Comparative analysis of microwave and ultrasound
extraction techniques for valorizing almond milk
byproducts

Mariam Taha, Krasimir Dimitrov, Jennifer Samaillie, Nicolas Blanchemain,
Celine Riviere

► To cite this version:

Mariam Taha, Krasimir Dimitrov, Jennifer Samaillie, Nicolas Blanchemain, Celine Riviere. Comparative analysis of microwave and ultrasound extraction techniques for valorizing almond milk byproducts. LWT - Food Science and Technology, 2025, LWT-Food Sci. Technol., 229, pp.118178. 10.1016/j.lwt.2025.118178. hal-05372331

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Distributed under a Creative Commons Attribution 4.0 International License
Comparative analysis of microwave and ultrasound extraction techniques
for valorizing almond milk byproducts

Mariam Taha<sup>a, *, ID</sup>, Krasimir Dimitrov<sup>a</sup>, Jennifer Samaillie<sup>a</sup>, Nicolas Blanchemain<sup>b</sup>,
Céline Rivière<sup>a</sup>

ARTICLE INFO

Keywords:

Almond milk by-product

Bioactive compounds

Ultrasound-assisted extraction

Microwave-assisted extraction

Polyphenols

Antioxidant activity

A B S T R A C T

This study explores the sustainable valorization of almond okara, a nutrient-rich by-product of almond milk production, through advanced green extraction technologies. Although conventional solid-liquid extraction (CSLE) has previously shown the potential of almond okara due to its high content of bioactive compounds, this research focuses on assessing and optimizing ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) to enhance the recovery of polyphenols and related phytochemicals. While UAE demonstrated significantly higher polyphenol yields than CSLE under comparable conditions, indicating superior extraction efficiency, MAE ultimately outperformed both methods, emerging as the most effective technique overall. Systematic optimization of MAE parameters, such as microwave power, extraction time, and solid-to-solvent ratio, resulted in significantly increased recoveries of total polyphenols (800 mg GAE/100 g dry okara), phenolic acids (770 mg CAE/100 g), tannins (670 mg GAE/100 g), and flavonoids (480 mg CE/100 g). Additionally, antioxidant activity peaked at 1280 mg AAE/100 g under optimal MAE conditions (400 W, 1:50 g/mL, 90 min). Moreover, UHPLC-UV analysis revealed a more diverse and abundant polyphenol profile in MAE extracts. These results highlight MAE as a powerful, efficient, and eco-friendly extraction method, endorsing the sustainable use of almond okara in functional foods, nutraceuticals, and cosmetics, aligned with green chemistry principles and the circular economy.

1. Introduction

The extraction of bioactive compounds from food processing by-products has emerged as a crucial strategy for promoting sustainability within the food industry. Almond okara, the nutrient-rich solid residue from almond milk production, represents a valuable yet underutilized by-product with significant potential for different applications. While whole almonds are widely recognized for their rich content of lipids and bioactive compounds, such as polyphenols and α-tocopherol, known for their antioxidant and anti-inflammatory properties (Chen et al., 2006; Mandalari et al., 2010), the chemical composition and specific utilization of almond okara have remained largely

unexplored. Recently, it has been revealed that almond okara contains substantial amounts of health-promoting compounds, highlighting its potential as a key ingredient in functional foods, nutraceuticals, and cosmetics (De Angelis et al., 2023; Lacivita et al., 2024; Taha et al., 2024).

Conventional solid-liquid extraction (CSLE) method was previously used to recover these bioactive compounds (Taha et al., 2024). While CSLE has demonstrated effectiveness, it also presents key limitations, including long extraction times, excessive solvent consumption, and moderate energy efficiency (Chemat et al., 2012). These issues highlight the need to explore alternative extraction methods, aligned with green chemistry principles, to improve overall efficiency and yield. In this

Abbreviations: CSLE, Conventional Solid-Liquid Extraction; MAE, Microwave-assisted extraction; UAE, Ultrasound-assisted extraction; UHPLC-UV, Ultra-High-Performance Liquid Chromatography–UltraViolet; GAE, gallic acid equivalent; CE, catechin equivalent; CAE, caffeic acid equivalent; AAE, ascorbic acid equivalent; MWP, microwave power.

E-mail address: mariam.wtaha@gmail.com (M. Taha).

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<footnote><sup>a</sup> Joint Research Unit 1158 BioEcoAgro, Univ. Lille, INRAE, Univ. Liège, UPJV, JUNIA, Univ. Artois, Univ. Littoral Côte d'Opale, ICV-Institut Charles Violette, 59650, Villeneuve-d'Ascq, France</footnote>

<footnote><sup>b</sup> INSERM, CHU Lille, U1008—Advanced Drug Delivery Systems, 59000, Lille, France</footnote>

<footnote>* Corresponding author. Joint Research Unit 1158 BioEcoAgro, Univ. Lille, INRAE, Univ. Liège, UPJV, JUNIA, Univ. Artois, Univ. Littoral Côte d'Opale, ICV-Institut Charles Violette, 59650, Villeneuve-d'Ascq, France</footnote>
context, eco-friendly extraction techniques, such as ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), have gained popularity (Backes et al., 2018; Biswas et al., 2023; Huma et al., 2018; Osorio-Tobón, 2020; Usman et al., 2023). UAE uses ultrasonic waves to generate cavitation, enhancing solvent penetration and mass transfer, which increases extraction efficiency while reducing environmental impact (Bouchez et al., 2020; Chemat et al., 2017; Dimitrov et al., 2022; Khalaf et al., 2024). Meanwhile, MAE employs microwave energy to evenly heat the solvent and plant matrix, disrupting cell walls and facilitating the release of bioactive compounds. This method is especially valued for its fast extraction times, lower solvent consumption, and higher yields (Bouchez et al., 2023; Chemat & Cravotto, 2013). Both UAE and MAE not only enhance extraction performance but also provide potential environmental benefits, making them promising options for the sustainable valorization of food industry by-products.

