# Continuous production of antioxidant extracts from olive leaves and branches through twin-screw extrusion: a feasibility study

P. Evon, L. Labonne, C. Vialle, Caroline Sablayrolles, M. del Mar Contreras, A. Vidal, E. Castro

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## To cite this version:

P. Evon, L. Labonne, C. Vialle, Caroline Sablayrolles, M. del Mar Contreras, et al., Continuous production of antioxidant extracts from olive leaves and branches through twin-screw extrusion: a feasibility study. Industrial Crops and Products, 2025, 228, pp.120886. 10.1016/j.indcrop.2025.120886. hal-05165484

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Submitted on 16 Jul 2025

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# Continuous production of antioxidant extracts from olive leaves and branches through twin-screw extrusion: a feasibility study

P. Evon<sup>a,*</sup>, L. Labonne<sup>a</sup>, C. Vialle<sup>a</sup>, C. Sablayrolles<sup>a</sup>, M. del Mar Contreras<sup>b</sup>, A. Vidal<sup>b</sup>, E. Castro<sup>b</sup>

## ARTICLE INFO

**Keywords:**

Olive

Leaves

Branches

Twin-screw extrusion

Aqueous extract

Freeze-drying

Antioxidant

## ABSTRACT

Twin-screw extrusion has recently seen its uses extended to the continuous green extraction of biomolecules. Here, it was successfully used for the extraction of antioxidants using subcritical water from two olive co-products, i.e., leaves (OL), and leaves plus branches (OLB). Extraction yields obtained were 52.5 % and 65.6 %, respectively, in proportion to the initial water-solubles. Total phenolic contents and antioxidant activities in the two freeze-dried extracts were 8.9 and 9.6 g gallic acid eq. per 100 g, and 9.8 and 12.1 g Trolox eq. per 100 g. These are of the same order of magnitude as those obtained in laboratory scale studies in which the extracts were obtained using ethanol rather than water as the extraction solvent, whether by maceration, Soxhlet or ultrasound. Twin-screw extrusion also enabled a high proportion of sugars to be co-extracted and, to a lesser extent, proteins, acetyl groups, and ash. For the future, this continuous industrial-scale technology represents a robust and economical methodology for the green extraction of antioxidants from olive leaves and branches. These extracts could find applications in agriculture or horticulture for plant protection, for food applications, or even in the cosmetics industry.

## 1. Introduction

### 1.1. Twin-screw extrusion applied to biomass: general overview

Twin-screw extrusion is well known for its uses in plastic compounding, where plant fillers are mixed into thermoplastic matrices as mechanical reinforcement, e.g., sunflower bark fibers into poly(lactic acid) (PLA) or polypropylene (PP) (Evon et al., 2023), miscanthus or bamboo fibers into PLA (Gamon et al., 2013), and coriander straw into PP or biopolyethylene (BioPE) (Uitterhaegen et al., 2018). Plastic compounding applications also include the plasticization of biopolymers such as starch (Chabrat et al., 2012) or proteins (Evon et al., 2021a). Twin-screw extrusion is also largely used for extrusion-cooking applications, e.g., snacks, cereals, pet food, fish feed (Chaabani et al., 2022), meat analogues, etc. This continuous and versatile technology can also be used in a more original way for the thermo-mechanical (TM) or thermo-mechano-chemical (TMC) refining of lignocellulosic fibers prior to the molding of bio-based fibrous materials such as (binderless) fiberboards, for example, from coriander straw (Uitterhaegen et al.,

2017), barley straw (Theng et al., 2019), linseed flax shives (Evon et al., 2021b) or amaranth bark (Evon et al., 2021c), or prior to the production of energy, e.g., second generation bioethanol (from dehydrated sweet corn co-products, sugarcane bagasse, sawdust of *Eucalyptus grandis*, etc.) in combination with a bio-catalytic action (bioextrusion) (Vandenbossche et al., 2016), or biogas (for example, from sweetcorn by-products, whole triticale, corn stover and wheat straw) (Chevalier et al., 2023).

