In vitro adsorption of Fumonisin B1 by multiple algae-modified clay formulations

In vitro adsorption of Fumonisin B1 by multiple algae-modified clay formulations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vitro adsorption of Fumonisin B1 by multiple algae-modified clay formulations Letícia Aliberti Galego Alves da Silva, Morgane Malard, Patricia Aparecida de Campos Braga, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8407612/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2026 Read the published version in Mycotoxin Research → Version 1 posted 9 You are reading this latest preprint version Abstract Mycotoxins are toxic secondary metabolites produced by fungi, and frequently encountered in cereals that compose a major part of livestock diets. Fumonisin B1 (FB 1 ) is one of the most prevalent toxins in feed, posing a risk to animal health and productivity. Considering mycotoxin mitigation strategies, adsorbents are an advantageous alternative for reducing mycotoxin uptake by animals. In this context, the main objective of this study was to develop an in vitro protocol for FB 1 adsorption and assess the binding efficacy of five formulated products composed of inorganic clay and algae extracts. For this purpose, algae-based formulations were provided by Olmix (Bréhan, France), and multiple parameters were evaluated for in vitro testing, such as pH and mycotoxin concentration. After the selection of adequate conditions, the adsorption capacities of five algae-based products were compared. Results indicate that the adsorption capacity of the algae-based products is mainly linked to the presence of algae, especially green algae; which present a high polysaccharide content in their cell walls as binding sites for mycotoxins. The use of algae for mycotoxin adsorption remains underexplored, but the findings of the present work indicate that algae-based products are effective for FB 1 control in animal feed. Adsorption mycotoxin mitigation feed physical methods Figures Figure 1 Figure 2 Introduction Mycotoxins are toxic secondary metabolites produced by filamentous fungi, including species within the Aspergillus , Penicillium and Fusarium genera. These microorganisms are known to infect cereals at multiple stages of production, ranging from field to storage; and result in a persistent risk of mycotoxin contamination throughout the entire production chain (Daou et al. 2021 ; Xu et al. 2023 ). Considering that cereals are major components of regular livestock diets, animals become vulnerable to the ingestion of contaminated grains. This exposure may lead to reduced performance, such as lower feed intake, milk yield, and growth efficiency and/or fertility (Vila-Donat et al. 2018 ; Xu et al. 2022a ; Kihal et al. 2022 ). Among the most commonly detected toxins in feed are Aflatoxins (AFs), Ochratoxin A (OTA), Trichothecenes, Zearalenone (ZEN) and Fumonisins (FBs) (Awuchi et al. 2022 ). Considering FBs, Fumonisin B 1 (FB 1 ) is the predominant compound encountered, especially in maize and its by-products (Beccaccioli et al. 2021 ; Pamphile and Azevedo 2002 ). Therefore, the occurrence of FB 1 in animal feed has been documented worldwide, with reports from Europe, the Americas, Asia and Africa showing contamination levels ranging from 24.5% to 100% (Gao et al. 2023 ). The presence of FB 1 in products intended for animal consumption is concerning, as it has been linked with equine leukoencephalomalacia and porcine pulmonary edema. Additionally, its ingestion can also result in the reduction of feed intake, lower egg production, hepatic necrosis, and thymic cortical atrophy in poultry (Awuchi et al. 2021 ; Chen et al. 2021 ; Schrenk 2022; Yang et al. 2020 ). Effects on ruminants are briefly described, with previous studies showing lower milk yield after FB 1 ingestion by dairy cattle; whereas adult beef cattle seem to be more resistant, exhibiting only mild hepatic necrosis (Diaz et al. 2000; Osweiler et al. 1993 ; Smith 2018 ). Therefore, economic pressures, particularly in the livestock industry, are motivating producers to seek effective solutions to prevent the adverse effects of mycotoxins on animal health and productivity. However, if grain contamination has already occurred, preventative measures are no longer feasible. Consequently, the implementation of mitigation strategies intended for the removal, degradation or inactivation of toxins becomes crucial. Therefore, integrated approaches that combine multiple strategies are essential to guarantee safe mycotoxin levels in animal feed (Shi et al. 2018 ; Xu et al. 2022a ). Described methods include chemical, biological and physical treatments of grain. Chemical agents, such as ammonia, hydrogen peroxide, and organic acids, are highly effective in reducing mycotoxin levels. Nevertheless, the use of such reagents can make raw materials inedible for animals and contributes to environmental pollution. Biological strategies involve the use of enzyme-producing microorganisms to degrade or bio-transform toxins, which presents challenges