findings confirm that the OASD + SSCO2 system achieved high intracellular recovery of antioxidants and phenolic compounds (>70% across all cases) while maintaining cell wall integrity. In contrast, conventional methods such as high-pressure extraction [73] or chemical extraction [74] often cause structural damage and compromise compound stability. This advantage was evident in both BSGs, with their pliable cell walls, and cocoa shells, with their more rigid composition. Importantly, the method proved effective irrespective of biomass type, successfully addressing both the pliable cell walls of BSGs and the more rigid composition of cocoa shells. This demonstrates the system’s strong selectivity, efficiency, and broad applicability for bioactive compound recovery.

4. Conclusions and Future Work

A major challenge in the extraction of bioactive compounds from agro-industrial residues lies in the structural variability of biomass. Hard materials such as cocoa shells, with high fiber content and rigid cell walls, typically require different extraction conditions compared to softer residues like BSGs. Most conventional extraction methods are optimized for a single biomass type, which limits their broader applicability.
This study demonstrates that the proposed two-stage (OASD + SSCO2) system can overcome this limitation. By tailoring the extraction strategy to the structural characteristics of each material, the system enabled efficient recovery of bioactive compounds from both soft and rigid agricultural residues using a unified, non-toxic, and green approach. The use of water and supercritical CO2 minimizes environmental impact while maintaining high efficiency, aligning with sustainable development goals. Compared to conventional methods, the two-stage approach represents a more sustainable and environmentally friendly alternative for the valorization of agricultural by-products.
While the present study focused on BSGs and cocoa shells, the results suggest that the system holds potential as a broadly applicable platform for processing diverse plant-based residues. Further research should explore its use with additional feedstocks, assess techno-economic feasibility, and evaluate environmental performance. Although SSCO2 showed strong extraction efficiency, its high operating cost must be considered when evaluating industrial translation. Future investigations should therefore aim to balance performance with cost-efficiency to support the development of practical large-scale applications.
This work provides a foundation for advancing waste-to-resource technologies that support global sustainability initiatives and contribute to circular economy objectives.

Author Contributions

Conceptualization, T.M. and H.O.; Methodology, T.M.; Investigation, H.-Y.I.C., X.Z. and Y.W.; Resources, T.M.; Data curation, H.-Y.I.C.; Writing—original draft, H.-Y.I.C.; Writing—review & editing, T.M. and H.O.; Visualization, H.-Y.I.C. 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.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

BSGBrewers’ Spent Grain
SSCO2Supercritical CO2 Extraction
MAEMicrowave-Assisted Extraction
UAEUltrasound-Assisted Extraction
SWESubcritical Water Extraction
OASDOhmic-Accelerated Steam Distillation
HPLCHigh-Performance Liquid Chromatography
CLSMConfocal Laser Scanning Microscopy
pHPotential of Hydrogen