Several studies have demonstrated the successful application of UAE and MAE to extract bioactives from by-products such as grape pomace, pomegranate peel, citrus peel, black bean waste, papaya, black choke-berry, and coconut and olive pomace cakes, as sources of bioactive compounds for functional foods and nutraceuticals (Aiello et al., 2022; Backes et al., 2018; Dimitrov et al., 2019; Drosou et al., 2015; Galvan d'Alessandro et al., 2012; Kaderides et al., 2019; Mali & Kumar, 2023; Rodríguez et al., 2022; Sandhu et al., 2021). These findings highlight these green techniques' capacity to significantly improve bioactive compounds' recovery while adhering to sustainable practices.

This study aims to evaluate and compare the effectiveness of UAE and MAE for recovering bioactive compounds from almond okara. The underlying assumption is that these advanced techniques can improve extraction efficiency and yield while reducing environmental impact, supporting the principles of green chemistry and the circular economy. Initially, UAE was explored as an energy-efficient alternative to CSLE and demonstrated improved extraction yields of total polyphenols (TP). Subsequently, MAE was investigated as another advanced technique with the potential for even higher extraction efficiency and enhanced antioxidant activity. At the first stage, a comparative analysis between CSLE, UAE, and MAE was conducted, focusing specifically on TP recovery.

Given the promising preliminary results obtained with MAE, a detailed optimization study was undertaken to further improve its efficiency. Key operating parameters, microwave power, extraction time, and solid-to-solvent ratio, were systematically optimized. During this phase, a more comprehensive phytochemical analysis was carried out, assessing not only the yields of total polyphenols but also the yields of tannins, flavonoids, and phenolic acids, as well as the antioxidant activity of the extracts. Additionally, UHPLC-UV analysis was performed to characterize and compare the polyphenolic profiles of the different extracts tested (CSLE, UAE, and MAE), further confirming the advantages of MAE.

# 2. Materials and methods

## 2.1. Chemicals and reagents

Gallic acid, caffeic acid, (+)-catechin, epicatechin, L-ascorbic acid, ammonium molybdate, sodium nitrite, sodium molybdate, sodium phosphate, sodium hydroxide, and HCl were sourced from Sigma-Aldrich (St. Louis, MO, USA). The Folin-Ciocalteu reagent, utilized for quantifying total polyphenols, was obtained from VWR (Fontenay-sous-Bois, France). Aluminum chloride was acquired from Panreac Química (Barcelona, Spain). Ethanol and acetonitrile (LC-MS grade) for UHPLC analyses were provided by Carlo Erba Reagents® (Val de Reuil, France). Ultra-pure water was obtained using a Millipore Integral 5 (Merck™, Trosly-Breuil, France) water purification system with a resistivity of not less than 18 MΩ·cm⁻¹. Formic acid was provided by Merck™ (Darmstadt, Germany).

## 2.2. Sample preparation: production of almond okara

The preparation of almond okara followed the method described by Taha et al. (Taha et al., 2024). Briefly, organic Spanish almonds, sourced from a local organic store, were processed to produce almond milk, and the resulting okara was harvested fresh. The okara was promptly used for dry matter analysis to compare the three extraction methods and optimize the conditions for quantifying their bioactive compounds.

## 2.3. Dry matter determination of okara

The dry matter (DM) content of freshly obtained okara was determined by drying it in a ventilated oven at 105 °C. A specific amount of the sample was weighed and dried for 24 h or until a constant weight was obtained (AOAC, 1995).

## 2.4. Extraction procedures

### 2.4.1. Conventional solid-liquid extraction (CSLE)

The kinetics of CSLE were studied over 120 min using a solid-to-water ratio of 1:50 g/mL at 70 °C, with the aqueous okara mixtures agitated at 150 rpm. Extract samples were collected at multiple time intervals (5–120 min) and centrifuged at 3046 × g (4350 rpm in a TX-150 rotor, Model Megar Star 600, VWR) at room temperature for 10 min and analyzed with Folin-Ciocalteu method to determine the total polyphenol yield ($Y_{TP}$), expressed as mg of gallic acid equivalent (GAE) per 100 g of dry almond okara (Cheaib et al., 2018; Taha et al., 2024).

### 2.4.2. Ultrasound-assisted extraction (UAE)

UAE was carried out in a 0.8 L agitated contactor (Lab750 for pipe, SinapTec, France) working at 22 kHz and a maximal power of ultrasound of 400 W. At time zero, the okara, at a 1:50 solid-liquid ratio, was placed in the water at room temperature, 22 ± 2 °C, and both agitation and sonication were started. The mechanical agitation of the extraction mixture (160 rpm) was provided by a blade stirrer that allowed the maintenance of all particles in suspension and the same ultrasound treatment of whole okara and maximal contact between the 2 phases. The power input of the ultrasound in the extraction media provoked a temperature increase. A regulation of the sonication (on/off with temperature control) allowed to respect the operating limit of the equipment (75 °C maximum). Hence, initially, ultrasound was applied in continuous mode till the temperature reached 72 °C. At this moment the sonication was stopped and then restarted when the temperature decreased to 68 °C. Thus, in this period of discontinuous sonication, the temperature was maintained at 70 ± 2 °C. Samples were taken at 0, 5, 10, 15, 30, 45, 60, 90, and 120 min, then centrifuged at 3046×g (4350 rpm in a TX-150 rotor, Model Megar Star 600, VWR) at room temperature for 10 min and analyzed with the Folin-Ciocalteu method to determine the total polyphenol yield ($Y_{TP}$).