for in-field application due to environmental interactions. Lastly, physical methods include sorting and separation, floating and segregation by density, irradiation, ultrasound treatment, dehulling, milling and adsorption (Luo et al. 2018 ; Peng et al. 2018 ). Adsorption involves the use of an adsorbing agent (AA), which forms mycotoxin–adsorbent complexes through direct binding with the toxins. This process reduces mycotoxin bioaccessibility and, therefore, intestinal absorption; as the formed complexes are excreted via feces. As a consequence, this also leads to a decrease in mycotoxin uptake and in its spread to target organs (Boudergue et al. 2009 ; Xu et al. 2022a ). In contrast with other mitigation strategies, which aim to diminish mycotoxin levels during feed production, AAs are used as feed additives. Additionally, the main advantages of such binders are their cost, safety and ease of administration through inclusion in feed (Peng et al. 2018 ). Among employed AAs are organic (yeast cell wall, yeast cell wall beta-D-glucan fraction, oat and alfalfa fibers) and inorganic (bentonites, montmorillonites, zeolite and activated carbon) compounds (Kihal et al. 2022 ). In general, inorganic binders are highly efficient in adsorbing AFs, due to the high polarity and small molecule size of these toxins. However, such products are relatively inefficient in adsorbing other mycotoxins, especially Fusarium toxins, such as Deoxynivalenol (DON) and ZEN. In this context, the use of organic binders is recommended (Vila-Donat et al. 2018 ). Considering the FB 1 molecule size and its structural conformation, adsorption may be a challenging issue, especially with inorganic products. To address this limitation, adsorption can be increased through the incorporation of biological components, such as algae extracts, to inorganic binders. This modification enhances the interlayer spaces of clays, increasing surface area and resulting in more binding sites for mycotoxins (Cai et al. 2024; Oguz et al. 2022 ; Rasheed et al. 2020 ; Wang et al. 2023 ; Xu et al. 2022b ). Algal extracts are appealing options for AAs, as they are well-known for their biosorption/bioremediation of heavy metals (Cheng et al. 2019 ; Lin et al. 2020 ). These organisms’ cell walls contain a wide range of proteins and polysaccharides, such as β-D-glucans, that are potential binding sites for mycotoxins. Additionally, other algal compounds could also be responsible for adsorption capacity, such as chlorophyll and chlorophyllin; which may also form complexes with mycotoxins, reducing their bioaccessibility (Simonich et al. 2007 ). Furthermore, the inclusion of algae as feed additives may not only help prevent the adverse effects of mycotoxins but also promotes improved animal health (Perali et al. 2020 ). Algae display high bioactive compound content, which can act as prebiotics and therefore enhance animal immunity. In addition, production performance (i.e. weight gain, feed intake, and feed conversion rate) may also be increased, since algae are rich in proteins, vitamins, and minerals (Fraga-Corral et al. 2023 ; Makkar et al. 2016 ; Yadavalli et al. 2023 ). Estimating the adsorbent efficiency of different products is indispensable, since it depends on the type of adsorbent, its physico-chemical properties, and the target mycotoxins. In vivo testing of mycotoxin binding efficacy of a large number of adsorbents is challenging, due to the complexity and cost; making in vitro analyses powerful tools for assessing and ranking the efficacy of various AAs. Nevertheless, experimental conditions for in vitro tests reported in the literature may vary widely, making it difficult to establish reliable protocols (Boudergue et al. 2009 ; Faucet-Marquis, 2014). Considering the information cited above, the aim of this study is to develop an in vitro protocol for FB1 adsorption to evaluate and compare the binding efficacy of products composed of inorganic clay and algae extracts, in order to identify the most effective formulation for FB 1 adsorption in livestock feed. Material and Methods Mycotoxin binders Five AA formulations were provided by Olmix (Bréhan, France) for the development of this study. All products consisted of inorganic clay mixed with algae extracts, including red and green algae. They were each labeled with a code (A1 to A5) and their characteristics are summarized in Table 1. For the development and validation of the in vitro method, only A2 was employed; with the other formulations used only for efficacy testing. Additionally, activated charcoal (AC) was used as a positive control for adsorption tests. [Please, insert Table 1 file here] Chemicals and reagents FB 1 external standards were acquired from Sigma-Aldrich (Fallavier, France), with stock solutions prepared in acetonitrile/water (50/50; v/v) for long-term storage at -20 ºC and further dilution (calibration curves and experiments). The solvents used for the FB 1 external standard dilution, mobile phase and buffer preparation for subsequent analysis by high-performance