# Application of enzyme-assisted extraction on the brown seaweed Fucus vesiculosus Linnaeus (Ochrophyta, Fucaceae) to produce extracts for a sustainable agriculture

Manon Choulot, Chirelle Jabbour, A.S. Burlot, Lun Jing, Maja Welna, Anna Szymczycha-Madeja, Cecile Le Guillard, Izabela Michalak, N. Bourgougnon

► To cite this version:

Manon Choulot, Chirelle Jabbour, A.S. Burlot, Lun Jing, Maja Welna, et al.. Application of enzyme-assisted extraction on the brown seaweed Fucus vesiculosus Linnaeus (Ochrophyta, Fucaceae) to produce extracts for a sustainable agriculture. Journal of Applied Phycology, 2025, 10.1007/s10811-024-03436-2. http://dx.doi.org/10.1007/s10811-024-03436-2

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# Application of enzyme-assisted extraction on the brown seaweed *Fucus vesiculosus* Linnaeus (Ochrophyta, Fucaceae) to produce extracts for a sustainable agriculture

Manon Choulot<fnref n="1" />,<fnref n="4" />,<fnref n="5" /> · Chirelle Jabbour<fnref n="4" /> · Anne-Sophie Burlot<fnref n="4" /> · Lun Jing<fnref n="2" /> · Maja Welna<fnref n="3" /> · Anna Szymczycha-Madeja<fnref n="3" /> · Cécile Le Guillard<fnref n="5" /> · Izabela Michalak<fnref n="1" /> · Nathalie Bourgougnon<fnref n="4" />

Received: 25 October 2024 / Revised: 20 December 2024 / Accepted: 27 December 2024 / Published online: 28 January 2025
© The Author(s) 2025

## Abstract

The agricultural sector plays a crucial role in sustaining population growth and ensuring our well-being. However, as this sector faces numerous challenges due to environmental conditions and the increasing population, it is essential to identify natural alternatives that promote sustainable farming practices and protect the environment. Seaweed-based extracts have gained popularity in agriculture because of their numerous benefits for plant growth and health. This study focuses on *Fucus vesiculosus* Linnaeus, a brown seaweed species prevalent along the Brittany coast. The research involved comparing enzyme-assisted extraction (EAE) with conventional aqueous extraction methods for the purpose of determining the effectiveness of EAE in producing enriched extracts that could have an interesting agricultural application. The results indicate that enzymatic extraction of *F. vesiculosus* significantly increased the content of neutral sugars by 34% and reducing sugars by 21% in the extracts, compared to conventional aqueous extraction (WE). Regarding plant growth regulators, the levels of isopentenyladenosine (iPR) and cis-zeatin (cZ) were enhanced by 6 times and 28 times, respectively, when using EAE instead of WE. Additionally, the total phenolic content was notably higher in EAE extracts, showing a twofold increase over WE extracts. Furthermore, the various extracts demonstrated superior antioxidant activity compared to raw *F. vesiculosus* powder. Thus, this study confirms that EAE is an effective method for enriching *F. vesiculosus* extracts with various compounds that can play a vital role in agriculture.

**Keywords** Brown seaweed · *Fucus vesiculosus* · Enzyme-assisted extraction · Biochemical composition · Elements · Plant growth regulators

## Introduction

Over the last few years the agricultural sector is facing numerous challenges due to a burgeoning global population, climate warming, severe weather events, the over use of mineral fertilizers and in the same time the necessity to produce food in more efficient and sustainable ways. This context led again to a broader interest in natural alternatives to preserve environment and produce quality food. One of the most popular, intensively developing groups of biostimulants are seaweed extracts. Biostimulants are the most promising short-term markets for seaweed, projected to reach US$ 4.4 billion by 2030. According to Global Seaweed New and Emerging Markets Report (World Bank 2023), seaweed-based biostimulants can expect to see significant growth over the next few years as additional investment goes into research and development for improving efficacy, and as

<figure><img src="image_2.png" /></figure>

<footnote>Nathalie Bourgougnon
nathalie.bourgougnon@univ-ubs.fr</footnote>

<footnote n="1">¹ Faculty of Chemistry, Department of Advanced Material Technologies, Wrocław University of Science and Technology, Wrocław, Poland</footnote>

<footnote n="2">² Centre Mondial de L'Innovation of Groupe Roulier, Phys-Chem & Bioanalytics Department, 35400 Saint-Malo, France</footnote>

<footnote n="3">³ Faculty of Chemistry, Department of Analytical Chemistry and Chemical Metallurgy, Wrocław University of Science and Technology, Wrocław, Poland</footnote>

<footnote n="4">⁴ Université Bretagne Sud, EMR CNRS, LBCM, 56000 Vannes, IUEM, France</footnote>

<footnote n="5">⁵ Centre Mondial de L'Innovation de Groupe Roulier, Plant Nutrition R&D Department, 35400 Saint-Malo, France</footnote>
more and more seaweed processors are exploiting seaweed biomass as a whole in a biorefinery approach to create multiple added-value products.

For centuries, coastal populations in various regions across the globe have harvested a diverse range of seaweeds, source of livelihood and survival (Jacob et al. 2012). These seaweeds were first used for domestic purposes, such as human health, consumption, or glass production (Arzel 1987). Seaweeds have also been employed as fertilizers, soil enhancers, and for soil pH adjustment. In regions such as Brittany and along the French Atlantic coast, seaweed was traditionally collected by coastal farmers using large rakes following storms and directly applied to fields. This traditional practice persists on the island of Batz in northern Brittany and the Rhuys peninsula in southern Brittany in France (Choulot et al. 2022). The application of seaweed-based products in agriculture has been gaining momentum in crop production systems over the past 30 years. Today, seaweed used for biostimulants is mainly brown wild-harvested especially *Durvillaea potatorum* and *Ecklonia maxima* (Laminariales) or *Ascophyllum nodosum* (Fucales) (Rousseau et al. 2023). Various liquid products are present on the market such as Seasol with *Durvillaea potatorum*, Kelpak with *Ecklonia maxima*, or Fertileader, AlgaGreen and Maxicrop with *Ascophyllum nodosum* (Choulot et al. 2022). Rich in wide variety of compounds – macronutrients and micronutrients, alginates and oligosaccharides, Plant Growth Regulators (PGRs), vitamins, and osmolytes – seaweeds extracts are used as agricultural input products in different forms, such as biofertilizers, liming materials, soil improvers, plant biostimulants, and fertilizing product blends (e.g., Bikovens et al. 2017; Michalak and Baśladyńska 2021; Mzibra et al. 2021; Choulot et al. 2022; Krautforst et al. 2023; Spain et al. 2024).

Among the harvested algae in France, *Fucus vesiculosus* Linnaeus, commonly known as bladder wrack, is most commonly found on sheltered rocky shores subject to some degree of disturbance, such as scour (Bunker et al. 2017). In Brittany, *F. vesiculosus* is particularly abundant, forming extensive intertidal zones characteristic of the mid and low-mid intertidal zones. This species can grow to sizes ranging from tens centimeters to one meter in length, with a lifespan typically between 2 and 5 years (Knight and Parke 1950). The biomass of *Fucus* can now reach several hundred to several thousand fresh tonnes per year (Mesnildrey et al. 2012; CSAVM 2023, personal communication), continuing to play a significant role in local economies for various industries such as cosmetics and agriculture. In agriculture, due to its unique chemical composition, *Fucus* biomass is being tested for the use in agriculture as a soil additive, positively influencing plant growth, for example: oat (Bikovens et al. 2017), garden cress (Michalak and Baśladyńska 2021) or sorghum (Krautforst et al. 2023).

For the production of seaweed based biostimulants, alkaline extraction using potassium hydroxide is currently the most common method, constituting approximately 80% of all biostimulant production (Choulot et al. 2022; World Bank 2023). However, some bioactive compounds such as PGRs, vitamins, and antioxidants are susceptible to degradation under harsh extraction conditions (extreme pH or/and high temperatures) (Sharma et al. 2014). The complexity of the cell wall, with strong interactions between fibrillar, matrix polysaccharides, polyphenols, and proteins, hampers the release of compounds during extraction. Numerous physicochemical and biological methods have been developed to recover active compounds from algal biomass (Kadam et al. 2013; Michalak and Chojnacka 2014). In the context of climate change and socio-environmental concerns, the blue circular economy, selecting an appropriate extraction method must balance metabolites selectivity and recovery, cost-effectiveness, and eco-friendly processes based on green and sustainable chemistry and engineering.