### 2.4.3. Microwave-assisted extraction (MAE)

The MAE of okara was conducted in a 1 L glass boiling flask using deionized water as the solvent. Each experiment was performed in triplicate in an ETHOS X microwave oven (Milestone, Italy), equipped with dual magnetrons capable of delivering up to 1900 W of power at a frequency of 2.45 GHz. A rotating diffuser ensured uniform microwave distribution throughout the chamber. The boiling flask was positioned inside the oven and connected to a condenser integrated with a water-cooling system (Smart H150-2100 chiller, Lab Tech, Boston, MA, USA), set at 8 °C to facilitate efficient condensation.

The microwave power ranged from 100 W to 800 W, and continuous agitation was maintained through a magnetic stirring mechanism integrated into the oven. The process started at an ambient temperature of 24 ± 2 °C without additional temperature regulation. Instead, the solvent reached and was maintained at its boiling point, with vapors being continuously condensed and recycled, ensuring a stable solvent
**volume** throughout the extraction. The compact ETHOS X terminal enabled precise control of power output, stirring parameters, and real-time temperature monitoring using an infrared sensor.

After extraction, the liquid extracts were centrifuged at 3046 × g (4350 rpm, TX-150 rotor, Model Megar Star 600, VWR) for 10 min at room temperature, effectively separating the solid residues from the solution. The resulting supernatant was then analyzed to determine key phytochemical parameters, including total polyphenol yield (Y<sub>TP</sub>), tannin content (Y<sub>T</sub>), flavonoid yield (Y<sub>F</sub>), total phenolic acid yield (Y<sub>TPA</sub>), and antioxidant activity (AA).

### 2.4.4. Extraction kinetics: comparison of CSLE and UAE at different powers for polyphenol extraction under optimized conditions

This preliminary kinetic study aimed to evaluate and compare the performance of aqueous CSLE with UAE to better understand their effectiveness. The study's kinetics were conducted over a 2-h period, during which supernatants were collected at 0, 5, 10, 15, 30, 45, 60, 90, and 120 min. For UAE, two power levels were tested: 200 W and 400 W at a maximum temperature of 70 °C. To maintain consistency, CSLE was conducted at 70 °C, a temperature not tested in the previous study (Taha et al., 2024). For both methods, a solid-to-solvent ratio of 1:50 g/mL was used as the previous results indicated that this ratio provided optimal polyphenol recovery for CSLE. Focusing on these conditions, the study aimed to assess the total polyphenol yield (Y<sub>TP</sub>) and compare the effectiveness of each technique under similar parameters. The analytical method is detailed in Section 2.5. Results were expressed as milligrams of gallic acid equivalent (GAE) per 100 g of dry almond okara.

$$Y_{TP} : \left( \frac{mg \ TP}{100 \ g \ dry \ okara} \right) = \frac{C^*V}{M} \qquad \text{Equation 1}$$

Where C is the concentration of total polyphenols of the extract (mg GAE/mL), V is the volume of the solvent (mL), and M is the dry weight of the sample (g).

### 2.4.5. Comparison of UAE and MAE at various power levels

To further investigate and compare the extraction efficiency of the two green extraction techniques, UAE at 200 W was first compared with MAE at five different microwave power levels (100, 200, 400, 600, and 800 W), with all extractions conducted for 90 min and using a constant solid-to-solvent ratio of 1:50 g/mL.

The choice of 200 W for UAE was based on preliminary optimization experiments (described in Section 2.4.2), which showed that 200 W provided the same total polyphenol yield as the UAE at 400 W. In the first phase, the total polyphenol yield was used as the primary benchmark to compare UAE at 200 W with MAE at the five power levels. In the second phase, to gain more detailed insights into the performance of MAE at different power levels, additional analyses were carried out on the five MAE extracts only (from the 100–800 W experiments). These included quantification of tannins, flavonoids, phenolic acids, and antioxidant activity, following the methods described in Sections 2.5.2 to 2.5.5.

### 2.4.6. MAE parameters optimization: solid-to-solvent ratio and extraction time

In addition to thoroughly understanding the efficiency of MAE, the influence of other crucial factors was studied, including extraction time (15, 30, 45, 60, 90, and 120 min) and solid-to-solvent ratio (1:10, 1:30, and 1:50 g/mL). The optimal parameters were determined by comparing the total yields of polyphenols, tannins, flavonoids, and phenolic acids, as well as antioxidant activity.

## 2.5. Determination of the total polyphenol and tannin yields in aqueous extracts

For total polyphenol and tannin quantification, the Wiesneth and

Jürgenliemk (2017) microplate spectrophotometric method was used. The full methodological details are given in Taha et al. (2024).

### 2.5.1. Determination of the total polyphenol yield (Y<sub>TP</sub>)

The total polyphenol yield was determined using a modified Folin-Ciocalteu method (Liu et al., 2009). Briefly, 25 µL of aqueous extracts or gallic acid (GA) (0–200 mg/L) were added to a 96 well UV-transparent well-bottom microplate. A 1/10 dilution of Folin-Ciocalteu reagent (125 µL) was added, followed by incubation in the dark at 40 °C for 2 min. Then, 100 µL of a 4 % sodium carbonate (Na₂CO₃) solution was added, and the plate was incubated at 40 °C for 2 h. Absorbance was measured at 690 nm using a UV-2450-spectrophotometer (Spectrostar Nano, BMG Labtech, Germany). The total polyphenol yield was expressed as gallic acid equivalents (mg GAE/100 g dry okara).