liquid chromatography coupled with a triple quadrupole mass spectrometry (LC-MS/MS), were HPLC grade and purchased from J.T. Baker (Phillipsburg, USA). In addition, validation of the in vitro method was performed using citrate buffer (C 6 H 8 O 7 • H 2 O and C 6 H 5 O 7 Na 3 • 2H 2 O; 0,1 M) containing 10% methanol, adjusted to pH 3, 5 and 7. Chromatographic conditions and method validation LC-MS/MS for mycotoxin quantification was performed using the Agilent 1290 Infinity LC-System (Agilent Technologies, Santa Clara, USA) coupled to a 6460 triple quadrupole (Agilent Technologies, Santa Clara, USA) mass spectrometer, at the Food Toxicology Laboratory, Food Engineering School, State University of Campinas (UNICAMP). The Zorbax SB-C8 column (3.0 mm x 100 mm; 1.8 µm) was used for chromatographic separation at 25 ºC. The analyses were performed in isocratic mode, using 30% of 0.1% (v/v) formic acid in water (mobile phase A) and 70% of 0.1% (v/v) formic acid in methanol (mobile phase B), with a flow rate of 0.35 mL/min and injection volume of 3 µL. Mass spectrometry conditions were: Electrospray Source Ionization in the positive mode (ESI+) with gas temperature at 300 °C, gas flow at 10 L/min, nebulizer at 35 psi, sheath gas flow at 10 L/min, sheath gas temperature at 350 °C, capillary voltage at 3.0 kV and nozzle voltage at 0.5 kV. Ultrapure nitrogen was used in the nebulizer and as collision gas. For FB 1 , m/z 722 (precursor), m/z 353 (quantifier transition) and m/z 334 (qualifier transition) were monitored. The fragmentor applied was 185 V and collision energies were 40 eV for both quantifiers and qualifier transitions. Data acquisition and analyses were performed using MassHunter software workstation version 7.00 (Agilent Technologies, Santa Clara, USA) in selected reaction monitoring (SMR) mode, using a dwell time of 100 ms per channel. The linearity of FB 1 was obtained based on calibration curves in the matrices (Figure S1; citrate buffer 0.1 M with 10% methanol at pH 3, 5, and 7). FB 1 standard was prepared at seven concentrations (0.5, 1, 2.5, 5, 7.5, 10, and 12.5 µg/mL) in triplicates, and subsequently analyzed under the conditions previously described (Sun et al. 2018). All linearities (R 2 ) obtained were ≥ 0.99 at pHs 3, 5, and 7 (Figure S1). Recovery was evaluated in triplicates on the same day as the analysis, with results ranging from 95% to 110% for citrate buffer at pH 3, 5, and 7 (EC 2006). The limits of detection (LODs) and the limits of quantification (LOQs) for each matrix are provided in Table S1; these were calculated through the minimum concentration detected with a 3x and a 10x signal-noise ratio, respectively. Furthermore, precision was assessed at three FB 1 concentration levels (2.5 µg/mL, 5 µg/mL and 7.5 µg/mL) by analyzing five replicates of each level in citrate buffer pH 5, without the presence of adsorbent. Intra-day precision was assessed by analyzing all replicates on the same day under the same conditions. Inter-day precision was determined by repeating the procedure on three different days. The results were expressed as the coefficient of variation (CV%) for each level (Table S2). All CV% values were within the acceptable limits of ≤ 20%, in accordance with SANTE/11312/2021 (EC 2021) (Table S2). The selectivity of the method was assessed by analyzing blank and fortified samples (2.5 µg/mL of FB 1 ) in citrate buffer (pH 3, 5, and 7). The fortified samples exhibited well-defined peaks in the qualifier transition (m/z 334) with consistent retention times ranging from 1.928 to 1.955 minutes (Figure S2). No interfering peaks were observed in the blank samples at the corresponding retention times, demonstrating the selectivity of the method for FB 1 analysis (Figure S2). Effect of pH and FB 1 concentration on adsorption capacity pH Three different pH levels (3, 5 and 7) were evaluated, to determine the optimal level to proceed with the trials. For this purpose, three FB 1 working solutions (5 µg/mL) were prepared in citrate buffer with 10% methanol at pH 3, 5 and 7, separately. Next, 5 mL of each solution was pipetted into 50 mL polypropylene tubes containing either: 0.1% (w/v) of A2, according to manufacturer instructions; 0.1% (w/v) of activated charcoal (AC; AC positive control); or no adsorbent (control without adsorbent); in triplicates (Faucet-Marquis et al. 2014) Samples were then incubated (30 minutes; 37ºC; 8 x g) and centrifuged (15 minutes; 8586 x g). Subsequently, 1 mL of supernatant from each sample was retrieved, filtered by syringe filter (0.22 µm) and analyzed with HPLC-MS/MS (item 2.3.). After chromatographic analyses, the adsorption percentage of each test was calculated based on equation 1: (1) where C ads is the adsorbed mycotoxin yield (µg/mL) and C 0 is the concentration of FB 1 in the controls without adsorbent (µg/mL) (Joannis-Cassan et al. 2011). FB 1 concentration Four FB 1 concentrations (1 µg/mL, 2.5 µg/mL, 5 µg/mL, and 10 µg/mL) were prepared in 5mL of pH 5 citrate buffer (10% methanol) as solvent. Considering the evaluated