### 1.2. Emerging use of twin-screw extrusion for the green extraction of biomolecules

More recently, twin-screw extrusion has seen its uses extended to the continuous green extraction of biomolecules (Evon et al., 2018; Vandenbossche et al., 2019; Bittencourt Moraes Duarte Silva et al., 2025). For such configuration, a filtration module is positioned along the barrel, usually in the penultimate position, enabling a filtrate containing the extracted molecules (liquid extract) and a solid residue, known as the raffinate or extrudate, to be collected separately (Fig. 1). For example, it

<footnote><sup>a</sup> Laboratoire de Chimie Agro-Industrielle, LCA, Université de Toulouse, INRAE, INPT, Toulouse, France</footnote>

<footnote><sup>b</sup> Department of Chemical, Environmental and Materials Engineering, Institute of Biorefineries Research, Universidad de Jaén, Jaén, Spain</footnote>

<footnote>* Corresponding author.
E-mail address: philippe.evon@toulouse-inp.fr (P. Evon).</footnote>
was already used for:

- The mechanical pressing of vegetable oils such as sunflower (Evon et al., 2009), jatropha (Evon et al., 2013) or coriander (Uitterhaegen et al., 2015).

- The aqueous extraction of sunflower oil (Evon et al., 2007, 2009).

- The aqueous extraction of polyphenols and antiradicals from wood by-products, e.g., maritime pine knots and stumps, and aspen knots and barks, in subcritical conditions, i.e., temperature above 100 °C and pressure above 1 bar (Celhay, 2013; Celhay et al., 2014).

- More recently, the extraction of biostimulatory molecules (e.g., flavonoids) from sunflower by-products (stalks and heads), still using subcritical water (Li et al., 2022), including the optimization of the twin-screw extrusion process (Li et al., 2025).

In parallel, many studies dealt with the extraction of antioxidants from olive co-products but only at laboratory scale, with ethanol or a hydroalcoholic solution as the extraction solvent, whether by maceration (Lama-Muñoz et al., 2019), Soxhlet (Lama-Muñoz et al., 2020) or ultrasound (Contreras et al., 2019, 2021). However, none of these studies involved a continuous extraction process, and none of them proposed scaling up the extraction procedure. In addition, although ethanol is considered to be a green solvent, compatible with the 12 principles of green chemistry, and may even be of biosourced origin, ethanol is highly flammable, toxic, and it is also classified since 2007 as a group 1 human carcinogen by the International Agency for Research on Cancer (IARC) (INRS, 2019).

The aim of the present study was thus to validate the feasibility of using the twin-screw extrusion technology for the continuous extraction of antioxidant biomolecules from two olive co-products, i.e., leaves, and leaves plus branches, using subcritical water as a greener solvent with health benefits, no flammability, and no toxicity rather than ethanol or a hydroalcoholic solution.

## 2. Materials and methods

### 2.1. Materials

Two different olive co-products were used in this study, i.e., leaves (OL), and leaves plus branches (OLB). They originated from the region of Jaén in Spain, and they were first grinded using a Electra (France) Goulu N hammer mill fitted with a 6 mm grid before their twin-screw extrusion treatment. Most of the chemicals and reagents utilized were analytical grade. These reagents, i.e., Folin-Ciocalteu's reagent, gallic acid (≥98 %), sodium carbonate, sulfuric acid, 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) and sugars (>98 %), were purchased from Sigma-Aldrich (St. Louis, MO, USA). High purity standards of oleuropein (≥98 %) and hydroxytyrosol (≥98 %) were acquired from Extrasynthese (Genay, France) and Sigma-Aldrich, respectively. Iron (III) chloride 6-hydrate

pure, hydrochloric acid 37 %, and acetonitrile (HPLC grade) were obtained from AppliChem Panreac (Darmstadt, Germany). VWR Chemical (Gliwice, Poland) provided methanol (HPLC grade). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was from Acros Organics (New Jersey, USA). A Bradford Protein Assay was purchased from Bio-Rad (Hercules, CA, USA). Ultrapure water was obtained using a Milli-Q system (Millipore, Bedford, MA, USA).