### 2.5.2. Determination of the tannin yield (Y<sub>T</sub>) with hide powder method

The tannin yield was determined by the hide powder method. Briefly, 0.5 mL of the extracts was mixed with 10 mg of hide powder (Sigma-Aldrich Chemie GmbH, Germany) to precipitate the tannins. The mixture was stirred and incubated in the dark for 60 min, then centrifuged at 3046 g (4350 rpm in a TX-150 rotor, Model Megar Star 600, VWR) for 5 min at room temperature. The supernatant was then analyzed using the Folin-Ciocalteu method for total polyphenol determination. The tannin yield (Y<sub>T</sub>) was calculated by subtracting the tannin-free polyphenol content from the total polyphenol content of the extract and was expressed as gallic acid equivalents (mg GAE/100 g dry okara).

## 2.6. Determination of the total flavonoid yield (Y<sub>TF</sub>) in extracts

The method detailed by Taha et al. (2024) consists of adding 1.5 mL of the extract to a mixture of 450 µL of 5 % sodium nitrite (NaNO₂), 900 µL of 10 % AlCl₃-H₂O, and 4 mL of 1 M sodium hydroxide (NaOH). The mixture was stirred and incubated for 5 min before each addition. The final volume was adjusted to 15 mL with ultrapure water, and the absorbance was measured at 510 nm. The results obtained were expressed as mg (+)-catechin equivalent (CE)/100 g dry okara.

## 2.7. Determination of the total phenolic acid yield (Y<sub>TPA</sub>) in extracts

The total phenolic acid content of almond okara extracts was assessed using the nitrite-molybdate reagent method, which was adapted from the protocol by Zhishen et al. (1999) and later refined by Kim et al. (2003) (Kim et al., 2003; Zhishen et al., 1999). Briefly, 1 mL of the extract was combined with 1 mL of 0.5 M hydrochloric acid (HCl) and 1 mL of a 10 % NaNO₂ solution. This mixture was allowed to stand at room temperature for 5 min. Afterward, 1 mL of a 10 % sodium molybdate (Na₂MoO₄) solution was introduced, and the reaction was incubated for an additional 5 min. To stabilize the reaction, 2 mL of an 8.5 % NaOH solution was added, and the absorbance of the resulting yellow complex was measured at 470 nm using an UV-Vis spectrophotometer. A calibration curve was generated using caffeic acid as a standard (0–100 µg/mL), and the total phenolic acid content was reported as milligrams of caffeic acid equivalents (CAE) per gram of dry weight (mg CAE/100 g of dry okara).

## 2.8. Evaluation of total antioxidant activity by the phosphomolybdenum method

The total antioxidant capacity of the extracts was evaluated using the method described by Aquino et al. (Aquino et al., 2001), and adapted by Taha et al., 2024 (Taha et al., 2024). Briefly, 0.1 mL of the sample extract was mixed with 1 mL of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The mixture was incubated at 95 °C in a water bath for 90 min. After cooling
to room temperature, the absorbance of each sample was measured at 695 nm against a blank using a spectrophotometer (Spectrostar Nano, BMG Labtech, Ortenberg, Germany). The antioxidant activity was expressed as ascorbic acid equivalents (mg AAE/100 g dry okara).

## 2.9. Instrumentation and chromatographic conditions

The presence of (+)-catechin and epicatechin in the extracts obtained by the different extraction methods (CSLE, UAE, MAE) was determined by UHPLC-UV-MS by comparison with the retention times, UV spectra and mass spectra of commercial standards. UHPLC-UV-MS analyses were carried out on a Waters Acquy equipment (Waters® Acquy UPLC® H-Class, Guyancourt, France) consisting of two independent pumps, an automatic injector, a controller, a diode array detector (PDA) and a quadrupole (QDA) mass spectrometer with ESI as the ionization source. The software used was Empower 3.

The chromatographic separation was performed using a Waters® Acquy BEH Phenyl column (2.1 × 100 mm, 1.7 µm) maintained at 30 °C and connected to a 0.2 µm in-line filter. A gradient elution was applied using solvent A (water with 0.1 % formic acid, v/v) and solvent B (acetonitrile with 0.1 % formic acid, v/v). The gradient program was as follows: initial conditions at 2 % B, held isocratically for 1 min; a linear increase to 20 % B from 1 to 5 min; isocratic mode at 20 % B from 5 to 6 min; a linear increase to 100 % B from 6 to 10 min; isocratic mode at 100 % B from 10 to 11.5 min, followed by a re-equilibration time of 2.5 min. The flow rate was maintained at 0.3 mL/min throughout the analysis. UV detection was performed at 280 nm, as this wavelength is commonly used for detecting phenolic compounds, especially flavan-3-ols like catechin and epicatechin.

Mass spectrometry detection was performed in both positive and negative ionization modes, with a mass range of 100–1000 Da. The cone voltage was set at 15 V, and the capillary voltage was 0.8 kV. Sample solutions were prepared from the filtrates obtained after solid-liquid extraction with a 1:10 ratio (solid/solvent), followed by filtration through a 0.45 µm PTFE filter. A mixture of ethanol/water (50:50, v/v) was used as a blank. Standard solutions were prepared at a concentration of 0.1 mg/mL. The injection volume was 2 µL for standards and 4 µL for samples.

## 2.10. Statistical analysis

All experiments and all measurements were performed in triplicate. The results are presented as mean ± standard deviation. Analyses were performed using two-way and one-way ANOVA followed by a Tukey post hoc test to compare the extracts. Statistical analyses were performed using GraphPad Prism 10.

# 3. Results

## 3.1. Extraction kinetics: A comparison between CSLE and UAE

In a previous work (Taha et al., 2024), conventional solid-liquid extraction (CSLE) at 60 °C with a 1:50 solid-to-solvent ratio yielded approximately 200 mg GAE/100 g of dry okara. To ensure a fair comparison with ultrasound-assisted extraction (UAE), the CSLE temperature in the present study was increased to 70 °C, corresponding to the highest temperature used for UAE at different power levels. However, as shown in Fig. 1, the total polyphenol yield ($Y_{TP}$) of CSLE at 70 °C remained similar to that obtained at 60 °C, with no significant improvement.