Currently, enzyme-assisted extraction (EAE), microwave-assisted extraction (MAE) or ultrasound-assisted extraction (UAE) are among the techniques most mentioned in the literature for natural biomass processing, used alone or combined (Wijesinghe and Jeon 2012; Michalak and Chojnacka 2014; Choulot et al. 2022; Le Guillard et al. 2023; Krautforst et al. 2023; Choulot et al. 2023; Spain et al. 2024). The processes are described as feasible and efficient alternatives to traditional extraction methods and might be transposed on a large scale for seaweed processing in some industrial sectors.

Due to the highly site-specific activity of the hydrolytic enzymes, enzymatic hydrolysis process has been an excellent, environmentally-friendly method to produce some active compounds. EAE effectively extracts valuable components from seaweed without the need for denaturing conditions such as solvents or high temperatures. The effectiveness of EAE in seaweed liquefaction has been demonstrated through the use of two types of enzymes: proteases and carbohydrates. Research has consistently shown that the use of enzymes can significantly enhance the value of seaweed extracts or compounds (Hardouin et al. 2014a, b; Michalak and Chojnacka 2014; Choulot et al. 2023; Spain et al. 2024). EAE is currently used in various industrial applications, including energy (methanation), agriculture, feed, and food (Wijesinghe and Jeon 2012; Michalak and Chojnacka 2014; Terme et al. 2020; Choulot et al. 2023). Several works explore EAE for the production of molecules of interest from seaweeds (Hardouin et al. 2014a, b; Jousselin et al. 2023; Spain et al. 2024) but no work reports the use of this technique for the production of biostimulants with *F. vesiculosus* biomass.

<figure><img src="image_3.png" /></figure>
This study explores the potential benefits of employing enzyme-assisted extraction on *F. vesiculosus* (Ochrophyta, Fucaceae) by analyzing specific compounds pertinent to agriculture, including plant growth regulators, polyphenols and macro/micronutrients.

## Material and methods

*Fucus vesiculosus* powder (<904 µm) was purchased from Groupe Roullier (Saint-Malo, France). Seaweeds were collected in Brittany (France) in July 2019 and dried in an industrial drying tunnel at 100 ± 20 °C. Right after drying, *F. vesiculosus* was ground and stored in opaque plastic buckets at room temperature for further experiments.

## Enzyme-Assisted Extraction (EAE) and Water Extraction (WE) of *Fucus vesiculosus*

A proximate analysis was conducted to aid selection of appropriate enzymes for seaweed hydrolysis. Three commercial enzymatic preparations were selected – two glycosidases, G1 and G3, and one protease, P2 (Table 1) with respective activities based on the supplier's datasheets.

EAE was performed as described by Hardouin et al. (2014a, b), with minor modifications. The extraction was performed with a 2.5% solid/liquid ratio (w/w) (25 g of dried seaweeds powder was added to 1 L of distilled water) in a jacketed glass reactor vessel. The pH was adjusted for each enzyme using either hydrochloric acid (HCl) or sodium hydroxide (NaOH). Enzymes added at a concentration of 5% enzyme/seaweed ratio (w/w) (1.25 g of enzyme in accordance with the 25 g of seaweed powder used) were placed in a 50 °C water bath with 150 rpm of continuous tangential agitation.

After 17 h, the enzymes were denatured by raising the temperature to 90 °C for 15 min. Water extracts (WE) were obtained from seaweed powder processed in the same conditions (including the denaturation step) without enzyme addition. The extracts (WE and EAE) were centrifuged (5000×g for 15 min at 20 °C) to remove the undigested residue, and then freeze-dried. Each extraction, WE and EAE, was performed in triplicate. The dry matter extraction yield

(EY) was obtained by calculating the ratio between the total weight of dry extract ($m_1$) and the total weight of dry matter in the reactor ($m_2$), and results are expressed in percentage (Eq. 1).

$$ EY(\%) = \frac{m_1(g)}{m_2(g)} \times 100 \quad (1) $$

## Analysis of biochemical composition

Analyses were carried out on seaweed powder and on extracts (EAE and WE). Firstly, for some analyzes (total neutral sugars and proteins content) the *F. vesiculosus* powder was subjected to acid hydrolysis to improve their recovery and quantification according to Hardouin et al. (2014a, b). Thus, 5 mL of 1 M HCl were added to 10 mg of dry seaweed and incubated for 2 h at 100 °C in a dry bath. Then, the solution was neutralized by adding 5 mL of 1 M NaOH. All chemical analyses were performed in triplicate (n=3).

Secondly, for other analyses (including total phenolic content and antioxidant activity), a multistep extraction process was conducted on *F. vesiculosus* powder. Initially, 5 mg of *F. vesiculosus* powder was mixed with 1 mL of distilled water and incubated for 2 h at 100 °C in a thermoshaker set to 1000 rpm. The resulting supernatant was then collected. The remaining precipitate underwent a second extraction with the addition of 1 mL of 1 M HCl, followed by another 2-h incubation under the same conditions. The supernatant from this second extraction was combined with the first and neutralized using 1 mL of 1 M NaOH. Subsequently, the precipitate was resuspended in 1 mL of 5 M NaOH and incubated again. The final supernatant was collected and neutralized with 1 mL of 5 M HCl. All chemical analyses were performed in triplicate (n=3).

## Total sugars

Analysis of total sugars content was performed according to the method described by Dubois et al. (1956). 1 mL of tested sample or standard (glucose) (Avocado Research Chemicals Limited) was inserted into a hemolysis tube. 15 µL of 75% phenol (Prolabo) was added. After stirring the solution,

<table><caption>**Table 1** Characteristics of the enzymatic preparations used for extraction of *Fucus vesiculosus*</caption><thead><tr><th>Enzyme</th><th>Activity</th><th>Source</th><th>Form</th><th>Operational pH</th><th>Operational temperature</th></tr></thead><tbody><tr><td rowspan="2">Glycosidase</td><td>G1</td><td>β-glucosidase</td><td>Aspergillus niger</td><td>Powder</td><td>4</td><td>50–65 °C</td></tr><tr><td>G3</td><td>β-1,3-glucanase and Botrytis glucanase</td><td>Trichoderma longibrachiatum</td><td>Liquid</td><td>5</td><td>50 °C</td></tr><tr><td>Protease</td><td>P2</td><td>Protease (neutral) (endo)</td><td>Bacillus amyloliquefaciens</td><td>Powder</td><td>7</td><td>50 °C</td></tr></tbody></table>
2.5 mL of 96% H₂SO₄ was added to the tube. The solution was cooled in an ice bath for 10 min and then placed in a water bath at 30 °C for 10 min. After stirring, the solutions were ultrasonicated for 5 s to remove micro air bubbles. The absorbance was read at 490 nm and the results are expressed in percentage of dry seaweed (d.s.) or percentage of dry extract (d.e.). The standard glucose range was performed at 20, 40, 60, 80 and 100 µg mL⁻¹.

## Reducing sugars

This analysis was carried out on extracts (WE and EAE) according to the Miller method (1959). First, a solution of 3,5-dinitrosalicylic acid (DNS) was prepared by dissolving 1 g of DNS in 70 mL of 0.8 M NaOH while heating. After that, 30 g of sodium potassium tartrate was added before making up the solution to 100 mL with distilled water, which was then kept in the dark. 500 µL of the sample or standard solution (glucose) (Avocado Research Chemicals Limited) were added to 500 µL DNS reagent in Eppendorf tubes. The sample solutions were heated in a water bath for 10 min at 95 °C and cooled immediately in an ice bath for 5 min. Then, the samples were incubated 15 min in the dark before taking a 200 µL aliquot and measuring its absorbance at 540 nm on a 96-well plate using a Multiskan GO plate reader (Thermo Scientific). The results are expressed in percentage of dry mass of extract (d.e.). The glucose concentrations were determined using a range of standard solutions at 50, 100, 150, 200, 250, 300, 400 and 500 µg mL⁻¹.

## Uronic acids

The composition of uronic acids was analyzed using the colorimetric method originally developed by Blumenkrantz and Asboe-Hansen (1973) and later modified by Filisetti-Cozzy and Carpita (1991). When exposed to hot concentrated strong acid, uronic acids dehydrate and then cyclize, forming derivatives of 5-formylfuroic acid. These derivatives react with meta-hydroxydiphenyl (MHDP) to produce a pink chromophore that absorbs light at λ=525 nm. In addition, the coloration is brighter in the presence of borate, but remains very sensitive to interference from neutral sugars, which stain in the presence of concentrated sulphuric acid. For this reason, potassium sulfamate is added to limit these interferences (Pierre 2010). 200 µL of the tested sample or standard (glucuronic acid from Acros Organics) was placed into a hemolysis tube. Subsequently, 20 µL of 4 M sulfamic acid (Alfa Aesar) was added. After stirring the mixture, 1.2 mL of a 75 M sodium tetraborate solution (from Merck) was carefully added. The tubes were then capped and incubated at 80 °C for 20 min. Following incubation, the tubes were cooled in ice for 5 min. 40 µL of a 0.15% aqueous solution of meta-hydroxydiphenyl (MHDP) (Acros Organics) was

then added. The reaction was allowed to proceed for 10 min before measuring the optical density at 525. The glucuronic acid standard curve was prepared with concentrations of 20, 40, 60, 80, and 100 µg mL⁻¹. The results were expressed as mg g⁻¹ of dry mass of seaweed (d.s.) or as a percentage of dry mass of extract (d.e.). The concentration and content of uronic acids were calculated in the same manner as for total sugars.