### 2.2. Analytical methods

The moisture and ash contents were determined according to ISO 665:2000 and 749:1977, respectively. Measuring the mass loss of the test sample after 1 h in boiling water made it possible to determine the content in water-soluble components. Determinations were all carried out in triplicate.

### 2.3. Twin-screw extrusion

The extraction of antioxidants from OL and OLB using subcritical water was conducted on an industrial-scale machine (Cleextral Evolum HT 53, France), using a previously optimised screw profile (Li et al., 2022, 2025). This co-rotating and co-penetrating twin-screw extruder had a barrel length of 1.9 m. It consisted of eight modules, each 4 D in length (D is the screw diameter, i.e., 53 mm), except for module 1, which had an 8 D length. A filter section consisting of six hemispherical dishes with 1 mm diameter perforations outfitted on module 7 enabled filtrate collection.

During the liquid/solid fractionation process, OL and OLB were fed at the level of module 1 using a gravimetric feeder, and water was injected at the end of module 2 thanks to a piston pump. Following operating conditions were applied during the extrusion process: 100 °C and 110 °C, respectively, for the extraction and pressing temperatures, 200 rpm for the screw rotation speed, around 10 kg/h for the incoming flow rate of raw material, and 5.5 for the expected L/S ratio. In particular, under conditions of temperature above 100 °C and pressure above 1 bar, which can be achieved with twin-screw extrusion, the polarity, surface tension and viscosity of water can be greatly reduced, to the point of approaching the properties of solvents conventionally used for extracting polyphenols (Celhay, 2013). For example, in such subcritical state, water has proved highly effective in extracting polyphenols and antioxidants from various plant materials, including birch bark (Co et al., 2012), spruce knots (Co et al., 2011), poplar knots (Hartonen et al., 2007), and also maritime pine knots and stumps, and aspen knots and barks (Celhay, 2013; Celhay et al., 2014).

For both raw materials treated, sampling was made once the twin-screw extruder was operating at steady state. Two 10-minute samples were then collected to assess the mean outlet flow rates of liquid extract and solid raffinate. Extraction yields and specific mechanical energy (SME) were calculated according to the formulas given in Li et al.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Schematic representation of a twin-screw extruder used as a liquid/solid (L/S) extractor and separator for the continuous green extraction of biomolecules (Vandenbossche et al., 2019).</figcaption></figure>
(2022), (2025).

## 2.4. Treatment of the liquid extracts

During extrusion processing, liquid extracts were collected at the bottom of the filter section. Sampling was followed by centrifugation to remove small solid particules in suspension, concentration via partial water evaporation, and finally freeze-drying to produce stable dry powder products for chemical composition and antioxidant activity assessment. Centrifugation was conducted using a centrifugal dryer (RC50PXR, Rousselet-Robatel, France) equipped with a 10 µm mesh bag at a rotation speed of 2000 rpm, with liquid extracts processed at a flow rate of approximately 40 kg/h.

Concentration was achieved using a 300 L pilot unit (K9955 Tournaire, France), with partial water evaporation carried out under vacuum (80–100 mbar) and heating in a bain-marie to approximately 35 °C. The water evaporation rate was around 20 L/h, and the mass reduction of the clarified extract post-concentration was approximately 3–3.5 times. Finally, freeze-drying was performed using a 38 L pilot freeze-dryer (Cryonext, France), with the freezing temperature set at –30 °C and a 0.3 mbar vacuum applied.