In contrast, UAE demonstrated significantly higher efficiency, with TP yields reaching approximately 550 mg GAE/100 g of dry okara at 200 W and 400 W. The superior performance of UAE is attributed to the cavitation effect, where ultrasonic waves generate microbubbles that collapse, disrupting plant cell walls and enhancing solvent penetration. This process facilitates the release of polyphenols more effectively than

CSLE (Chemat et al., 2017; Sandhu et al., 2021).

Fig. 1 also highlighted the effect of extraction time; UAE was faster and more efficient than CSLE, especially at 400 W. TP yield increased rapidly with extraction time, peaking at 15–30 min (550 mg GAE/100 g of dry okara), after which further extraction did not significantly improve the yield. This trend was consistent across both UAE power levels, with higher power levels achieving maximum yields in shorter times. A two-way ANOVA test, followed by Tukey's post hoc test, confirmed statistically significant differences in TP yields between CSLE and UAE and across UAE power levels ($p < 0.05$). Similar findings were reported by Carciochi et al. (2021) (Carciochi et al., 2021), who demonstrated that applying ultrasound at 70 °C significantly enhanced caffeine extraction from guarana seeds while reducing extraction time compared to conventional methods.

The kinetics of UAE showed that the yields obtained at 15 min at 400 W were very close to the maximal ones, while at 200 W, more time was needed to get similar yields. The faster kinetics at 400 W could be related to both mechanical and thermal effects of ultrasound assistance. Indeed, the higher cavitation effect at the higher power (400 W) could provoke stronger and faster detexturation of the okara matrix and, therefore, improved accessibility of the solvent to the extractible molecules in the matrix, resulting in higher mass transfer of polyphenols. The higher power density at 400 W also provoked a more rapid increase in the temperature of the extraction mixture and, respectively, faster decrease of solvent viscosity, favoring the mass transfer. These findings align with previous studies demonstrating the efficiency of the UAE in enhancing polyphenol recovery while reducing extraction time (Abdallah et al., 2023; Garcia-Castello et al., 2015; Ghitescu et al., 2015). However, the higher power of 400 W did not provide significantly higher maximal extraction yields. Indeed, the same maximal temperature was used in the experiments of UAE at 200 W and at 400 W, as a result of the temperature regulation used in the used UAE equipment (sonication off/on cycles to not exceed 70 °C).

The optimal conditions for UAE from almond okara were found to be 200 W ultrasonic power for 30 min, maximizing polyphenol recovery while maintaining an energy-efficient and sustainable process.

## 3.2. Comparison between the total polyphenol yield obtained by UAE and MAE

One-way ANOVA followed by Tukey's post hoc tests revealed significant differences ($p < 0.0001$) between UAE and MAE regarding TP yield, particularly at higher microwave power levels. The maximum TP yield was achieved with MAE at 400 W with 800 mg GAE/100 g of dry okara, after which the yield declined slightly (Fig. 2).

Microwave assistance provoked a heating of the extraction mixture. Respectively, the speed of temperature increased, and the maximal temperatures of the experiments at different powers varied. Hence, at 100 W, the temperature reached 41 °C, at 200 W it reached 71 °C, and at 400 W and above, the solvent reached its boiling temperature. After this moment, the extraction temperature was about 96 ± 2 °C. Even under the mild conditions of 100 W, the yield of total polyphenols obtained by MAE was almost twice as high as the yield obtained by CSLE at 70 °C. The superior performance of MAE can be attributed to microwave energy's rapid and uniform heating, which promotes cell wall disruption and the release of intracellular compounds. In addition, microwave energy generates internal heating within the plant matrix, leading to more efficient cell wall breakdown and compound release than CSLE (Biswas et al., 2023). The very similar yields obtained using UAE and MAE at 200 W, both in extractions with maximal temperatures of about 70 °C, suggested that these two assistances allowed to disrupt the okara matrix and to solubilize and extract polyphenols with very similar efficiency. Then, the results obtained in MAE at higher powers could be related to the higher maximal temperatures (about 94 °C at 400 W) under these conditions, providing higher solubility of some phenolic compounds, in particular, less polar (Antony & Farid, 2022). The
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Kinetics of CSLE and UAE in water expressed as total polyphenol yield (mg GAE/100 g dry okara) (solid-to-solvent ratio of 1:50 g/mL). The amounts are presented as mean ± SD. Different letters (A–J) indicate significant differences between the extracts within a column (Tukey post-test, p < 0.05), with values not sharing the same letter being statistically different.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Comparison of aqueous CSLE, UAE at 200 W, and MAE at different power levels on the total polyphenol yield expressed as mg GAE/100 g dry okara (120 min of extraction, solid-to-solvent ratio of 1:50 g/mL). The amounts are presented as mean ± SD. Different letters (A–E) indicate significant differences between the extracts within a column (Tukey post-test, p < 0.05), with values not sharing the same letter being statistically different.</figcaption></figure>

significantly higher TP yields obtained using MAE are consistent with previous studies reporting the superior performance of MAE compared to UAE in polyphenol extraction (Dahmoune et al., 2015; Drosou et al., 2015; Gligor et al., 2019; Kaderides et al., 2019). Moreover, the TP yield

decline over MAE' 400 W aligns with existing literature, which suggests that excessive microwave power may lead to thermal degradation of certain polyphenols, reducing the overall efficiency (Alara & Abdurahman, 2019; Albuquerque et al., 2017; Chy et al., 2024; Dahmoune et al.,
2015; Li et al., 2013). Indeed, in the present study, the maximum temperatures were almost the same as those of MAE at 400, 600, and 800 W, but the higher input power and longer time at maximal temperature at 600 and 800 W might provoke some thermal degradation (Antony & Farid, 2022; Taha et al., 2024). The obtained results highlight MAE at 400 W as the most effective method for extracting polyphenols from almond okara.