product should demonstrate a high affinity and capacity to adsorb mycotoxins at its low inclusion rate in livestock diets, 0.1% (w/v) of product A2 was employed. The tests were performed in triplicates, using 0.1% (w/v) of AC as positive control (AC positive control) and mycotoxin working solution without adsorbent as control without adsorbent. Next, the tubes were incubated (30 minutes; 37 ºC; at 8 x g) and centrifuged (15 minutes; at 8586 x g). After, approximately 1 mL of the supernatant was withdrawn, filtered by syringe filter (0.22 µm), and taken for HPLC-MS/MS analysis using the conditions described above (item 2.3.); and adsorption percentage was calculated based on Formula 1. Isotherm curve Considering the previously determined optimal pH (item 2.4.1.), 5 mL of increasing amounts of FB 1 (0.5, 1, 2.5, 5, and 7.5 µg/mL) were incubated in 50 mL polypropylene tubes with 0.1% (w/v) of adsorbent (A2), 0.1% (w/v) of AC (AC positive control) or without adsorbent (control without adsorbent). All tests were performed in triplicates and incubation conditions were 37 ºC, with rotation 8 x g for 30 min. Next, all samples were centrifuged (15 minutes; 8586 x g) and 1 mL of their supernatants were withdrawn separately, filtered by syringe filter (0.22 µm), and taken for HPLC-MS/MS analysis; as formerly described (item 2.3.). This step was performed to determine the ideal intermediate concentration of FB 1 , to evaluate the adsorption capacity of different algae-based products. For this purpose, the adsorption percentage of each concentration was calculated based on Formula 1 (item 2.4.1). Moreover, an isotherm curve was also elaborated by plotting the variations in the residual FB 1 concentration after adsorption in µg/mL ( C eq ) against the quantity of adsorbed FB 1 per gram of adsorbent ( Q eq ), calculated using Formula 2: (2) where C 0 is the concentration of FB1 in the blank controls (µg/mL), m is the mass of adsorbent in grams and V is the final volume of the solution in liters. Additionally, the data obtained for the isotherm curve was fitted into two different models, Freundlich and Hill (Joannis-Cassan et al. 2011). In this regard, the first assumes the sorption occurs on a heterogeneous surface and is described by Formula 3: (3) where K F is a constant representing the capacity of the adsorbent to bind to the mycotoxin (milligram 1 - ( 1 ∕ n F ) × liter 1 ∕ n F per gram) and n F is a constant concerning the affinity of the adsorbent (Joannis-Cassan et al. 2011). As for the Hill model, it is usually employed to describe the adsorption of diverging compounds onto a heterogeneous substrate (Joannis-Cassan et al. 2011), using Formula 4: (4) where Q Hmax is the maximal mycotoxin adsorption corresponding to the site saturation (mg/g), K D symbolizes the Hill constant (mg/L) and n H corresponds to the cooperativity coefficient of the interaction among product and toxin (Joannis-Cassan et al. 2011). Evaluation of adsorption capacity of five algae-based products The ideal conditions (pH and FB 1 concentration) obtained from the last steps (item 2.4) were applied, to compare FB 1 adsorption capacities of five different algae-based products (Table 1). Tests were performed using 0.1% (w/v) of each product (A1-A5), 0.1% (w/v) of AC (AC positive control) and no adsorbent (control without adsorbent) in triplicates. FB 1 working solutions (2.5 µg/mL) were prepared in citrate buffer, pH 5 (0.1 M) with 10% methanol, and pipetted (5 mL) into 50 mL polypropylene tubes containing either the test products, AC (AC positive control), or no product (control without adsorbent). All tubes were incubated (30 minutes; 37 ºC; 8 x g) and centrifuged (15 minutes; 8586 x g), followed by the removal of 1 mL of each supernatant. Samples were then filtered by syringe filter (0.22 µm) and taken for HPLC-MS/MS analysis according to item 2.3. Adsorption efficacy was calculated based on Formula 1 (item 2.4.1). Statistical analysis Mean and standard deviation calculations of assay replicates were performed in Microsoft Excel 2016. The equilibrium point isotherm with two model fits (Freundlich and Hill model) was elaborated by plotting the experimental data and other resources in R software (R Core Team 2025). Lastly, unpaired t-test results for the adsorption of red algae and green algae-based products were obtained from GraphPad Prism software version 8, with no correction for multiple comparisons (GraphPad, 2018, v. 8.0.1). Results Effect of pH and FB 1 concentration on adsorption capacity Effect of pH The pH level is an influential parameter for in vitro adsorption tests (Faucet-Marquis et al. 2014). Thus, three different assays were performed: at pH level 3, 5, and 7. All of the AC positive control samples showed adsorption levels above 90%, as expected (Table 2). Conversely, the A2 product adsorption performance varied greatly according to the pH. At a lower pH level (3), FB 1 adsorption was the highest (88.06%±0.011) and the closest to the AC positive control samples. When pH was increased to 5, the adsorption