## 2.5. Chemical composition and antioxidant activity of the extracts

The total phenolic contents were determined by Folin-Ciocalteu method. The antioxidant activities were determined by Ferric Reducing Antioxidant Power (FRAP), according to Lama-Muñoz et al. (2020) and Contreras et al. (2021), respectively. In both assays, the absorbance was measured by a microplate reader, model Bio-Rad iMark (Hercules, CA, USA). Contents in hydroxytyrosol and oleuropein were determined by HPLC-UV with standards. A C18 column model BDS Hypersil (Thermo Scientific, Waltham, MA, USA) (25 cm of length; 4.6 mm of internal diameter; 5 µm of particle size), and a liquid chromatograph (Shimadzu Corp., Kyoto, Japan) were used for this analysis at 280 nm. Contents in sugars (i.e., glucose, galactose, and mannitol) and acetyl groups were determined following the National Renewable Energy Laboratory (NREL) methodology, technical report NREL/TP-510-42618 (Sluiter et al., 2008). A column CARBOSep CHO-782 PB (Transgenomic Inc, Omaha, USA) equipped in a Waters HPLC device (2695 model) (Milford, MA, USA) was used for the quantification of the sugars, according to Lama-Muñoz et al. (2020). Acetyl groups were measured using an ICSep ICE-COREGEL-87H3 column (Transgenomic, Inc., Omaha, NE, USA) in an Agilent liquid chromatograph 1260 (Agilent Technologies, Santa Clara, CA, USA) (Contreras et al., 2021). Protein contents were determined by the Bradford assay using bovine serum albumin (BSA) as standard with the aforementioned microplate reader, according to Contreras et al. (2021).

## 2.6. Statistical analyses

All determinations were conducted in duplicate or triplicate, and data are expressed as means ± standard deviations. The means were compared by the use of a single factor analysis of variance (ANOVA) using the GLM procedure of the SAS data analysis software. The comparison between the different individual means was performed using the Duncan's multiple range test at a 5 % probability level.

# 3. Results and discussion

Table 1 mentions the material balance of the two samplings (i.e., from OL and OLB), including the treatment of the two filtrates after twin-screw extrusion, as well as the associated energy costs. Extraction yield obtained was 16.2 ± 0.0 % for OL, and 19.8 ± 0.4 % for OLB (yield expressed as a percentage of the initial dry matter), or 52.5 ± 0.1 % and 65.6 ± 1.4 %, respectively, relative to the initial quantity of water-solubles. These promising extraction yields are in fact in line or

<caption>Table 1</caption>

Material balance of the two samplings (i.e., from OL and OLB), including the post-treatment of the filtrates after twin-screw extrusion, and energy costs (results are expressed as mean value ± standard deviation, n = 2).