### 3.3. Effect of microwave-assisted extraction power on the yields of tannins, flavonoids, phenolic acids, and the antioxidant activity of okara extracts

The effect of microwave power (MWP) on the extraction efficiency of bioactive compounds from almond okara was thoroughly investigated. For tannins, the yield increased steadily with power up to 400 W with 670 mg GAE/100 g of dry okara, beyond which a slight decline was observed in Fig. 3a. This suggests that while moderate to high power enhances extraction by improving cell wall permeability, excessive energy input may degrade tannins by overheating (Ciuperca (Apreutesei) et al., 2023). Recent studies have shown that hydrolysable tannins content (HCT), which is part of the almond composition with an average of 82.1 mg/100 g (Bolling, 2017), decreases significantly with increasing microwave power. This reduction likely results from the thermal degradation of high molecular weight hydrolysable tannins, which break down into smaller molecules like gallic acid and glucose at elevated temperatures (Joshi et al., 2023; Shende et al., 2024). Additionally, another study indicates that hydrolysable tannins are particularly unstable in aqueous solutions when subjected to higher temperatures, contributing to their degradation (Y.-H. Wang et al., 2019). Similarly, the total condensed tannins content (TCT), also an important part of almonds with an average of 162 mg/100 g, initially increases with longer treatment times but ultimately decreases. This trend is attributed to the thermal degradation of tannins during

prolonged exposure to high power levels (Das et al., 2020; Sebestyén et al., 2019).

Regarding the flavonoid yield, Fig. 3b shows a significant increase as the power increases from 100 W to 400 W, reaching a yield of 480 mg CE/100 g of dry okara. Beyond this point, the yield decreases, suggesting a saturation threshold beyond which additional energy input does not improve extraction efficiency. The decrease in flavonoid yield at higher power levels suggests that 400 W is the optimal extraction condition, as excessive power offers no additional benefits and may lead to unnecessary energy consumption and degradation. The literature supports this, indicating that flavonoid extraction efficiency peaks beyond which degradation or equilibrium effects are observed (Gligor et al., 2019).

As with flavonoids, the yield of phenolic acids increased up to a power of 400 W and then decreased, confirming that a higher energy input does not significantly improve extraction (Fig. 3c). A one-way ANOVA revealed statistically significant differences among 400 W, 600 W, and 800 W, indicating a saturation effect. Due to their structural diversity, phenolic acids exhibit varying thermal stabilities. The observed decline suggests that once a certain power level is reached, most of the extractable phenolic acids have been released, and additional power may accelerate solvent heating, leading to a reduction in yield (Spigno et al., 2007).

Antioxidant activity followed the same power-dependent trend as the polyphenolic yields, increasing to 1280 mg AAE/100 g of dry okara at 400 W, before a slight decline (Fig. 3d). Excessive heating at higher power levels may degrade heat-sensitive antioxidants. Previous studies highlight similar trends, suggesting that beyond a certain MWP, a decline in TP yield and antioxidant activity has been observed (Chy et al., 2024; Lin et al., 2020; Vallejo-Castillo et al., 2019).

This reduction could be attributed to the thermal degradation of certain polyphenols. Furthermore, if microwave power exceeds a certain threshold, high temperatures generally above 100 °C (Liazid et al., 2007), can lead to the degradation of heat-sensitive substances, damage

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** The effect of aqueous microwave-assisted extraction (MAE) at different power levels on (a) tannin content (expressed as mg GAE/100 g dry okara), (b) total flavonoid content (expressed as mg CE/100 g dry okara), (c) phenolic acid content (expressed as mg CAE/100 g dry okara), and (d) the antioxidant activity of the extracts (expressed as mg AAE/100 g dry okara). Extraction conditions: 120 min, solid-to-solvent ratio of 1:50 g/mL. Data are presented as mean ± SD. Different letters (A–D) indicate significant differences between the extracts within a column (Tukey post-test, p < 0.05), with values not sharing the same letter being statistically different.</figcaption></figure>
to plant cells, and ultimately reduce extraction process yields.

The comparison of the effect of MWP on different groups of phenolic compounds showed that this effect is less pronounced for phenolic acids than for total flavonoids and tannins. Thus, if the objective is to obtain an extract with a relatively high yield of phenolic acids, milder extraction conditions with low energy consumption could be used. On the other hand, if the objective is to obtain high yields of flavonoids, tannins, or maximum antioxidant activity from okara extracts, a higher MWP will be needed.

This study confirmed that, under the studied conditions, the MAE of polyphenols from almond okara is superior to the UAE. While increasing MAE power improved the yields of tannins, flavonoids, phenolic acids, and antioxidant activity up to a certain threshold, excessive power did not provide any additional benefit. It may lead to the degradation of compounds. According to the results, 400 W represented the optimal range for polyphenol extraction, equilibration efficiency, and compound stability. The results underscore the importance of optimizing MAE

parameters to balance yield maximization with compound stability, reinforcing the concept that “more power” does not equate to “better extraction”.

### 3.4. Effect of microwave-assisted extraction time on the yields of total polyphenols, tannins, flavonoids, phenolic acids, and the antioxidant activity of okara extracts

Analysis of Fig. 4 highlighted the effect of time on the yield of total polyphenols, tannins, flavonoids, phenolic acids, and antioxidant activity for MAE at a power of 400 W and a solid-to-solvent ratio of 1/50 g/mL. Each figure revealed a consistent trend: yield or activity increases with extraction time up to an optimum point, after which further extraction time results in a plateau.