was reduced almost by half to 48.06%±0.032. Lastly, at pH 7, FB 1 adsorption was the closest to 0% (2.8%±0.018), and showed a very high coefficient of variation, ruling it out as an alternative. Between pH levels 3 and 5, the former presented a lower coefficient of variation and standard deviation. However, its adsorption level is too high and similar to AC, which could hinder comparisons among products. Alternatively, pH 5 presented an ideal and intermediate adsorption rate, with an adequate coefficient of variation and higher recovery, when compared to pH 3. In this context, all further assays were carried out at pH 5. [Please, insert Table 2 file here] Effect of FB 1 concentration To evaluate the effect of FB 1 concentration on the adsorption capacity, 1 µg/mL, 2.5 µg/mL, 5 µg/mL, and 10 µg/mL of FB 1 were tested, considering 0.1% (w/v) of the product A2 and citrate buffer at pH 5. All the recovery tests were acceptable, ranging between 97% and 109% (EC, 2006). AC positive control assays exhibited the highest adsorption rates (Table 3). Moreover, FB 1 adsorption by A2 ranged from 33%±0.01 (1 µg/mL), to 45%±0.04 (5 µg/mL). In this context, higher adsorption was observed for 2.5 µg/mL of FB 1 (Table 3), with no statistical difference when compared to FB 1 at 5 µg/mL. Nevertheless, when FB 1 concentration was 10 µg/mL, mean adsorption dropped by 10%, indicating that higher FB 1 concentrations might cause saturation in the binding sites of the product (i.e. 10 mg of FB 1 :1 g of the product). [Please, insert Table 3 file here] Equilibrium point and isotherm curve After selecting pH 5 for the in vitro adsorption test, an isotherm curve was elaborated with different FB1 concentrations (0.5, 1, 2.5, 5, and 7.5 µg/mL). As a result, the experimental data were used to build an equilibrium isotherm curve (Figure 1), which shows an exponential relationship among C eq , Q eq and non-saturation of the adsorbent’s binding sites. Considering the two models applied for curve fitting, the Freundlich model demonstrates agreement with the experimental data; predominantly at lower FB 1 concentrations. In contrast, the Hill model displays a better fit at higher mycotoxin levels. Thus, the Freundlich model seems to be the most suitable for the acquired experimental data due to its significant n factor; which reflects the non-linear nature of the adsorption. Additionally, the Hill model is mostly related to the binding of multiple species and presents many uncertainties in the conditions of the present study; contributing to its unsuitability to the experimental data (Joannis-Cassan et al. 2011). The highest adsorption percentage was observed at 7.5 µg/mL (61%±0.03), followed by 5 µg/mL (40%±0.01), 1 µg/mL (32%±0.01), 2.5 µg/mL (30%±0.04) and 0.5 µg/mL (29%±0.01). Despite observing higher adsorption rates for FB 1 at concentrations of 7.5 and 5 µg/mL during this experiment, the recovery tests approached the upper limit of 110% for concentrations of 0.5, 1, 5, and 7.5 µg/mL. In contrast, a recovery rate of 97% was achieved at 2.5 µg/mL. Consequently, this concentration was selected for the comparison of the five different algae-based products. In addition, while conducting the previous experiment, the FB 1 concentration of 2.5 µg/mL achieved a mean adsorption of 44%±0.02; therefore, further corroborating this choice for future product comparisons. [Please insert Figure 1 file here] Adsorption capacity of five algae-based products To compare five different algae-based formulations, in vitro adsorption tests that employed the ideal conditions from previous analyses (pH 5; 2.5 µg/mL FB1) were performed, and the results are shown in Figure 2. Disregarding the AC positive control, the greatest adsorption performance was achieved by A2 (48%±0.05), followed by A3 (42%±0.04), A1 (38.5%±0.03), A4 (38%±0.03) and A5 (34%±0.05). Additionally, the Q eq of the tested products showed the same pattern as the adsorption percentage, where A2 exhibited the highest values (1.022 µg of FB 1 adsorbed per mg of product) and A5 the lowest (0.718 µg of FB1 adsorbed per mg of product). Three green algae-based and two red algae-based formulas were tested. In this regard, a significant difference (p < 0.05) was found between the adsorption performance of products containing green algae (mean adsorption = 43.06 ±0.06) and red algae (mean adsorption = 36.25±0.05); suggesting that green algae-based formulations may be more efficient in adsorbing FB1. [Please insert Figure 2 file here] Discussion In vitro adsorption tests are generally used as a screening method for the efficacy assessment of mycotoxin binders. Some of the main advantages of performing such assays are their lower cost and quicker results, when compared to in vivo analyses. However, most of the studies on in vitro adsorption trials do not present standardized and repeatable conditions, especially regarding the pH levels and mycotoxin concentration (Faucet-Marquis et al. 2014 ; Kihal et al. 2022 ). This is particularly important, as these assays represent the gold