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Figure 1. Schematic illustration of the sustainable two-stage extraction system (OASD + SSCO2) for enhanced recovery of bioactive compounds from cocoa shells and Brewer’s spent grain.
Figure 1. Schematic illustration of the sustainable two-stage extraction system (OASD + SSCO2) for enhanced recovery of bioactive compounds from cocoa shells and Brewer’s spent grain.
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Figure 2. Schematic diagram of ohmic-accelerated steam distillation (OASD) apparatus.
Figure 2. Schematic diagram of ohmic-accelerated steam distillation (OASD) apparatus.
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Figure 3. Process flow diagram of supercritical CO2 extraction (SSCO2) system.
Figure 3. Process flow diagram of supercritical CO2 extraction (SSCO2) system.
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Figure 4. Comparison of antioxidant and phenolic compound extraction efficiencies from Brewer’s spent grains and cocoa shells using a two-stage system (OASD + SSCO2).
Figure 4. Comparison of antioxidant and phenolic compound extraction efficiencies from Brewer’s spent grains and cocoa shells using a two-stage system (OASD + SSCO2).
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Figure 5. HPLC peak profiles of extracts from BSGs by two-stage (OASD + SSCO2): (A) fatty acids, and (B) antioxidants. Details of peak 1–peak 27 on Table 1.
Figure 5. HPLC peak profiles of extracts from BSGs by two-stage (OASD + SSCO2): (A) fatty acids, and (B) antioxidants. Details of peak 1–peak 27 on Table 1.
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Figure 6. HPLC peak profiles of extracts from cocoa shells by two-stage (OASD + SSCO2): (A) fatty acids, and (B) antioxidants. The details of peaks information are in Table 3.
Figure 6. HPLC peak profiles of extracts from cocoa shells by two-stage (OASD + SSCO2): (A) fatty acids, and (B) antioxidants. The details of peaks information are in Table 3.
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Figure 7. Confocal laser scanning microscopy (CLSM) images showing cell wall integrity and distribution of antioxidants, phenolic acid, and polyphenolic acid before and after extraction from BSG.
Figure 7. Confocal laser scanning microscopy (CLSM) images showing cell wall integrity and distribution of antioxidants, phenolic acid, and polyphenolic acid before and after extraction from BSG.
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Figure 8. Confocal laser scanning microscopy (CLSM) images showing cell wall integrity and distribution of antioxidants, phenolic acid, and polyphenolic acid before and after extraction from cocoa shell.
Figure 8. Confocal laser scanning microscopy (CLSM) images showing cell wall integrity and distribution of antioxidants, phenolic acid, and polyphenolic acid before and after extraction from cocoa shell.
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Table 1. Comparison of bioactive compound yields and recovery rates from BSGs using a two-stage system (OASD + SSCO2).
Table 1. Comparison of bioactive compound yields and recovery rates from BSGs using a two-stage system (OASD + SSCO2).
S.NoCompounds Have Been Extracted Soxhlet Extraction Yield Reference Concentration (mg/L)Extraction Yield (mg/L) from Two-Stage (OASD + SSCO2)Extraction Rate for Two-Stage (OASD + SSCO2)(%)S.NOCompounds Have Been ExtractedSoxhlet Extraction Yield Reference Concentration (mg/L)Extraction Yield (mg/L) from Two-Stage (OASD + SSCO2)Extraction Rate for Two-Stage (OASD + SSCO2) (%)
1Caffeic Acid160.05102.43464%15Lecithin80.322.313%
2Ferulic Acid162.70 132.01782%16Phospholipids10.4320.22022%
3p-Coumaric Acid159.86153.13895%17Triglycerides1.02320.102410%
4Gallic Acid160.56150.92994%18Coumarins8.9210.935110%
5Quercetin161.02139.21686%19Lignans8.0210.92111%
6Kaempferol161.8389.00955%20Resveratrol9.1230.7137%
7Syringic Acid1.210.8827472%21Tocopherols E16.0210.86725%
8Vanillic Acid0.20010.018069%22Tocopherols K1.59880.080745%
9Linoleic Acid, C18:21.9421.1105457%23Kaempferol10.4320.70256%
10Oleic Acid, C18:11.8131.3024872%24Apigenin10.3240.2011%
11Palmitic Acid, C16:01.83421.4214877.5%25Luteolin0.945320.03904%
12Stearic Acid, C18:01.29320.322425%26Naringenin6.1321.70927%
13Alpha-Linolenic Acid, C18:32.01210.053123%27Curcumin10.4321.81917%
14Carotenoids15.4327.43249%
Table 2. Comparison of recovery rates of selected compounds from BSGs, alongside samples with comparable cell wall stiffness.
Table 2. Comparison of recovery rates of selected compounds from BSGs, alongside samples with comparable cell wall stiffness.
CompoundSampleInsoluble Fiber in SampleRecovery MethodRecovery RateReference
p-Coumaric acidBSG15–30%Two-stage (OASD + SSCO2)95%
Peach pomace14% [54]High-pressure processing (HPP)21%[53]
Gallic acidBSG15–30%Two-stage (OASD + SSCO2)94%
Liquidambar formosana leaves1–3% [55]Microwave-assisted simultaneous hydrodistillation68%[56]
Ferulic acidBSG15–30%Two-stage (OASD + SSCO2)82%
Wheat bran43% [57]Microwave water extraction (PMWE)78%[58]
QuercetinBSG15–30%Two-stage (OASD + SSCO2)87%
Onion peel14.2% [59]MAE89%[60]
Table 3. Extraction and quantification of bioactive compounds from cocoa shell.
Table 3. Extraction and quantification of bioactive compounds from cocoa shell.
S.No.Extracted CompoundsSoxhlet Extraction Yield Reference Concentration (mg/L)Extraction Yield (mg/L) from Two-Stage (OASD + SSCO2)Extraction Rate for Two-Stage (OASD + SSCO2)(%)S.No.Extracted CompoundsSoxhlet Extraction Yield Reference Concentration (mg/L)Extraction Yield (mg/L) from Two-Stage (OASD + SSCO2)Extraction Rate for Two-Stage (OASD + SSCO2) (%)
1Quercetin141105.374%13Theobromine16.09.123257%
2Catechin18015383%14Caffeine12080.23167%
3Epicatechin161.27.5%15Theophylline1.641.0161%
4Ferulic acid1.30.862%16Oleic acid2.930.029311%
5Caffeic acid1216050%17Linoleic acid1.270.050313%
6Gallic acid13112093%18Palmitic acid1.240.235218%
7Chlorogenic acid13012693%19Stearic acid1.360.08426%
8Vanillin15713281%20β-Sitosterol1.250.02112%
9Epicatechin1.680.3029318%21Campesterol1.800.978354%
10Cyanidin1.610.921357%22Stigmasterol1.670.04213%
11Delphinidin1.600.9124257%
12Methylxanthines1.620.7312145%
Table 4. Comparison of recovery rates of selected compounds from cocoa shells using several extraction techniques, alongside samples with comparable cell wall stiffness.
Table 4. Comparison of recovery rates of selected compounds from cocoa shells using several extraction techniques, alongside samples with comparable cell wall stiffness.
CompoundSampleInsoluble Fiber in SampleRecovery MethodRecovery RateReference
Chlorogenic acidCocoa shells50–64%Two-stage (OASD + SSCO2)94%
Spent coffee beans60–66% [62]Solvents extraction (tetrahydrofuran, itaconic acid) 92%[61]
Gallic acidCocoa shells50–64%Two-stage (OASD + SSCO2)93%
Rosehip seed waste62–66% [63]Pretreatment with SSCO2 and ultrasound-assisted extraction (UAE)9.5%[64]
CatechinCocoa shells50–64%Two-stage (OASD + SSCO2)83%
Açaí (E. oleracea)59–65% [65]Energized dispersive guided extraction (EDGE)27%[66]
VanillinCocoa shells50.1–64.1%Two-stage (OASD + SSCO2)81%
Vanilla pods [67]60–71%Deep eutectic solvent (NADES)43%[68]
Table 5. Quantitative analysis of fluorescence intensity (area measurements) from CLSM images, showing values before and after extraction for BSGs (corresponding to Figure 7) and for cocoa shells (corresponding to Figure 8).
Table 5. Quantitative analysis of fluorescence intensity (area measurements) from CLSM images, showing values before and after extraction for BSGs (corresponding to Figure 7) and for cocoa shells (corresponding to Figure 8).
Sample Cell WallAntioxidantsPhenolic AcidPolyphenolic Acid
BSGBefore extraction (Area)4878.1 ± 27899.1 ± 56941.5 ± 54215.2 ± 3
After (Area)3054.2 ± 3854.2 ± 3612.4 ± 3421.1 ± 2
Cocoa shellsBefore extraction (Area)4015.1 ± 23124.6 ± 13012.6 ± 52243.5 ± 1
After extraction (Area)3842.0 ± 3842.5 ± 2624.5 ± 8567.5 ± 2
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Chu, H.-Y.I.; Zhang, X.; Wang, Y.; Miri, T.; Onyeaka, H. Sustainable Extraction of Bioactive Compounds from Cocoa Shells Waste and Brewer’s Spent Grain Using a Novel Two-Stage System Integrating Ohmic-Accelerated Steam Distillation (OASD) and Supercritical CO2 Extraction (SSCO2). Sustainability 2025, 17, 7373. https://doi.org/10.3390/su17167373