Regarding the TP yield in Fig. 4a, the results indicated a peak around 90 min of extraction time, with a $Y_{TP}$ of 770 mg GAE/100 g of dry okara; after this point, the yield reached a plateau. Numerous studies suggest

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** The effect of aqueous microwave-assisted extraction (MAE) at different extraction time on (a) tannin content (expressed as mg GAE/100 g dry okara), (b) total flavonoid content (expressed as mg CE/100 g dry okara), (c) phenolic acid content (expressed as mg CAE/100 g dry okara), and (d) the antioxidant activity of the extracts (expressed as mg AAE/100 g dry okara). Extraction conditions: MWP 400 W, solid-to-solvent ratio of 1:50 g/mL. Data are presented as mean ± SD. Different letters (A–D) indicate significant differences between the extracts within a column (Tukey post-test, $p < 0.05$), with values not sharing the same letter being statistically different.</figcaption></figure>
that extending extraction time improves the yield (Huang et al., 2017; Karami et al., 2015; Pimentel-Moral et al., 2018). Since extraction involves mass transfer, prolonged irradiation periods are expected to increase both the extraction yield and the amount of polyphenols obtained until equilibrium is reached.

In the case of tannins, the yield increased gradually from 15 to 60 min to reach a $Y_T$ of approximately 590 mg GAE/100 g of dry okara, after which it reached a plateau. The optimal extraction time for tannins in this study seems to be between 60 and 90 min. For flavonoids, the yield increased with extraction time up to 90 min with $Y_{TF}$ 460 CE/100 g

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** The effect of aqueous microwave-assisted extraction (MAE) at different solid-to-solvent ratios on (a) tannin content (expressed as mg GAE/100 g dry okara), (b) total flavonoid content (expressed as mg CE/100 g dry okara), (c) phenolic acid content (expressed as mg CAE/100 g dry okara), and (d) the antioxidant activity of the extracts (expressed as mg AAE/100 g dry okara). Extraction conditions: MWP of 400 W, 120 min of extraction time. Data are presented as mean ± SD. Different letters (A–C) indicate significant differences between the extracts within a column (Tukey post-test, $p < 0.05$), with values not sharing the same letter being statistically different.</figcaption></figure>
of dry okara, after which it reaches a plateau Fig. 4c. This study's optimal extraction time for flavonoids was approximately 90 min. The graph in Fig. 4d for phenolic acids yield showed a similar trend, with yield increasing up to an optimal time (around 60 min) with 770 mg CAE/100 g of dry okara before plateauing. Finally, for antioxidant activity, Fig. 4e demonstrated an increase in activity up to 60 min, with an antioxidant activity level of 1200 AAE per 100 g of dry okara.

In conclusion, the results underlined that optimizing MAE time is essential to maximize the yield and activity of bioactive compounds. The literature consistently emphasizes the importance of precise control of extraction conditions to balance efficiency and compound stability. This study indicates that the optimal extraction time is between 60 and 90 min. After this, a plateau is systematically observed.

### 3.5. Effect of microwave-assisted extraction solid-to-solvent ratio on the yields of total polyphenols, tannins, flavonoids, phenolic acids, and the antioxidant activity of okara extracts

The solid-to-solvent ratio significantly impacted MAE efficiency. The results in Fig. 5 indicated that the extraction efficiency increased as the solid-to-solvent ratio decreased, achieving its maximum yield with 1:50 g/mL. This trend aligns with the principle of equilibrium, where a larger solvent volume enhances the solubilization and diffusion of bioactive compounds, a phenomenon well-documented in the literature (Bener et al., 2022; Tomasi et al., 2023).

For TP, the highest yield of 770 mg/100 g dry okara was observed at a 1:50 g/mL ratio, nearly double the yield obtained with a 1:10 ratio. This finding is consistent with previous MAE studies (Huma et al., 2018; Pimentel-Moral et al., 2018), which report that increasing the L/S ratio leads to higher yields due to improved solvent availability, facilitating better solubilization and diffusion of polyphenolic compounds.

Tannin extraction followed a similar trend, with the highest yield being 580 mg GAE/100 g of dry okara with a solid-to-liquid ratio of 1:50 g/mL. This result aligns with Zhang et al. (Zhang et al., 2011), which highlighted the importance of solvent volume in maximizing tannin extraction. As relatively large and complex molecules, tannins require sufficient solvent to release them from the plant matrix without excessive aggregation or degradation.

The yield progressively increased for flavonoids as the solid-to-solvent ratio decreased, reaching its peak at a ratio of 1:50 g/mL. This finding is supported by (Kaur et al., 2012; X. Wang et al., 2024; Xiao et al., 2008) those who reported that a greater volume of solvent enhances the solubility and diffusion of flavonoid compounds, thereby leading to improved extraction efficiency. The observed increase in yield indicated that flavonoids exhibit high solubility under optimal MAE conditions, facilitating efficient recovery.

Similarly, phenolic acids showed their highest yield at 1:50 g/mL, which is consistent with Dahmoune et al.'s findings that increasing the solvent volume improves phenolic acid extraction without degradation (Dahmoune et al., 2015). Finally, the antioxidant activity was optimized with a ratio of 1:50 g/mL, reinforcing the role of solvent volume in maximizing the recovery of antioxidant compounds, as it prevents compound oxidation and ensures efficient diffusion.