standard used by most companies developing adsorbents for livestock feed, since they mimic the animal gastrointestinal system. During digestion, mycotoxins ingested with feed can be bound by the adsorbent, reducing their absorption and subsequent biotransformation in the liver. Consequently, lower toxin levels reach systemic circulation, decreasing their transfer through the food chain into animal-derived products such as milk, eggs or meat (Avantaggiato et al. 2006; Kihal et al. 2023). Thus, validating a robust adsorption protocol is essential to ensure both animal and food safety. The adsorption capacity of mineral products, such as bentonites, is directly linked to their physico-chemical characteristics, especially their interlayer spaces’ conformation/charge and cation exchange capacity. The latter is highly influenced by the pKa of the adsorbent (point zero charge) and the medium’s pH level. In this regard, when the pH level of the medium is lower than the pKa of the adsorbent, it results in a loss of charge and lower cation exchange capacity. Conversely, when the medium’s pH level is higher than the bentonite’s pKa, it becomes more electronegative and adsorption capacity is increased (Du et al. 2021 ; Kihal et al. 2022 ). Considering that FB 1 is a highly polar compound, minor pH alterations can result in structural modifications as well as protonation/deprotonation of carbonyl groups. In acidic conditions, the FB 1 molecule is protonated, while in neutral-alkaline pH it exists as an anion (Momany and Dombrink-Kurtzman 2001 ; Šegvić and Pepeljnjak 2001 ). The results of the present study, show that adsorption was higher at pH 3, which is not consistent with the physico-chemical characteristics of FB 1 and bentonites. At this pH range, the cation exchange capacity and electronegativity of the AA should be low; which considerably reduces the binding performance when only bentonite is utilized (Barrientos-Velázquez et al. 2016 ). The results obtained from the current study suggest that the adsorption of FB 1 by the algae-based product complex occurs mainly due to the presence of algae in the interlayers of bentonites. Algae mycotoxin adsorption mechanisms are not yet well understood. However, it is known that algal cell walls contain polymers, such as β-D-glucans, xylans, galactanes and mannans. These components are likely key contributors to the adsorption capacity of algae, as they can bind to mycotoxins through Van der Walls interactions or hydrogen bonds (Cheng et al. 2019 ; Fraga-Corral et al. 2023 ; Jouany et al. 2005 ; Yiannikouris et al. 2006 ). Such interactions are not based on charge exchange but rather depend on the structural stability of the polysaccharide molecules. In the case of β-D-glucans and other glucose-based polymers, this stability is pH-dependent. Under acidic conditions (pH 3), the structural rigidity of these polysaccharides increases, which improves their ability to bind to mycotoxins. Conversely, at neutral pH levels (5–7), the molecules are less stable and can suffer conformational changes, which might hinder mycotoxin adsorption (Faucet-Marquis et al. 2014 ; Yiannikouris et al. 2004 ; Yiannikouris 2006). Therefore, the low FB 1 adsorption observed at pH 5–7 likely reflects the reduced activity of the algae component, due to pH-sensitive structural changes. Simultaneously, the bentonite may not have attained sufficient surface electronegativity to effectively contribute to adsorption at this pH interval. Altogether, this suggests that pH 5–7 likely represents an intermediate zone, where neither components perform optimally, leading to the observed decrease in overall adsorption efficiency. Moreover, our results also support the evidence that the modification of inorganic mineral products (i.e. bentonites) by adding organic extracts may increase their adsorption capacity; since previous reports indicate FB 1 adsorption by clays alone is moderate to low (Elliott et al. 2020 ). In this respect, Rasheed et al. ( 2020 ), through the addition of orange peel extracts to pure bentonite, obtained efficient in vitro adsorption of Aflatoxin B1 (AFB 1 ), FB 1 , and OTA. In their study, FB 1 adsorption exceeded 80% in buffered solutions at pH 2.5 and pH 7, as well as in simulated gastric fluids (acidic conditions). However, when the reaction was performed in simulated intestinal fluids (near-neutral conditions), the performances was considerably reduced to under 20% (Rasheed et al. 2020 ). These observations are consistent with our results, in which higher adsorption efficiency was also observed at acidic pH 3. Structurally, the organic components were found within the interlayers of the clay, like the algae-based products, generating additional binding sites for mycotoxins. The authors suggested that FB 1 might have formed peripheral interactive forces, especially Van der Waals interactions with the organic fraction of the product (Rasheed et al. 2020 ). Additionally, Oguz et al. ( 2022 ) evaluated the adsorption efficacy of clays, plant extracts, and