## Proteins

The total amount of proteins was determined using the colorimetric method developed by Smith et al. (1985). 25 µL of sample or standard solution were placed in a 96-well flat-bottom microplate, to which was added 200 µL of the kit reagent (Pierce BCA Protein Assay Kit, Thermo Scientific). The microplate was incubated at 37 °C for 30 min before reading at 562 nm on a Thermo Scientific Multiskan GO microplate spectrophotometer. Bovine serum albumin (BSA) was used as the standard, with concentrations of 50, 100, 250, 500, 1000 µg mL⁻¹. The results were expressed in percentage of dry mass of seaweed (d.s.) or percentage of dry mass of extract (d.e.).

## Ash

The mineral matter content was determined for *F. vesiculosus* powder according to the method of Hardouin et al. (2014a, b). The sample was placed in a crucible and calcined by ignition. The ash content was determined thermometrically after calcination of 100 mg of seaweed powder followed by a passage for 2 h in a Carbolite CSF Muffle Furnace (UK) at 585 °C. The final mass corresponds to the mineral matter in the sample and was expressed in percentage of dry mass of seaweed (d.s.).

## Multi-elemental composition

The total contents of elements in *F. vesiculosus* and extracts (WE and EAE) were determined using inductively coupled plasma-optical emission spectrometry (ICP-OES) (Agilent bench-top simultaneous optical emission Ar-ICP spectrometer (model 720)) following microwave-assisted closed-vesSEL digestion (Multiwave PRO microwave reaction system (Anton Paar GmbH)). For this purpose, 0.25 g of sample was placed in a PTFE (polytetrafluoroethylene) vessel, to which were added 5 mL of 65% HNO₃ (Merck, Germany). Then, the mixture underwent digestion for 60 min at a maximum temperature of 190 °C. After cooling at room temperature, samples were adjusted to 25 g with deionized water and placed in a cool chamber until measurement.

Analysis of nitrogen was realized using an elemental FLASH 2000 CHNS analyzer (Thermo Fisher Scientific, USA). Briefly, 2.5 mg of the sample was put in a Universal Soft Tin Containers (OD: 5 mm; H=8 mm, V=157 µL from

<figure><img src="image_4.png" /></figure>
Thermo Fisher Scientific). Calibration was released using a standard of aspartic acid (Merck). Analysis was conducted with the instruction of the manufacturer.

The results were expressed as mg kg⁻¹ of dry mass of seaweed (d.s.) or mg kg⁻¹ of dry mass of extract (d.e.). Analyses were performed in duplicate.

## Plant Growth Regulators (PGRs)

The analysis of plant growth regulators was performed on *F. vesiculosus* raw material without acidic pretreatment and extracts (EAE and WE). Methods for the quantification of plant growth regulators have been previously described (Haddad et al. 2018; Réthoré et al. 2021). 20 mg of sample was suspended in H₂O (Milli-Q, 18.2 MΩ·cm, Millipore) containing isotope-labelled internal standards. The extracts were then purified by SPE (solid phase extraction) using Evolute express CX (cation exchange), Biotage express plates and ABN columns (Acidic, Basic and Neutral) for cytokinins and other PGRs according to the manufacturer's protocol.

The analyses of PGRs were performed by LC-MS using a Nexera X2 UHPLC system (Shimadzu) coupled to a QTrap 6500 + mass spectrometer (Sciex). The separation was carried out by injecting a 2 µL sample into a Kinetex Evo C18 core-shell column (100×2.1 mm, 2.6 µm, Phenomenex) at a flow rate of 0.7 mL min⁻¹, with a column temperature maintained at 40 °C. The mobile phases were composed of solvent A Milli-Q water containing 0.1% formic acid, and solvent B acetonitrile LCMS grade containing 0.1% formic acid. The analyses were performed in scheduled MRM mode. An external calibration curve was established for each PGR. A quality control sample containing all external and internal standards was injected every 10 samples to assess the system stability. The calculated concentration was corrected by the internal standard recovery rate. The results were expressed in µg kg⁻¹ of dry mass of seaweed (d.s.) or dry mass of extract (d.e.).

## Total phenolic content (TPC)

Folin-Ciocalteu method is commonly used for measuring the total phenolic content (TPC). This assay is based on the reduction–oxidation (redox) reactions, which are usually considered to be relatively stoichiometric and on the redox potential of the phenolic hydroxyl group (Hagerman and Butler 1989). This analysis was performed directly on *F. vesiculosus* raw material, on extracts after EAE and on WE from *F. vesiculosus*. Briefly, in a 96-well microplate, 20 µL of samples (1 mg mL⁻¹) or standard (Phloroglucinol from Sigma Aldrich) was added to 100 µL of Folin-Ciocalteu reagent (2N) followed by an incubation of 5 min at 30 °C. Then, 80 µL of Na₂CO₃ solution (7.5%; m/v) was added. The microplate was incubated in the dark for 30 min at 40 °C. Optical density was measured at 760 nm (Waterhouse 2002; Singleton et al. 1999). Phloroglucinol was

used as standard prepared in different concentrations ranging from 0 to 1000 µg mL⁻¹. The results were expressed in milligram of Phloroglucinol Equivalent (PE) per g of dry mass of seaweed (d.s.) or dry mass of extract (d.e.).

## Evaluation of antioxidant activities—DPPH Free radical scavenging assay

DPPH with stable groups is a typical reagent for evaluating antioxidant activity and can be used as a representative to appraise the scavenging activity of antioxidant compounds against free radicals. This analysis is based on modified method of Guo et al. (2012). A series of ascorbic acid solutions, served as control, was prepared in different concentrations (0–30 µg mL⁻¹). 100 µL of 0.25 mM DPPH solution was introduced to 100 µL sample solution or control in a 96-well microplate. Sample solutions from EAE and WE extracts were prepared in different concentrations by diluting the stock solution in methanol (0–1000 µg mL⁻¹). Before reading the optical density at 517 nm, all samples were incubated at 40 °C for 30 min in the dark. Percentage of inhibition was calculated using the following formula (2):

$$I(\%) = [(A_C - A_S)/A_C] \times 100 \quad (2)$$

where: *I (%)* = inhibition (expressed in %), *A_C* = absorbance of control, and *A_S* = absorbance of samples. *IC₅₀*, corresponding to the concentration sufficient to obtain 50% of a maximum scavenging capacity of samples, was determined based on the regression obtained from dose-response curve.

## Statistical analyses

Statistical analyses were carried out using R software and RStudio interface, 2023.09.01 version, to evaluate the statistically significant differences (for *p* < 0.05) between examined groups. Multiple comparison analyses were used for this comparative study between extracts obtained from *F. vesiculosus*. They include parametric (ANOVA) and non-parametric (Kruskall-Wallis) tests after checking the normality and equal variances of data. These analyses used various packages such as rstatix, RcmdrMisc, multcomp, rcompanion and FSA (Holander et al. 1973; Chambers and Hastie 1992; Fox et al. 2024).

# Results

## Fucus vesiculosus powder

### Proximate composition

Figure 1 presents the biochemical composition of the *F. vesiculosus* raw material. Although *F. vesiculosus* is a
very common seaweed species in the Brittany coast, little research has been conducted on the study of its biochemical composition.

*Fucus vesiculosus* was mainly composed of neutral sug-
ars which constitute 22% of d.s. Proteins were the second
important component found in *F. vesiculosus* constituting
17% of d.s. Uronic acids constituted 12% of d.s. however
there is no information was found in the literature concern-
ing the content of uronic acids in *F. vesiculosus* biomass.
In addition, the raw *F. vesiculosus* presented 39.8 ± 9.8 mg
eq PE g⁻¹ d.s. of total phenolic content which consists of
approximately 4% of dry weight.