AMA Style

Chu H-YI, Zhang X, Wang Y, Miri T, Onyeaka H. Sustainable Extraction of Bioactive Compounds from Cocoa Shells Waste and Brewer’s Spent Grain Using a Novel Two-Stage System Integrating Ohmic-Accelerated Steam Distillation (OASD) and Supercritical CO2 Extraction (SSCO2). Sustainability. 2025; 17(16):7373. https://doi.org/10.3390/su17167373

Chicago/Turabian Style

Chu, Hao-Yu Ivory, Xinyu Zhang, Yuxin Wang, Taghi Miri, and Helen Onyeaka. 2025. "Sustainable Extraction of Bioactive Compounds from Cocoa Shells Waste and Brewer’s Spent Grain Using a Novel Two-Stage System Integrating Ohmic-Accelerated Steam Distillation (OASD) and Supercritical CO2 Extraction (SSCO2)" Sustainability 17, no. 16: 7373. https://doi.org/10.3390/su17167373

APA Style

Chu, H.-Y. I., Zhang, X., Wang, Y., Miri, T., & Onyeaka, H. (2025). Sustainable Extraction of Bioactive Compounds from Cocoa Shells Waste and Brewer’s Spent Grain Using a Novel Two-Stage System Integrating Ohmic-Accelerated Steam Distillation (OASD) and Supercritical CO2 Extraction (SSCO2). Sustainability, 17(16), 7373. https://doi.org/10.3390/su17167373

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