glucomannans, individually and in combination, using in vitro methods. For FB 1 , the authors reported moderate binding levels for clays and glucomannans alone. However, when plant extracts and glucomannans were added to a mixture of clays, there was a 20% increase in the adsorption capacity of the product, particularly under acidic conditions (pH 3), where FB 1 adsorption reached 54.61%. (Oguz et al. 2022 ). These findings corroborate the results of the current study, in which FB 1 adsorption by algae-modified clay was greatest at pH 3. As for in vivo studies, Tsiouris et al. ( 2021 ), by adding yeast cell walls and silymarin to clays, observed the reduction of adverse effects related to AFB 1 and OTA in broiler chicks. Treated animals also showed healthier intestinal conditions after ingesting the formulated product, which could hinder the development of gut pathogens, such as Escherichia coli , Clostridium perfringens , Salmonella spp. and Campylobacter spp. (Tsiouris et al. 2021 ). In addition, El-Nekeety et al. ( 2017 ) also formulated an organically modified clay and, as a result, the product was able to efficiently adsorb FB 1 and ZEN, reducing their toxic effects in rats. An isotherm curve was constructed to characterize FB 1 adsorption by one algae-based formulation, to select an optimal concentration for testing different products (Boudergue et al. 2009 ; Joannis-Cassan et al. 2011 ). The shape of the curve resembled those observed in other studies involving FBs and organically modified clays (Baglieri et al. 2013 ). According to Baglieri et al. ( 2013 ) and Lemke et al. ( 1998 ), such a profile indicates the binding of the mycotoxin to specific sites. Furthermore, the data revealed that binding site saturation was not achieved at the tested concentrations; however, our preliminary results suggest that an FB 1 concentration of 10 µg/mL combined with 0.1% (w/v) of adsorbent may saturate the binding sites of the product. Additionally, two different models were applied for isotherm curve fitting: the Freundlich model and the Hill model. The Freundlich model is applicable to non-linear multilayer adsorption, showing the exponential distribution of the active binding sites and their energies. In contrast, the Hill model describes cooperative interactions in biological systems and is mainly related to the binding of different species. Therefore, the Freundlich model appears to better fit the conditions and experimental data of the current study when compared to the Hill model (Al-Ghouti and Da’ana 2020 ; Rajahmundry et al. 2021 ). After determining the FB 1 concentration to be 2.5 µg/mL from the isotherm curve, five different algae-based products were tested. The results showed adsorption percentages from 48% (A2) to 34% (A5). Moreover, green algae-based products exhibited significantly higher adsorption compared to those derived from red algae. This contrast may be attributed to the distinct compositions of the red and green algae cell walls (Romera et al. 2008). Red algae cell walls contain sulfated polysaccharides composed of galactanes serving as potential adsorption sites, whereas green algae have glycoprotein-rich walls with diverse functional groups (amino, carboxyl, sulfate, hydroxyl) acting as binding domains (Romera et al. 2008). At present, there are no studies comparing mycotoxin adsorption by multiple products containing different types of algae. However, both algal extracts and live algae have been widely used for heavy metal biosorption, with results showing that green algae generally display higher adsorption capacity than red algae, consistent with our findings (Boukarma et al. 2024 ; Romera et al. 2006 ). Considering the use of only algae or algae-based products for mycotoxin adsorption, Perali et al. ( 2020 ) tested the binding efficacy of Lithothamnium calcareum , for the adsorption of AFB 1 in broiler chicks. As a result, the authors showed not only a reduction of adverse effects related to mycotoxin ingestion, but also an improved body weight, weight gain, and feed intake in the animals treated with L. calcareum (Perali et al. 2020 ). Conversely, another study involving DON and algae-modified clay, showed that the AA was not effective in adsorbing the toxin nor in avoiding the adverse effects related to DON in nursery pigs; possibly due to a low affinity of the product to this mycotoxin (Frobose et al. 2016 ). Altogether, when preventive measures against mycotoxin contamination in animal feed are ineffective, adsorbents are recommended to reduce toxin uptake and prevent productivity losses (Boudergue et al. 2009 ; Jouany, 2007 ; Vila-Donat et al. 2018 ). FB 1 , one of the most commonly found toxins in feed, has a complex and elongated structure, which makes its adsorption challenging. Therefore, it is essential to test and evaluate novel AAs to mitigate its adverse effects and enhance livestock performance (Gao et al. 2023 ; Oguz et al. 2022 ; Shi et al. 2018 ; Xu et al. 2022a ). In this context, incorporating algae into livestock diets may simultaneously contribute to