Multi-element analysis

Multi-element composition of *F. vesiculosus* is presented in Table 2. Elements are classified as macroelements (essential for plant growth and development) and microelements (necessary for a number of physiological functions). The three main elements, in term of content are potassium and sodium with 25 993 mg kg⁻¹ d.s. and 25 497 mg kg⁻¹ d.s., respectively, and calcium with 15 966 mg kg⁻¹ d.s.

Plant growth regulators

According to Table 3, three PGRs are most represented in *F. vesiculosus*. Salicylic acid was detected in large quantity with up to 112.7 µg kg⁻¹ of d.s., followed by auxin (53.5 µg kg⁻¹ d.s.) and the ethylene precursor – ACC (1-amino-cyclopropane-1-carboxylic acid) (36.9 µg kg⁻¹ d.s.).

<table><caption>**Table 2** Multi-element composition of *Fucus vesiculosus* (mg kg⁻¹ d.s.) (mean ± SD; n = 2)</caption><thead><tr><th>Element</th><th><i>F. vesiculosus</i><br>powder material</th></tr></thead><tbody><tr><td><strong>Macroelements</strong></td><td></td></tr><tr><td>Potassium</td><td>25 993 ± 312</td></tr><tr><td>Calcium</td><td>15 966 ± 335</td></tr><tr><td>Sulphur</td><td>10 818 ± 834</td></tr><tr><td>Nitrogen</td><td>8 831 ± 762</td></tr><tr><td>Magnesium</td><td>8 603 ± 155</td></tr><tr><td>Phosphorus</td><td>716 ± 9</td></tr><tr><td><strong>Microelements</strong></td><td></td></tr><tr><td>Iron</td><td>569 ± 11</td></tr><tr><td>Boron</td><td>118 ± 3</td></tr><tr><td>Manganese</td><td>106 ± 2</td></tr><tr><td>Zinc</td><td>25 ± 1</td></tr><tr><td>Copper</td><td>2 ± 0</td></tr><tr><td>Another element</td><td></td></tr><tr><td>Sodium</td><td>25 497 ± 312</td></tr></tbody></table>

Fucus vesiculosus-derived extracts

Biochemical composition

Figure 2 presents the dry matter extraction yield (Fig. 2(a)) and the biochemical composition of the extracts (Fig. 2(b-f)) obtained from *F. vesiculosus* after EAE and WE, according to the operational pH of each enzyme (one protease P2 at

<figure><img src="image_5.png" /><figcaption>**Fig. 1** Proximate composition of *Fucus vesiculosus* raw material (percentage of d.s.)</figcaption></figure>

<figure><img src="image_6.png" /></figure>
<table><caption>**Table 3** Content of plant growth regulators in *Fucus vesiculosus* (µg kg⁻¹ d.s.; mean ± SD; n = 3)</caption><thead><tr><th>Plant Growth Regulators</th><th>Content in F. vesiculosus</th></tr></thead><tbody><tr><td>Salicylic acid (SA)</td><td>113 ± 3</td></tr><tr><td>Auxin</td><td></td></tr><tr><td>Indole-3-acetic acid (IAA)</td><td>54 ± 1</td></tr><tr><td>1-amino-1-cyclopropane-carboxylic acid (ACC)</td><td>37 ± 3</td></tr><tr><td>Dihydrojasmonic acid (DJA)</td><td>8 ± 0</td></tr><tr><td>Abscisic acid (ABA)</td><td>2 ± 0</td></tr><tr><td>Cytokinins</td><td></td></tr><tr><td>Cis-zeatin (cZ)</td><td>&lt;0.1</td></tr><tr><td>Isopentenyladenine (iP)</td><td>1 ± 0</td></tr><tr><td>Isopentenyladenosine (iPR)</td><td>1 ± 0</td></tr><tr><td>Trans zeatin (tZ)</td><td>0 ± 0</td></tr></tbody></table>

pH 7, two different glucosidases G1 and G3 at respectively pH 4 and pH 5).

Results obtained for the dry matter extraction yield (Fig. 2(a)) showed that pH influences the recovery of compounds from *F. vesiculosus*. Significantly higher dry matter extraction yields were observed with WE at pH 4 and pH 7, showing an increase of 18% (*p* < 0.001) and 14% (*p* = 0.003) respectively, compared to WE at pH 5. Notably, only G3 enzyme enhanced the dry matter extraction yield, presenting a statistically significant increase of 13% (*p* 0.006) over WE at pH 5. Among the enzymes tested, G1 achieved the highest dry matter extraction yield at 60%, outperforming both enzyme G3 (*p* = 0.026) and P1 (*p* = 0.024).

Regarding total sugars (Fig. 2(b)), only the enzyme G3 allowed statistical increase (+34%) in the extraction of these compounds in comparison with the corresponding WE at pH 5. The extract obtained with G3 contained the highest amount of total sugars (38 ± 4% of d.e.) in comparison with the two other enzymes G1 and P2 with statistically significant differences between these groups (i.e., G1 and G3 and P2) with results multiplied by 1.8 (*p* = 0.0008) and by 2 (*p* = 0.0004), respectively.

For both glycosidases (G1 and G3) significant higher amounts of reducing sugars were obtained (Fig. 2(c)) equal to 21% and 10% d.e., respectively with significant differences in comparison with WE at pH 4 and WE and pH 5. Finally, even if extracts obtained with G1 had lower content of total sugars in comparison with G3, they contained twice more reducing sugars in comparison with G3 (*p* = 0.0002).

The uronic acid content (Fig. 2(d)) varied between 7 and 10% d.e., with no significant differences among the different extracts, except for those with P2 at pH 7. These latter extracts had the lowest uronic acid content at 6.8 ± 0.4% d.e., while the WE pH 5 extracts had the highest at 9.5 ± 0.8% d.e.

Regarding proteins, the conventional water extracts contained 39 ± 3% d.e. of proteins at pH 5, and this increased to 53 ± 3% d.e. at pH 4 (Fig. 2(e)). Statistically significant

differences were observed between WE at pH 4 and pH 5 (*p* 0.004), and between WE at pH 7 and pH 5 (*p* < 0.005). Extracts obtained with G1 and P2 had higher protein content compared to G3, with statistically significant differences.

Figure 2(f) shows the total phenolic content (TPC) of the different extracts. The results indicate that the highest TPC was found for the extracts obtained by EAE carried out with P2 enzyme at pH 7 (116 ± 7 mg PE g⁻¹ d.e.) with twice more TPC in comparison with its WE at pH 7 (54 ± 11 mg PE g⁻¹ d.e.). On the other hand, extract produced with G1 enzyme at pH 4 yielded a higher TPC (71 ± 11 mg PE g⁻¹ d.e.) than its WE at the same pH (58 ± 11 mg PE g⁻¹ d.e.). Finally, G3 enzyme extract at pH 5 showed a TPC content of 57 ± 8 mg PE g⁻¹ d.e. while the WE extract at pH 5 yielded a TPC content of 50 ± 9 mg PE g⁻¹ d.e.

## Multi-element analysis

Table 4 summarizes the multi-element composition of extracts obtained from *Fucus vesiculosus* by EAE and WE.

Whatever the method and conditions of extraction, the most abundant elements are sodium, potassium and sulphur. It appears that conventional WE most often leads to similar or higher contents in macro- and microelements in aqueous extracts in comparison with extracts produced with EAE. Indeed, only few results show that enzymes allow for increasing the content of elements in the extract (d.e.), for example the content of nitrogen. The highest nitrogen content in the dry mass of the extract was obtained when P2 or G1 were used, leading to higher quantities that their respective WE at same pH.

## Plant growth regulators

Table 5 presents the content of PGRs in extracts obtained through WE and EAE. As observed for *F. vesiculosus* powder (Table 3), the three main PGRs present in *F. vesiculosus* extracts were SA, IAA and ACC. Regarding the comparison of PGRs detected in raw *F. vesiculosus* powder and extracts, Table 5 reveals that new PGRs were identified in the extracts, such as cZR and tZR which are precursors of cZ and tZ respectively. Conversely, DJA is not anymore detected in the extracts.