<table><thead><tr><th>Raw material</th><th>OL</th><th>OLB</th></tr></thead><tbody><tr><td>Screw rotation speed (rpm)</td><td>200.8<br>± 0.0<sup>a</sup></td><td>200.8<br>± 0.0<sup>a</sup></td></tr><tr><td>Inlet</td><td></td><td></td></tr><tr><td>Raw material moisture content (%)</td><td>10.5 ± 0.4<sup>b</sup></td><td>13.6 ± 0.2<sup>a</sup></td></tr><tr><td>Raw material water-soluble content (% dry matter)</td><td>30.8 ± 0.3<sup>a</sup></td><td>30.2 ± 0.8<sup>a</sup></td></tr><tr><td>Raw material inlet flow rate (kg/h)</td><td>10.4</td><td>8.6</td></tr><tr><td>Water inlet flow rate (kg/h)</td><td>55.0</td><td>55.0</td></tr><tr><td>Total inlet flow rate (kg/h)</td><td>65.4</td><td>63.6</td></tr><tr><td>L/S ratio</td><td>5.3</td><td>6.4</td></tr><tr><td>Device's filling coefficient (kg/h rpm)<sup>a</sup></td><td>0.052</td><td>0.043</td></tr><tr><td>Outlet</td><td></td><td></td></tr><tr><td>Solid raffinate moisture content (%)</td><td>38.9 ± 0.1<sup>a</sup></td><td>30.5 ± 0.6<sup>b</sup></td></tr><tr><td>Solid raffinate water-soluble content (% dry matter)</td><td>16.9 ± 0.2<sup>a</sup></td><td>14.9 ± 0.0<sup>b</sup></td></tr><tr><td>Solid raffinate outlet flow rate (kg/h)</td><td>6.5 ± 1.1<sup>a</sup></td><td>4.4 ± 0.4<sup>b</sup></td></tr><tr><td>Filtrate moisture content (%)</td><td>90.3 ± 0.2<sup>b</sup></td><td>92.4 ± 0.0<sup>a</sup></td></tr><tr><td>Filtrate outlet flow rate (kg/h)</td><td>55.4 ± 1.9<sup>a</sup></td><td>57.9 ± 3.0<sup>a</sup></td></tr><tr><td>Total outlet flow rate (kg/h)</td><td>61.8 ± 3.0<sup>a</sup></td><td>62.3 ± 3.4<sup>a</sup></td></tr><tr><td>Filtrate outlet flow rate as a proportion of total outlet flow rate (%)</td><td>89.6</td><td>93.0</td></tr><tr><td>Proportion of water evaporated during twin-screw extrusion (%)</td><td>6.5</td><td>2.4</td></tr><tr><td>Material temperature in the pressing zone (module 8) during sampling (°C)</td><td>107.3<br>± 0.1<sup>b</sup></td><td>108.6<br>± 0.8<sup>a</sup></td></tr><tr><td>Treatment of the liquid extracts through centrifugation</td><td></td><td></td></tr><tr><td>Centrifugation flow rate (kg/h)</td><td>41.5</td><td>36.2</td></tr><tr><td>Filtrate's foot (i.e., small solid particules in suspension in the filtrate) (%)</td><td>18.6</td><td>12.6</td></tr><tr><td>Clarified filtrate (%)</td><td>81.4</td><td>87.4</td></tr><tr><td>Clarified filtrate dry matter content (%)</td><td>3.4 ± 0.0<sup>a</sup></td><td>2.9 ± 0.1<sup>b</sup></td></tr><tr><td>Extraction yields</td><td></td><td></td></tr><tr><td>Yield expressed as a percentage of the initial dry matter (%)</td><td>16.2 ± 0.0<sup>b</sup></td><td>19.8 ± 0.4<sup>a</sup></td></tr><tr><td>Yield relative to the initial quantity of water-solubles (%)</td><td>52.5 ± 0.1<sup>b</sup></td><td>65.6 ± 1.4<sup>a</sup></td></tr><tr><td>Energy costs</td><td></td><td></td></tr><tr><td>Extruder's amperage (A)</td><td>21.6 ± 1.6<sup>b</sup></td><td>26.2 ± 1.4<sup>a</sup></td></tr><tr><td>SME (W h/kg raw material)</td><td>212.4<br>± 15.9<sup>b</sup></td><td>313.4<br>± 16.5<sup>a</sup></td></tr><tr><td>SME (kW h/kg dry extract)</td><td>1.46<br>± 0.11<sup>b</sup></td><td>1.83<br>± 0.10<sup>a</sup></td></tr><tr><td>Production cost (€/kg dry extract)</td><td>0.30<br>± 0.02<sup>b</sup></td><td>0.37<br>± 0.02<sup>a</sup></td></tr></tbody></table>

even higher than other ones from previous studies also dealing with the aqueous extraction of active molecules. A very recent study dealing with the extraction of biostimulants (e.g., flavonoids) from sunflower by-products revealed in the optimal twin-screw extrusion conditions a 13.6 % extraction yield in proportion to the initial dry matter, corresponding to a 64.4 % extraction yield in proportion to the initial water-solubles (Li et al., 2025). As another example, Celhay et al. (2014) also investigated the extraction of polyphenols and antiradicals from wood by-products through twin-screw extrusion. They evidenced much lower yields in proportion to the initial dry matter, i.e., a yield for total extractives (in g per 100 g dry matter) up to 1.8 % and 3.1 % for maritime pine knots and stumps, respectively, and up to 2.1 % and 6.9 % for aspen knots and barks, respectively. From the purities in polyphenols in the dry extracts, this corresponded to extraction yields for polyphenols (in g gallic acid eq. per g dry matter) of 0.21 %, 0.20 %, 0.16 %, and 0.78 %, respectively. SME values were 1.46 ± 0.11 and 1.83 ± 0.10 kW h/kg dry extract, respectively, making this continuous extraction operation quite inexpensive (i.e., 0.30 ± 0.02 and 0.37 ± 0.02 €/kg dry extract, respectively, considering a mean electricity price of 0.2047 €/kW h in the French energy mix in 2024). By means of comparison, much higher SME of 2.80 kW h/kg dry extract was obtained for the extraction of