The results indicated that a solid-to-solvent ratio of 1:50 g/mL ensures optimal recovery of polyphenols, tannins, flavonoids, and phenolic acids while maintaining high antioxidant activity. Future studies should determine whether increasing the solvent volume beyond 1:50 g/mL further enhances extraction or whether yields decrease beyond this point. However, excessive solvent use may compromise practical feasibility and increase extraction and processing costs. The ratio of 1:30 g/mL could be a good compromise offering relatively high extraction yields and phenolic compound concentrations and, therefore, lower energy consumption for extract drying, compared to MAE at a ratio of 1:50 g/mL.

### 3.6. Comparative UHPLC-UV-MS analysis of polyphenol extraction from almond okara using CSLE, UAE, and MAE

To provide a more detailed and qualitative comparison of the polyphenol profiles obtained by the different extraction techniques, UHPLC-UV-MS analyses were conducted on extracts obtained under the best conditions for each method (CSLE, UAE, and MAE), following the completion of the MAE parameter optimization (Section 3.3). The extracts were analyzed at the same concentration, and their chromatographic profiles were compared at 280 nm. CSLE appeared to have the lowest extraction efficiency, revealing lower peak intensities and a reduced number of detected compounds. At this same wavelength, UAE appeared to have improved extraction compared to CSLE, displaying slightly higher peak intensity and a wider extracted phenolic compound. MAE proved to be the most effective method, demonstrating the most intense and best-defined peaks, indicating a priori higher extraction of polyphenols, in particular, more apolar compounds with retention times greater than 5 min (Fig. 6). The chromatogram of the MAE extract at 280 nm mainly showed the presence of two peaks with the corresponding retention times $Rt = 5.4$ min and $Rt = 6.2$ min. Mass spectra for these two peaks gave molecular ions $[M-H]^{-}$ at m/z 289 and $[M+H]^{+}$ at m/z 291. The comparison of retention times, UV spectra, and mass spectra of these two peaks with reference standards confirmed the presence of (+)-catechin and (−)-epicatechin (≈6.0 min) in MAE extract. Their presence was at higher levels than in UAE extract. (+)-catechin was slightly detected in the CSLE extract, while (−)-epicatechin was not detected (Fig. S1). These data confirm the previous results that MAE is the most efficient extraction method for recovering some phenolic compounds, in particular, more apolar from almond okara. The superior performance of MAE in the extraction of catechin and epicatechin, two key flavonoids with well-documented antioxidant and anti-inflammatory properties, underscores its potential for almond okara valorization. These findings are supported by studies such as Zhang et al. (2011) and Alara and Abdurahman (2019), which highlight the ability of MAE to maximize yields of bioactive compounds while adhering to green chemistry principles.

## 4. Conclusion

This study evaluated two green extraction methods, ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), for recovering bioactive compounds from almond okara. Although conventional solid-liquid extraction (CSLE) was previously assessed, it was briefly included here as a baseline for comparison. At a maximum temperature of 70 °C, UAE outperformed CSLE, producing 2.75 times higher total polyphenol levels. While UAE at 400 W showed faster kinetics than at 200 W, the final yields were similar. Moreover, under the same temperature conditions, UAE and MAE at 200 W yielded comparable results. MAE was the most efficient at 400 W (maximum temperature of 94 °C), due to fast and effective heating that improved extraction and resulted in higher total polyphenol yields than UAE and CSLE. Excessive microwave power (over 400 W) reduced yields, likely because of thermal degradation, while longer extraction times (beyond 90 min) did not enhance results. These patterns remained consistent across all measured compounds (total polyphenols, flavonoids, tannins, phenolic acids) and the antioxidant activity of the extracts.

Overall, this research demonstrates the superior performance of MAE for almond okara valorization, both in terms of extraction efficiency and extract quality. It highlights MAE's potential as an efficient, scalable, and environmentally friendly method for producing bioactive-rich extracts and represents a meaningful step toward implementing circular economy principles within the agri-food sector (Esposito et al., 2020; Osorio et al., 2021).

Moreover, these results not only improve our understanding of how microwave power influences polyphenol extraction but also open new directions for both industrial applications and future scientific inquiry.
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Polyphenol chromatographic profiles obtained by UHPLC-UV at 280 nm of almond okara extracts using CSLE (a), UAE (b), and MAE (c), as well as chromatograms at 280 nm of catechin (d) and epicatechin (e).</figcaption></figure>

Scaling up this optimized MAE process holds promise for developing more sustainable and energy-efficient extraction methods across the food, nutraceutical, and cosmetics sectors (Boateng, 2023). To facilitate this transition, future research should include a techno-economic assessment to evaluate industrial feasibility, alongside a life cycle assessment (LCA) to determine the environmental impact (Pappas et al., 2023).

In parallel, further studies should explore the biological activities of MAE extracts, such as anti-inflammatory and antimicrobial properties, and assess their stability in real food and supplement matrices. Finally, combining MAE with complementary pre-treatments (e.g., enzymatic hydrolysis or fermentation) or hybrid extraction techniques could improve both the yield and specificity of valuable bioactive compounds (Nayak et al., 2024).

By unlocking the value of almond okara, this research advances green extraction technologies and supports broader efforts in circular bioeconomy innovation.

### CRediT authorship contribution statement

**Mariam Taha:** Writing – original draft, Visualization, Validation, Software, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Krasimir Dimitrov:** Writing – review & editing, Supervision, Methodology, Investigation, Conceptualization. **Jennifer Samailie:** Methodology, Investigation. **Nicolas Blanchemain:** Writing – review & editing, Resources, Funding acquisition. **Céline Rivière:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

### Funding

The authors are grateful to the Region Hauts-de-France, STaRS-Appel à projets "Accueil de jeunes chercheurs d'avenir" for this project's funding. The authors also thank the Region Hauts-de-France for CPER BiHauts Eco de France.

### Declaration of competing interest

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence the work reported in this paper.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.lwt.2025.118178.

### Data availability

The data that has been used is confidential.

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