## Biological anti-oxidant activity

The IC₅₀ values resulting from the DPPH test for the various *F. vesiculosus* powder and extracts are presented in Table 6. The IC₅₀ values across different extracts ranged between 177.3 and 212.6 µg mL⁻¹ and are not all significantly different, making challenging the comparisons between DPPH scavenging activity of EAE and WE extracts. The highest total phenolic content was observed in the protease extract at pH 7 (116.4 ± 6.8 mg PE g⁻¹ d.e.). Despite this, it did not exhibit the greatest antioxidant activity. Surprisingly, it was
<figure><img src="image_7.png" /><figcaption>a. Extraction yield (%)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>b. Total sugars (% d.e.)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>c. Reducing sugars (% d.e.)</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>d. Uronic acids (% d.e.)</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>e. Proteins (% d.e.)</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>f. Total Phenolic Content (mg eq phloroglucinol PE/g d.e.)</figcaption></figure>

**Fig. 2** (a) Extraction yield expressed as the mass of dry matter extracted per 100 g of dry *F. vesiculosus* and biochemical composition of *Fucus*-derived extracts expressed in percentage of dry extract

(d.e.) – (b) total sugars; (c) reducing sugars; (d) uronic acids (% of dry mass of extract (d.e.); (e) proteins; (f) Total phenolic content (mg eq phloroglucinol PE g⁻¹ d.e.) mean ± SD; n = 3

the WE at pH 4 that showed the highest antioxidant activity, despite having a lower total phenolic content (57.7 ± 11 mg PE g⁻¹ d.e.) with an IC₅₀ value of 177.3 µg mL⁻¹.

# Discussion

## Characterization of *Fucus vesiculosus* powder

Comparing the data obtained with existing literature is challenging. Indeed, various factors, including those related to the harvesting, as well as others such as biomass

pre-treatment (such as drying and grinding processes), selection of biochemical analysis techniques, contribute to significant variations. The main parameters responsible of variations in the biochemical composition of seaweed, including *F. vesiculosus*, are related to the harvest season, geographical location, environmental conditions like wave exposure and intensity, salinity, light availability, and reproductive phase or intra- and inter-specific competition (Paiva et al. 2018).

Based on Fig. 1, 33% of the non-detected compounds could be attributed to sulfated groups linked with fucose-rich polysaccharides, such as fucans and fucoidans

<figure><img src="image_13.png" /></figure>
<table><caption>**Table 4** Multi-element composition of extracts produced from *Fucus vesiculosus* by WE (pH 4 and pH 5) or EAE (enzymes G1, G3, P2) (mg kg⁻¹ d.e.; mean ± SD; n=2)</caption><thead><tr><th>Element</th><th>WE pH 4</th><th>WE pH 5</th><th>WE pH 7</th><th>G1 pH 4</th><th>G3 pH 5</th><th>P2 pH7</th></tr></thead><tbody><tr><td><strong>Macroelements</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Potassium</td><td>40 623 ± 325</td><td>39 643 ± 919</td><td>38 603 ± 46</td><td>37 861 ± 644</td><td>36 081 ± 589</td><td>39 005 ± 702</td></tr><tr><td>Sulphur</td><td>14 128 ± 48</td><td>14 465 ± 248</td><td>13 676 ± 11</td><td>13 534 ± 257</td><td>12 476 ± 73</td><td>12 627 ± 278</td></tr><tr><td>Magnesium</td><td>7 988 ± 19</td><td>7 223 ± 76</td><td>6 940 ± 38</td><td>7 544 ± 106</td><td>6 653 ± 65</td><td>6 694 ± 74</td></tr><tr><td>Nitrogen</td><td>4 878 ± 470</td><td>5 659 ± 40</td><td>5 330 ± 320</td><td>5 844 ± 210</td><td>5 767 ± 744</td><td>6 188 ± 288</td></tr><tr><td>Calcium</td><td>7 097 ± 38</td><td>4 980 ± 42</td><td>4 962 ± 42</td><td>6 469 ± 45</td><td>4 962 ± 2</td><td>4 805 ± 30</td></tr><tr><td>Phosphorus</td><td>644 ± 8</td><td>741 ± 1</td><td>782 ± 16</td><td>699 ± 6</td><td>752 ± 2</td><td>753 ± 9</td></tr><tr><td><strong>Microelements</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Boron</td><td>180 ± 1</td><td>190 ± 1</td><td>192 ± 5</td><td>168 ± 2</td><td>181 ± 1</td><td>181 ± 3</td></tr><tr><td>Iron</td><td>184 ± 1</td><td>139 ± 5</td><td>101 ± 2</td><td>179 ± 2</td><td>162 ± 4</td><td>96 ± 1</td></tr><tr><td>Manganese</td><td>91 ± 2</td><td>86 ± 1</td><td>83 ± 2</td><td>87 ± 1</td><td>83 ± 1</td><td>76 ± 1</td></tr><tr><td>Zinc</td><td>22 ± 0</td><td>23 ± 0</td><td>14 ± 0</td><td>20 ± 0</td><td>23 ± 1</td><td>14 ± 0</td></tr><tr><td>Copper</td><td>3 ± 0</td><td>3 ± 0</td><td>2 ± 0</td><td>2 ± 0</td><td>5 ± 0</td><td>2 ± 0</td></tr><tr><td><strong>Another element</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sodium</td><td>43 852 ± 614</td><td>42 913 ± 996</td><td>44 225 ± 388</td><td>47 397 ± 538</td><td>39 635 ± 692</td><td>45 035 ± 676</td></tr></tbody></table>

<table><caption>**Table 5** Plant Growth Regulators composition of the extracts produced from *Fucus vesiculosus* (µg kg⁻¹ d.e.; mean ± SD; n=3)</caption><thead><tr><th>Plant growth regulators</th><th>WE pH 4</th><th>WE pH 5</th><th>WE pH 7</th><th>G1 pH 4</th><th>G3 pH 5</th><th>P2 pH7</th></tr></thead><tbody><tr><td>Auxin</td><td>62 ± 6<sup>b</sup></td><td>88 ± 4<sup>b</sup></td><td>325 ± 34<sup>a</sup></td><td>58 ± 4<sup>b</sup></td><td>105 ± 12<sup>b</sup></td><td>289 ± 35<sup>a</sup></td></tr><tr><td>Indole-3-acetic acid (IAA)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Salicylic acid (SA)</td><td>213 ± 8<sup>b</sup></td><td>245 ± 17<sup>b</sup></td><td>289 ± 0<sup>a</sup></td><td>177 ± 3<sup>c</sup></td><td>245 ± 10<sup>b</sup></td><td>297 ± 5<sup>a</sup></td></tr><tr><td>1-amino-1-cyclopropane-carboxylic acid (ACC)</td><td>86 ± 35<sup>bc</sup></td><td>103 ± 4<sup>bc</sup></td><td>255 ± 29<sup>a</sup></td><td>57 ± 10<sup>c</sup></td><td>165 ± 45<sup>ab</sup></td><td>207 ± 22<sup>a</sup></td></tr><tr><td>Abscisic acid (ABA)</td><td>3 ± 0<sup>b</sup></td><td>3 ± 0<sup>b</sup></td><td>5 ± 0<sup>a</sup></td><td>2 ± 0<sup>b</sup></td><td>3 ± 0<sup>b</sup></td><td>4 ± 0<sup>a</sup></td></tr><tr><td>Cytokinins</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Isopentenyladenosin (iPR)</td><td>2 ± 1<sup>c</sup></td><td>1 ± 0<sup>c</sup></td><td>1 ± 0<sup>c</sup></td><td>12 ± 2<sup>a</sup></td><td>2 ± 0<sup>c</sup></td><td>4 ± 0<sup>b</sup></td></tr><tr><td>Isopentenyladenine (iP)</td><td>1 ± 0<sup>ab</sup></td><td>1 ± 0<sup>ab</sup></td><td>1 ± 0<sup>ab</sup></td><td>1 ± 0<sup>b</sup></td><td>1 ± 0<sup>ab</sup></td><td>1 ± 0<sup>a</sup></td></tr><tr><td>Cis-zeatin (cZ)</td><td>&lt; 0.1</td><td>&lt; 0.1</td><td>&lt; 0.1</td><td>28 ± 2</td><td>&lt; 0.1</td><td>&lt; 0.1</td></tr><tr><td>Trans-zeatin riboside (tZR)</td><td>&lt; 0.025</td><td>&lt; 0.025</td><td>&lt; 0.025</td><td>3 ± 1</td><td>&lt; 0.025</td><td>&lt; 0.025</td></tr><tr><td>Cis-zeatin riboside (cZR)</td><td>&lt; 0.05</td><td>&lt; 0.05</td><td>&lt; 0.05</td><td>2 ± 0</td><td>&lt; 0.05</td><td>2 ± 0</td></tr><tr><td>Trans-zeatin (tZ)</td><td>&lt; 0.05</td><td>&lt; 0.05</td><td>2 ± 0</td><td>&lt; 0.05</td><td>&lt; 0.05</td><td>&lt; 0.05</td></tr></tbody></table>