<footnote><sup>a</sup> Defined as the ratio of the raw material inlet flow rate to the screw rotation speed. Means in the same line with the same superscript letter (a-b) are not significantly different at p < 0.05.</footnote>
biostimulatory molecules from sunflower by-products (Li et al., 2025). However, these costs relate here solely to the production cost of the dry extract. They do not include the capital expenditure (CAPEX), i.e., the costs associated with purchasing the twin-screw extruder and its depreciation, and the operational expenditure (OPEX), i.e., the costs for the replacement of wear-prone parts of the extrusion machine like screw elements, sleeve modules, and filter grids, which may degrade over time due to abrasion. Before the process can be industrialised in the future, these extra-costs will have to be estimated and then taken into account to assess as accurately as possible the real cost of producing the antioxidant extracts from the olive co-products.

The two powders were then qualified in terms of their total phenolic content and antioxidant activity. The results are as follows: 8.9 ± 0.8 and 9.6 ± 0.5 g gallic acid eq. per 100 g for the total phenolic content in OL and OLB extracts, respectively, and 9.8 ± 0.1 and 12.1 ± 0.2 g Trolox eq. per 100 g for FRAP (Table 2). These are of the same order of magnitude as those obtained in laboratory scale studies, in which the extracts were obtained using ethanol or a hydroalcoholic solution rather than water as the extraction solvent, whether by maceration (Lama-Muñoz et al., 2019), Soxhlet (Lama-Muñoz et al., 2020) or ultrasound (Contreras et al., 2019, 2021). Twin-screw extrusion also enables a high proportion of sugars to be co-extracted (i.e., 22.0–24.5 g glucose, 1.8–2.3 g galactose, and 10.0–11.1 g mannitol per 100 g) and, to a lesser extent, proteins, acetyl groups, and ash (Table 3). Co-extracted compounds alongside phenolics provide additional opportunities for extract valorisation. Sugars (e.g., glucose) and proteins contribute nutritional value and can serve as precursors for platform compounds (such as lactic acid and ethanol) and amino acids, respectively. However, separating each component would require purification methods. Alternatively, the entire extract, rich in phenolic compounds, could be commercialized as a whole, similar to existing market products. Nonetheless, characterizing the other components is crucial, as this type of extract is inherently complex.

With such contents in phenolic compounds and such antioxidant activities, both freeze-dried extracts would be of real interest in agriculture or horticulture for plant protection, for food applications, or even in the cosmetics industry. These two extracts also contain high proportions of hydroxytyrosol, and especially oleuropein. According to the literature, these two compounds have significant market potential owing to their antioxidant, and health-related properties (Kashaninejad et al., 2020). Produced by green extraction using subcritical water and in a continuous mode through twin-screw extrusion, they offer clear advantages for future industrial applications. As a feasibility study, the present work will have however to be further optimised in the near future. As a reminder, the screw profile, and extraction and pressing

temperatures were chosen equal to those used for extracting bio-stimulants from sunflower by-products (Li et al., 2022, 2025). Thus, for future work, the screw and temperature profiles will have to be optimised to adapt to raw materials (i.e., olive leaves and branches) other than sunflower co-products. In particular, the temperature of 100 °C in twin-screw extrusion may be too high for the extraction of thermosensitive phenolic compounds (Co et al., 2012). It will therefore be necessary to study the effect of temperature on the quality of the extracted antioxidant molecules.

Twin-screw extrusion also exists from the lab scale to the industrial one (up to several hundreds of kg/h at the inlet in liquid-to-solid fractionation conditions). Scale-up operations are also facilitated thanks to a homothetic design of the Clextral twin-screw extruders from the Evolum range, allowing easy transposition of optimal conditions (e.g., screw and temperature profiles, inlet flow rate of raw material, machine's filling, L/S ratio, etc.) from the lab scale to the industrial one. In doing so, this technology could allow in the short term an intensification of the process of extraction of antioxidants from olive co-products.