<table><caption>**Table 6** Dry weight rate and antioxidant activity (IC₅₀) of the *Fucus vesiculosus* powder and extracts</caption><thead><tr><th>Sample</th><th>n</th><th>Dry weight rate (% f.w.)</th><th>Total phenolic content (mg PE g<sup>-1</sup> d.e. or d.s.)</th><th>Antioxidant activity (IC<sub>50</sub> µg mL<sup>-1</sup>)</th></tr><tr><th></th><th></th><th>Mean ± SD*</th><th>Mean ± SD*</th><th>Mean ± SD **</th></tr></thead><tbody><tr><td><i>F. vesiculosus</i> powder</td><td>6</td><td>91.0 ± 0.1<sup>d</sup></td><td>39.8 ± 9.8<sup>b</sup></td><td>309.2 ± 33.9<sup>c</sup></td></tr><tr><td>WE pH 4 extract</td><td>9</td><td>1.2 ± 0.0<sup>a</sup></td><td>57.7 ± 11.0<sup>abc</sup></td><td>177.3 ± 14.4<sup>a</sup></td></tr><tr><td>WE pH 5 extract</td><td>15</td><td>1.1 ± 0.0<sup>a</sup></td><td>50.2 ± 9.3<sup>ab</sup></td><td>189.6 ± 15.5<sup>a</sup></td></tr><tr><td>WE pH 7 extract</td><td>9</td><td>1.0 ± 0.0<sup>a</sup></td><td>54.2 ± 11.4<sup>abc</sup></td><td>212.6 ± 3.3<sup>b</sup></td></tr><tr><td>P2 pH 7 extract</td><td>6</td><td>1.1 ± 0.1<sup>a</sup></td><td>116.4 ± 6.8<sup>d</sup></td><td>188.2 ± 15.5<sup>a</sup></td></tr><tr><td>G1 pH 4 extract</td><td>9</td><td>1.4 ± 0.0<sup>b</sup></td><td>70.7 ± 10.6<sup>cd</sup></td><td>195.0 ± 12.7<sup>ab</sup></td></tr><tr><td>G3 pH 5 extract</td><td>15</td><td>0.7 ± 0.3<sup>c</sup></td><td>56.5 ± 8.0<sup>acd</sup></td><td>186.2 ± 13.5<sup>a</sup></td></tr></tbody></table>

* One-way analysis of means, not assuming equal variances, *p* < 0.05

** One-way analysis of means, not assuming equal variances, *p* < 0.05

IC₅₀ of ascorbic acid (control): 15.1 ± 1.1 µg mL⁻¹
(Rodríguez-Jasso et al. 2013; Obluchinskaya et al. 2022). *Fucus* species are known to be rich in minerals, proteins, antioxidant phloroglucinol-based phenolic compounds, and cell wall polysaccharides, including alginates and sulphated polysaccharides rich in fucose (fucoidan) (Bikovens et al. 2017; Paiva et al. 2018; Obluchinskaya et al. 2022; Rousseau et al. 2023). The main constituents of the brown seaweed fibers are three distinct polysaccharides, namely sulfated polysaccharides rich in fucose (fucoidan), alginic acid (consists of β-D-mannuronic (M) and α-L-glucuronic (G) acid units) and laminaran (Catarino et al. 2018). The contents may vary between 3.4 and 25.7% d.w. for fucoidan, 8.4 and 58.8% for alginic acid and 0.6 and 7.0% for laminaran. Obluchinskaya et al. (2022) showed the amounts of monosaccharides, phlorotannins, flavonoids, and the mineral composition significantly varied in *F. vesiculosus* with reproductive phase and geographic locations in different Arctic region. Paiva et al. (2018) also discovered seasonal and geographical differences in the nutritional composition (protein, carbohydrate, lipid and fatty acids, dietary fiber) and functional activity (total phenolic and flavonoid content, antioxidant activity) of *Fucus spiralis*.

Carbohydrates are the most abundant element of *Fucus* spp., involving in different roles including storage, defense responses, development, cell polarity or tissues integrity (Deniaud-Bouet et al. 2017). Carbohydrates can vary from 34–66% (d.w.) in *F. vesiculosus* (Catarino et al. 2018). The 22% of neutral sugars determined in *F. vesiculosus* in our study is quite similar to the result presented by Bikovens et al. (2017). Collected in March 2017 in Jurmala (Latvia), the total neutral sugars content of *F. vesiculosus* were analyzed using the same method and presented 24.6% d.w. Raw *Fucus* collected in Galicia (Spain) is rich in both soluble (10.52 ± 0.31%) and insoluble (48.63 ± 1.05%) fractions of dietary fibers (Díaz-Rubio et al. 2009). *Fucus vesiculosus* from Portugal is rich in carbohydrates (56.4 ± 0.4 g (100 g)⁻¹ seaweed d.w.) with fibers representing the main contribution (45.0 ± 0.1 g (100 g)⁻¹ d.w.). *Fucus vesiculosus* stood out by its high content of insoluble fiber compared to soluble fiber (Neto et al. 2018).

Proteins are the second most important compound in this *F. vesiculosus* powder with 17% of d.s. which is in accordance with the literature. Indeed, according to Fleurence (1999), most of the brown seaweeds industrially exploited (*Laminaria digitata*, *Ascophyllum nodosum*, *F. vesiculosus* and *Himanthalia elongata*) have a protein content lower than 15% d.w. It shows little variations, from 11% (Catarino et al. 2018) to 13% d.w. (Lorenzo et al. 2017), with up to 15% d.w. (Neto et al. 2018).

Within the Fucaceae phlorotannins are the main phenolic compounds found in brown algae, making them a valuable source of polyphenols (Obluchinskaya et al. 2022). In fact, phlorotannins have been identified as the

only phenolic compounds in *F. vesiculosus* (Catarino et al. 2017). The abundance of phlorotannins in brown marine algae, as documented in the literature, ranges from 1 to 14% of dry algal biomass (Ar Gall et al. 2015; Catarino et al. 2019; Maheswaria and Babu 2022). In our study, the total phenolic content in the raw *F. vesiculosus* consisted of approximately 4% of dry weight (39.8 ± 9.8 mg eq PE g⁻¹ d.s.) which is in accordance with the literature.

The mineral content in seaweed is very variable (8–40% d.w.). It is primarily attributed to the properties of their cell surface polysaccharides, which facilitate the retention of inorganic marine substances. In the particular case of *Fucus* spp., the ash (mineral) content may vary from 19 to 36% d.w. (Catarino et al. 2018). Obluchinskaya et al. in 2022 confirmed that the ash content of macroalgae varies significantly, depending on the species, geographic location, and reproductive phase. The authors showed averages ash values of 18.5% and 28.5% d.w. depending on reproductive phases and locations. In our study, *F. vesiculosus* contains low amount of minerals with only 12% of ash (Fig. 1).

*Fucus vesiculosus* contains several important minerals for plants such as P, K, Ca, Mg. According to Catarino et al. (2018), sodium and potassium are the major elements described in this genus. According to Balina et al. (2016), *F. vesiculosus* from Jurmala, Latvia, contains 21 500 mg kg⁻¹ calcium, 11 000 mg kg⁻¹ potassium, 9 300 mg kg⁻¹ magnesium, 6 300 mg kg⁻¹ sodium, 1 680 mg kg⁻¹ manganese, 1 400 mg kg⁻¹ phosphorus, 490 mg kg⁻¹ iron, 89 mg kg⁻¹ zinc and 12.7 mg kg⁻¹ copper. The amounts of iron and magnesium obtained in this study are quite similar by contrast, phosphorus, zinc, copper and manganese are much lower (by 49 to 94%, respectively). In addition, calcium is present in lower quantity in comparison with the results of literature (lower by 26%), while potassium and sodium are present in higher quantity (increase by 136 and 305%, respectively). These differences could be explained by the effect related to local harvesting conditions (i.e., Spain for Balina et al. (2016) and France in this study) and seasonal variations (i.e., in January for Balina et al. (2016) and in July in this study) as reported and confirmed by Allen et al. (2015). In addition, content of elements in seaweeds depend on the concentrations of metal compounds in the surrounding water and their ability to bioaccumulate such compounds (Adams et al. 2011).

Following the initial discovery of auxin in marine algae by Overbeek (1940), research attention shifted towards identifying plant growth regulators (PGRs) present in seaweeds. However, conclusive identification of auxin remained uncertain until the 1970s. During the past two decades, various PGRs (auxins, cytokinins, gibberellins, abscisic acid) were reported in different seaweed extracts (Stirk and Van Staden 2014).