For its side, the extrudate (solid raffinate), which represents up to 42.6 % of the initial dry matter, and constitutes the lignocellulosic co-product of the process, can be supplemented by the filtrate's foot. It could be used for a variety of applications, such as energy production by combustion or methanisation, as a source of organic soil improver in agriculture or horticulture, or for the production of biosourced materials such as fiberboards.

## 4. Conclusion

As a continuous industrial-scale technology, twin-screw extrusion constitutes for the near future a robust and economical tool for the green extraction of antioxidants with subcritical water (i.e., a non-flammable, and non-toxic solvent with health benefits) from olive leaves and branches, while minimizing the amount of extracting solvent. The olive extracts could find applications for plant protection, for food applications, or in the cosmetics industry. This would guarantee for the years to come an additional source of income for olive producers.

## Funding

This research was conducted in the frame of the project PID2023-149614OB-C21, which was funded by MCIU/AEI/10.13039/501100011033, and by FEDER (European Union).

<caption>**Table 2**</caption>

Contents of phenolic compounds, and antioxidant activity of the lyophilized OL and OLB extracts (results are expressed as mean value ± standard deviation, n = 3), and comparison with data from the literature (raw material, type of extraction and extracting solvent cited each time).

<table><thead><tr><th>Extract</th><th>Total phenolic content<sup>1</sup><br>(g gallic acid eq. per 100 g)</th><th>FRAP<sup>2</sup><br>(g Trolox eq. per 100 g)</th><th>Hydroxytyrosol<sup>3</sup><br>(g per 100 g)</th><th>Oleuropein<sup>3</sup><br>(g per 100 g)</th></tr></thead><tbody><tr><td>OL</td><td>8.90 ± 0.76<sup>a</sup></td><td>9.76 ± 0.12<sup>b</sup></td><td>0.24 ± 0.01<sup>a</sup></td><td>4.01 ± 0.06<sup>b</sup></td></tr><tr><td>OLB</td><td>9.65 ± 0.47<sup>a</sup></td><td>12.06 ± 0.16<sup>a</sup></td><td>0.26 ± 0.01<sup>a</sup></td><td>7.25 ± 0.04<sup>a</sup></td></tr><tr><td colspan="5">Data from the literature</td></tr><tr><td>Leaves</td><td></td><td></td><td></td><td></td></tr><tr><td>Maceration, 60.8 % ethanol (Lama-Muñoz et al., 2019)</td><td>12.5<sup>4</sup></td><td>13.2<sup>4</sup> (DPPH)</td><td>-</td><td>6.3<sup>4</sup></td></tr><tr><td>Soxhlet, 60 % ethanol (Lama-Muñoz et al., 2020)</td><td>12.0–14.4<sup>45</sup></td><td>19.2–25.6<sup>45</sup> (DPPH)</td><td>-</td><td>14.0–29.2<sup>45</sup></td></tr><tr><td>Ultrasound, ethanol (Contreras et al., 2019)</td><td>13.1<sup>4</sup></td><td>14.9<sup>4</sup> (TEAC)</td><td>-</td><td>12.7<sup>4</sup></td></tr><tr><td>Ultrasound, 47 % ethanol (Contreras et al., 2021)</td><td>14.0<sup>4</sup></td><td>14.9<sup>4</sup> (TEAC)</td><td>-</td><td>≈ 16<sup>4</sup></td></tr><tr><td>Extraction (50°C, 80 % ethanol (Kashaninejad et al., 2020)</td><td>11.3</td><td>69.4</td><td>0.3</td><td>11.8</td></tr><tr><td>Maceration, 80 % ethanol (Vidal et al., 2022)</td><td>-</td><td>-</td><td>-</td><td>5.7</td></tr><tr><td>Leaves plus branches</td><td></td><td></td><td></td><td></td></tr><tr><td>Ultrasound, ethanol (Contreras et al., 2019)</td><td>5.0<sup>4</sup></td><td>4.8<sup>4</sup> (TEAC)</td><td>-</td><td>1.8<sup>4</sup></td></tr><tr><td>Ultrasound, 47 % ethanol (Contreras et al., 2021)</td><td>13.1<sup>4</sup></td><td>18.7<sup>4</sup> (TEAC)</td><td>-</td><td>≈ 2<sup>4</sup></td></tr></tbody></table>

<footnote n="1">¹Determined by Folin-Ciocalteu method. ² Antioxidant activity determined by Ferric Reducing Antioxidant Power. ³ Determined by HPLC-UV with standards. ⁴ Estimated theoretical value considering the extraction yield. ⁵ Depending on the variety and origin. Means in the same column with the same superscript letter (a-b) are not significantly different at p < 0.05.</footnote>
<caption>**Table 3**</caption>

Contents of sugars, protein, acetyl groups and ash of the lyophilized OL and OLB extracts (results are expressed as mean value ± standard deviation, n = 3), and comparison with data from the literature (raw material, type of extraction and extracting solvent cited each time).

<table><thead><tr><th>Extract</th><th>Glucose<sup>1</sup><br>(g per 100 g)</th><th>Galactose<sup>1</sup><br>(g per 100 g)</th><th>Mannitol<sup>1</sup><br>(g per 100 g)</th><th>Protein<sup>2</sup><br>(g BSA per 100 g)</th><th>Acetyl groups<sup>1</sup><br>(g per 100 g)</th><th>Ash<br>(g per 100 g)</th></tr></thead><tbody><tr><td>OL</td><td>24.49 ± 0.04<sup>a</sup></td><td>2.30 ± 0.02<sup>a</sup></td><td>10.00 ± 0.09<sup>b</sup></td><td>1.39 ± 0.02<sup>a</sup></td><td>0.22 ± 0.01<sup>a</sup></td><td>8.27 ± 0.04<sup>a</sup></td></tr><tr><td>OLB</td><td>22.04 ± 0.33<sup>b</sup></td><td>1.75 ± 0.02<sup>b</sup></td><td>11.09 ± 0.11<sup>a</sup></td><td>1.20 ± 0.02<sup>b</sup></td><td>0.16 ± 0.01<sup>b</sup></td><td>7.75 ± 0.05<sup>b</sup></td></tr><tr><td colspan="7">Data from the literature</td></tr><tr><td>Leaves</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Soxhlet, 60 % ethanol (Lama-Muñoz et al., 2020)</td><td>1.5–4.1<sup>3d</sup></td><td>0.2–0.9<sup>3d</sup></td><td>8.6–19.0<sup>3d</sup></td><td>-</td><td>-</td><td>-</td></tr><tr><td>Extraction (50°C, 80 % ethanol (Kashaninejad et al., 2020)</td><td>3.9<sup>3</sup></td><td>-</td><td>17.4<sup>3</sup></td><td>-</td><td>-</td><td>-</td></tr><tr><td>Olive tree pruning</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Autoclave (120°C, water (Romero-García et al., 2022)</td><td>18.75<sup>3</sup></td><td>1.25<sup>3</sup></td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

¹ Determined following the NREL methodology. ² Determined by the Bradford assay using BSA as standard. ³ Estimated theoretical value considering the extraction yield. ⁴ Depending on the variety and origin. Means in the same column with the same superscript letter (a-b) are not significantly different at *p* < 0.05.

## CRediT authorship contribution statement

**P. Evon:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. **L. Labonne:** Methodology, Investigation. **C. Vialle:** Writing – review & editing, Validation, Formal analysis, Conceptualization. **C. Sablayrolles:** Writing – review & editing, Validation, Formal analysis, Conceptualization. **M. del Mar Contreras:** Methodology, Investigation. **A. Vidal:** Methodology, Investigation. **E. Castro:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Formal analysis, Conceptualization.

## 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.

## Data Availability

No data was used for the research described in the article.

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