The first mention of the potential presence of PGR-type compounds in *F. vesiculosus* dates back to 1971. Biological

<figure><img src="image_14.png" /></figure>
tests carried out on *Avena* coleoptiles revealed the release of several indole-type compounds by the alkaline hydrolysis of various seaweeds, including *F. vesiculosus* (Buggeln and Craigie 1971). Most recently, a study determined the content of PGRs in *F. vesiculosus* (Sharma et al. 2012). The acidic extracts obtained from *F. vesiculosus* collected in June contained up to 46.84 µg kg⁻¹ d.s. which is in the same range with the results in this study. Regarding other brown seaweed species, the profiles and contents of PGRs have been explored in *Sargassum horneri* (Li et al. 2016). The content of salicylic acid was two times lower and the amount of IAA (Indole-3-acetic acid) six times lower compared with the present study. These authors have detected 3.9 µg kg⁻¹ for iP (Isopentenyladenine); 2.4 µg kg⁻¹ of iPR (isopentenyladenosine) and 5.6 µg kg⁻¹ of ABA (abscisic acid). Yalcin et al. (2019) observed that the content of phytohormones is very low for two brown seaweeds from 3.5 µg kg⁻¹ for *Cladostephus spongiosum* to 15.4 µg kg⁻¹ for *Cystoseira barbata*. Stirk et al. (2003) showed a content of IAA of 2.04 µg kg⁻¹ for *Ecklonia maxima* and 1.7 µg kg⁻¹ for *M. pyrifera*. However, in the present work, iP, iPR, cZ and ABA contents are quite similar to the values published by these authors. Stirk et al. (2003) have measured an amount of iP at 0.042 µg kg⁻¹ for *E. maxima* and 0.035 µg kg⁻¹ for *M. pyrifera*; iPR at 0.006 µg kg⁻¹ and 0.003 µg kg⁻¹ respectively and cZR was under the limit of detection for both seaweeds.

In general, PGRs from seaweeds are extracted with an organic solvent (i.e., methanol) with or without sonication. Subsequent purification by SPE or affinity chromatography is usually applied and the identification and quantification of PGRs are then performed by LC-MS or GC-MS (Benítez García et al. 2020; Yalcin et al. 2019). In the present study, the PGRs from *F. vesiculosus* were purified after extraction and concentrated by C18 or cation-exchange SPE depending on the nature of the molecule of interest. The quantification was performed by UHPLC-QTRAP-MS/MS in MRM (Multiple reaction monitoring) mode to achieve high specificity and sensitivity. The combination of an advanced chromatographic separation technique with tandem mass spectrometry allows the detection of PGRs down to ng kg⁻¹. While there are a few studies that have determined the levels of PGRs in seaweed, the final quantification is evidently affected by the method of harvesting and processing the seaweed samples after collection, including whether or not the raw material is dried.

## Comparison of *Fucus vesiculosus* extracts derived from conventional aqueous extraction (WE) and Enzyme-Assisted Extraction (EAE)

The obtained results for the dry matter extraction yield are in accordance with previous studies (Hardouin et al. 2014a, b; Le Guillard et al. 2016). Regarding total sugars (Fig. 2(b)), it

was expected that extracts obtained with glycosidases would contain more sugars compared with protease due to the main activity dedicated to hydrolysis of osidic compounds such as oligo- and polysaccharides. Results demonstrated the efficient enzymatic hydrolysis of polysaccharides contained in the seaweed thanks to G3 which allowed an increase in total sugars. As a result, the extract obtained using G3 at pH 5 was richer in sugars compared to the conventional water extraction method. Although EAE can significantly enhance the extraction of polysaccharides from brown algae by breaking down the cell wall, no studies were found in the literature specifically comparing our results obtained with the glucosidase enzyme. It is possible to assume that even if G1 enzyme breakdowns more sugars molecules, such as polysaccharides, resulting in more reducing ends (Fig. 2(c)), the obtained polysaccharides are not sufficiently hydrolyzed to be quantified by the colorimetric method used here for the determination of total sugars. Seaweed-derived polysaccharides have the potential to act as plant biostimulants. Mzibra et al. (2021) showed that polysaccharide enriched extract obtained from *F. spiralis* significantly increased germination percentage, germination speed, reduced mean germination time of tomato seeds treated with this extract at a dose of 0.02 mg mL⁻¹. Moreover, the same extract applied to the soil and as a foliar spray enhanced tomato growth parameters such as leaves number, plant length, dry weight of shoot and root, and chlorophyll content. Polysaccharide enriched extract from *F. spiralis* applied to the soil improved plant growth parameters more successfully than applied as a foliar spray.

Regarding proteins (Fig. 2(e)), none of the tested enzymes allowed the recovery of more proteins in comparison with their respective WE, even for the protease P2. Agregan et al. (2017) have analyzed the protein content of *F. vesiculosus* water extracts obtained through UAE. They obtained results higher than those presented in this study with 62.1 ± 2.3% d.e.

A significant difference between the TPC (Fig. 2(f)) was found for the EAE extraction with P2 enzyme compared with WE at the different tested pH. Among these extractions, the EAE with P2 enzyme at pH 7 seems to be the most efficient for enriching total phenolic content in *F. vesiculosus* extracts. Our findings align with the existing literature data. In fact, several studies have demonstrated that EAE can enhance the extraction efficiency of polyphenols from brown algae (Hardouin et al. 2014a, b; Teixeira-Guedes et al. 2023). Specifically, the disruption of the algal cell wall during EAE leads to the release of intracellular constituents, including proteins, which are prone to complex with polyphenols, causing aggregation and eventual precipitation (Siriwardhana et al. 2008). These polyphenols may interact with proteins through various mechanisms such as hydrogen bonding, π-bonding,
hydrophobic interactions, as well as ionic and covalent linkages. Wang et al. (2010) investigated the application of proteases to enhance the yield of polyphenols and antioxidants from the red seaweed *Palmaria palmata*. They observed an increase in the extraction efficiency of phenolic compounds following protease treatment, attributing this improvement to the conversion of proteins into small peptides and free amino acids. Indeed, the protease degradation of proteins can liberate bound polyphenols, thereby improving access to polyphenols and facilitating their extraction (Wang et al. 2010).

According to Table 4, enzyme G1 increases phosphorus contents in enzymatic extracts in comparison with WE at pH 4 (increase by 9% respectively). The enzyme G3 permitted to increase the extraction of iron (increase by 17%) and copper (by 45%) in comparison with WE at pH 5. The comparison of the three enzymes shows that G1 allowed for improved extraction of calcium, iron, magnesium, manganese, sodium and sulphur reaching the highest content in the dry extract in comparison with the extraction with G3 and P2. On the other hand, enzyme G3 permitted to obtain the highest extraction of boron, copper, phosphorus and zinc in comparison with enzymes G1 and P2. P2 permitted to obtain the highest content of potassium and nitrogen in the extract in comparison with G1 and G3 and had an equivalent amount of phosphorus in the extract as for the enzyme G3. Finally, considering the three main macroelements mandatory for plant nutrition and growth (nitrogen, phosphorus and potassium), extracts obtained with P2 are promising with the highest amount of nitrogen 6188 ± 288 mg kg⁻¹ d.e., which can be correlated to the use of a protease.

Plants require specific essential minerals crucial for their structural integrity and metabolic processes. Inadequate supply of these elements can result in severe abnormalities in plant growth and development. Among the macroelements, nitrogen, phosphorus, and potassium are primary nutrients found in both mineral and organic fertilizers. Nitrogen deficiency adversely impacts plant productivity by reducing photosynthesis, leaf area, and the lifespan of green foliage. Phosphorus deficiency limits plant growth and crop yield, while potassium deficiency makes plants more susceptible to stress, leading to stomatal closure and inhibition of photosynthetic rates in various crop species (Wissuwa et al. 2003; Wang et al. 2013; Mu and Chen 2021). The current study demonstrates that EAE extracts contain a range of elements, including nitrogen, phosphorus, and potassium. Therefore, application of these extracts on the crops could supply essential minerals for healthy growth (Choulot et al. 2022). Furthermore, these elements may play a role in activating plant defense mechanisms (Shukla et al. 2016). Literature data indicate that extract obtained from *F. vesiculosus* by UAE, rich in mineral components, may have a positive effect on plant growth, increasing the plant length, weight of plants,

and chlorophyll content, in comparison with the control group (Krautforst et al. 2023). Michalak and Baśladyńska (2021) showed that the application of 5% *F. vesiculosus* extract, produced by hydrolysis with potassium hydroxide, on garden cress improved not only plant length and chlorophyll content, but also biofortified cultivated plants in Cu, Fe, Mn, Zn, Ca, S, P and K.

The increases of some PGRs (Table 5) such as iPR and cZ in the dry extracts obtained through EAE with G1 pH4 could be explained by a more efficient and specific breakdown of seaweed cell wall polymers, thus improving the release of these compounds. This is implied by the increase in reducing sugars in G1 and G3 extracts in comparison with the WE (Fig. 2(c)). In this way, the selective depolymerization of cell wall polysaccharides and proteins tends to increase the diffusion of PGRs into the soluble phase, thus leading to higher contents for some PGRs. Moreover, two additional PGRs were quantified in the extracts (WE and EAE) in comparison with the raw seaweed. This could be explained by the temperature of extraction, the pH or the longer duration of extraction allowing a better cell wall disruption.

Regarding SA, the highest amount in the extract was obtained at pH 7 (WE and EAE) with significant differences observed with extracts obtained at pH 4 and pH 5. The same tendency was observed for IAA, ACC and ABA leading to the conclusion that an acidic pH has a negative effect on the recovery of these PGRs.

About cytokinins, the enzymes G1 and P2 allowed statistically significant increases in the content of iPR in comparison with extract produced by WE under the same extraction conditions. The largest increase in the content of iPR was observed in the extract obtained using the enzyme G1, which contained seven times more iPR (*p* < 0.001) compared with WE pH 4. For cZ, tZ and their precursors, in most of the extracts the values were below the lower limits of quantification of the analytical method (<0.1; 0.05; 0.025 µg kg⁻¹ d.e.), except for the enzyme G1 which enabled the production of extracts with quantified amounts of cZ, cZR and Tz. If the enzyme efficiency is compared, it appears that the extract obtained with the protease is richer in PGRs than extracts obtained with glycosidases. Indeed, P2 allowed to increase the content of iP by 8%, SA by 21%, ACC by 26%, ABA by 36%, iPR by 132% and IAA by 175% in extracts in comparison with the G3 extract.

Results obtained for the brown seaweed *F. vesiculosus* in this study were in the range to those obtained by Wally et al. (2012) for the commercial extracts produced from brown seaweeds collected in different countries, including *A. nodosum* from France. The authors observed that the content of iP varied, ranging from 27.7 µg kg⁻¹ d.e. for South Pacific *Durvillea* to 1 µg kg⁻¹ for *A. nodosum*. It is not detected for the South African *Ecklonia* extract. cZ was detected at 1.7 µg kg⁻¹ in North Pacific *Macrocystis* extract, while it was
not detected in extracts from other countries, including *A. nodosum* extract. The concentration of tZ was 10 µg kg⁻¹ for Chinese *Sargassum* extract and 1 µg kg⁻¹ d.e. for South African *Ecklonia*, with no detection in extracts from other countries, including *Ascophyllum* extract. iPR level ranged from 58.4 µg kg⁻¹ for Irish soluble seaweed extract powder to non-detected for extracts from various other countries, including *Ascophyllum* extract. As for ABA, it ranged from 10 µg kg⁻¹ for Chinese *Sargassum* extract to non-detected for extracts from various other countries, including French *Ascophyllum* extract. The content of IAA ranged from 615 µg kg⁻¹ for Norwegian extract to 6 µg kg⁻¹ for *A. nodosum*. Based on this study, the amount of IAA in *A. nodosum* extracts seems to be strongly influenced by the harvesting location (country/continent) (Wally et al. 2012).

Another study analyzed the PGRs contents in commercial extracts produced from French Atlantic brown seaweed *A. nodosum* (Wally et al. 2012). The IAA and iP contents were 6 µg kg⁻¹ d.e. and 1 µg kg⁻¹ d.e., respectively, which are much lower than the values reported in the present study. The fact remains that *A. nodosum* has been recognized for many decades for its fertilizing and biostimulant properties.

Thus, it appears that *F. vesiculosus* holds promise as a raw material to develop seaweed-based product for enhancing plant growth and development. This potential stems from the diverse properties of several growth regulators found within these extracts, including their ability to improve germination, root development, and flowering, thereby supporting crucial cellular processes such as iodine uptake and energy flow within plants (Wally et al. 2012; Benkova 2016). Moreover, these regulators create conducive environments for plant growth, metabolism, and insect pollination (Koo et al. 2020). Mattner et al. (2018) conducted a study where a commercial seaweed extract derived from *D. potatorum* and *A. nodosum* was applied to strawberries during both nursery and production stages. Their findings demonstrated an increase in root length and density of plants during the production stage with the use of seaweed extracts. Consequently, Mattner et al. (2018) suggested that seaweed extracts may enhance plant water and nutrient utilization, implying that components of seaweed other than minerals, such as PGRs, may be involved in this process. Specifically, IAA, a major PGRs present in these extracts, acts as a growth stimulant, promoting increased plant size (Benkova et al. 2016). Additionally, ABA functions as a growth inhibitor, inducing seed dormancy and influencing processes such as floral transition, lateral root formation, and seedling growth (Yang et al. 2022). Salicylic acid, on the other hand, plays crucial roles in regulating plant disease resistance, thermogenesis, abiotic stress tolerance, DNA damage/ repair, fruit yield, seed germination, and other physiological processes (Dempsey and Klessig 2017).

## Evaluation of antioxidant activity with DPPH

Our findings presented in Table 6 did not reveal a straightforward relationship between antioxidant activity and TPC in *F. vesiculosus* extracts. This result aligns with Sanchez-Bonnet et al. (2021) who described a significant negative correlation between TPC and DPPH radical scavenging activity in *F. vesiculosus* extracts (conventional and ultrasonic assisted extraction). They found that many of their extracts containing higher and lower content of polyphenols gave lower and higher antioxidant activity, respectively (Sanchez-Bonnet et al. 2021). Similarly, Hermund et al. (2018) also reported a negative correlation between TPC and RSA for *F. vesiculosus* aqueous ethanol extracts. They proposed that the lack of a positive correlation suggests that the extraction process was not selective, and other co-extracted compounds, such as polysaccharides, pigments, proteins, peptides, or vitamins, may influence the overall antioxidant activity synergistically or antagonistically.

However, it is important to emphasize that *F. vesiculosus* powder showed a total phenolic content of 39.8 ± 9.8 mg PE g⁻¹ d.s. and exhibited the lowest antioxidant activity with an IC₅₀ of 309.2 µg mL⁻¹ compared to the different extraction techniques applied in this study. Since the total phenolic content found with P2 enzyme at pH 7 was significantly greater than the content in the seaweed powder, this suggests that EAE with P2 not only enhance the overall polyphenol content in the extracts, but also improve the antioxidant activity in *F. vesiculosus* by lowering their IC₅₀ value.

## Conclusion

This study aimed to develop and evaluate the effectiveness of two extraction methods — enzyme assisted extraction (EAE) and water extraction (WE) — for the valorization of *F. vesiculosus*. The findings indicated that pH influenced the recovery of compounds from *F. vesiculosus*, with EAE showing higher efficiency at pH 4 compared to pH 5 and pH 7. Although EAE at pH 4 had a higher extraction yield than WE at the same pH, the difference was not significant. Similarly, WE was more efficient at pH 4 than at pH 5 and pH 7. Furthermore, this study demonstrated that EAE was significantly more effective than WE in producing enriched extracts, enhancing the release of bioactive compounds such as total sugars, a few plant growth regulators (PGRs), and polyphenols. Additionally, the polyphenol content and antioxidant activity of the raw algal powder and the various extracts were compared. The results highlighted that the extracts exhibited superior antioxidant activity, which can play a crucial role in agriculture by enhancing plant health through protection against oxidative stress, thereby supporting sustainable and eco-friendly agricultural practices by reducing reliance on chemical inputs.
**Acknowledgements** The authors thank Damien Souquet for the LC-MS analyses of PGRs.

**Author contribution** Authors' contributions: Conceptualization: M.C., C.LG., I.M., N.B. Methodology: M.C., C.J., A-S.B, L.J; M.W., A.S-M., C.LG., I.M., N.B. Validation: C.LG., I.M., N.B. Formal analysis: M.C. Statistical analyses: C.J., A-S.B Investigation: M.C., C.J., C.LG., I.M., N.B. Data curation: M.C. Writing—original draft preparation: M.C. Writing—review and editing: M.C., C.J., C.LG., I.M., N.B. Visualization: M.C. Supervision: C.LG., I.M., N.B. Project administration: N.B.

**Funding** Open access funding provided by Université de Bretagne Sud. This research was supported by the National Science Centre in Poland [grant 2019/33/B/NZ9/01844]; the French government under the programme "Investissements d'Avenir"—France 2030; Agro Innovation International – Timac Agro; and the ISblue project [grant ANR- 17-EURE-0015].

**Data availability** The data that support the findings of this study are available from the corresponding author (Nathalie Bourgougnon).

## Declarations

**Competing interest** The authors declare no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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