mycotoxin adsorption and improve animal health and immunity (Fraga-Corral et al. 2023 ; Yadavalli et al. 2023 ). In vitro adsorption tests are useful tools for evaluating the adsorption capacity of multiple agents on a small scale, with lower cost and analysis time when compared to in vivo assays. However, adsorption mechanisms are complex, especially when dealing with organic-based products, with conditions such as pH, saturation of the adsorbent, and type of mycotoxin influencing the product performance being important. In this regard, the evaluation of multiple test parameters in the present study resulted in an adequate in vitro FB1 adsorption protocol to screen different algae-based products. The results also showed different adsorption capacities of the formulations evaluated, showing that green algae were more efficient than red algae-based products. Such findings may indicate that the different compositions of these organisms’ cell walls can influence their performance in FB 1 adsorption. In summary, the evaluated products, particularly those containing green algae, show promising potential for reducing FB 1 exposure in livestock. However, further research is needed to assess their efficacy across different feed matrices and under simulated digestion conditions, as well as through in vivo studies on FB 1 bioavailability. This is especially relevant given that the inclusion of algae in livestock diets has already been linked to improvements in animal health. Declarations Ethics declarations 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. Competing interests The authors declare no competing interests. Data statement Data supporting this study are available from the corresponding author (Rocha, L. O.) under request. Funding This work was financially supported by the Olmix Group (Bréhan, France); The São Paulo Research Foundation (FAPESP) [grant number 2023/16635-7]; and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant number 140978/2023-2]. Author Contribution Conceptualization: MG, LOR; Methodology: LAGAS, MM, PACB; Validation: LAGAS, MM; Investigation: LAGAS, MM, PACB; Data curation: LAGAS, MM; Visualization: LAGAS, MM; Resources: APAB, MG, PNC, JB; Writing – original draft: LAGAS; Writing – review and editing: LAGAS, MM, APAB, MG, PNC, JB, LOR; Supervision: MG, LOR; Project administration: MG, LOR; Funding acquisition: MG, PNC, JB, LOR. Acknowledgement The authors would like to thank the Olmix Group (Bréhan, France) for the partnership and funding, The São Paulo Research Foundation (FAPESP, Project number: 2023/16635-7) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Project number: 140978/2023-2). Data Availability Data supporting this study are available from the corresponding author (Rocha, L. O.) under request. References Al-Ghouti MA, Da’ana DA (2020) Guidelines for the use and interpretation of adsorption isotherm models: A review. 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Code Product description A1 Bentonite combined with dry green algae A2 Bentonite combined with green algae extract, under development A3 Bentonite combined with green algae extract, for commercial use A4 Bentonite combined with red algae extract, under development A5 Bentonite combined with red algae extract, for commercial use Table 2. Effect of different pH levels on FB 1 (5 µg/mL) adsorption by the A2 algae-based product. Product pH Mean adsorption (%) ± SD Mean recovery (%) ± SD A2 3 88.06± 0.011 95.63± 0.04 5 48.06±0.032 96.59±0.039 7 2.8±0.018 110.97±0.026 AC 3 91.8± 0.001 95.63± 0.04 5 91.72±0.002 96.59±0.039 7 95.12±0.001 110.97±0.026 Table 3. Effect of FB 1 concentration on the adsorption of A2 algae-based product at pH 5. Product FB1 (µg/mL) Mean adsorption (%) ± SD Mean recovery (%) ± SD A2 1 33±0.01 109±0.02 2.5 44±0.02 97±0.14 5 45±0.04 98±0.03 10 36±0.04 103.1±0.04 AC 1 86±0.0005 109±0.02 2.5 80±0.001 97±0.14 5 92±0.003 98±0.03 10 95±0.0002 103.1±0.04 Additional Declarations No competing interests reported. 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07:00:57","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149332,"visible":true,"origin":"","legend":"","description":"","filename":"7860e60249bc46fc9cbb295a5b4fc2d01structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8407612/v1/14ee25270cf30373fb4a18cb.xml"},{"id":99497892,"identity":"af75f218-b5b3-4bba-b11e-2a915e5ae835","added_by":"auto","created_at":"2026-01-05 07:00:58","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159554,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8407612/v1/d3c060625f8fd11ee61501ff.html"},{"id":99790422,"identity":"8901cb44-801b-44b3-87ac-0fbc811550f8","added_by":"auto","created_at":"2026-01-08 12:58:04","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":298646,"visible":true,"origin":"","legend":"\u003cp\u003eEquilibrium isotherm curve elaborated for FB\u003csub\u003e1\u003c/sub\u003e adsorption, with two different model fits: Freundlich model (green line) and Hill model (purple line).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8407612/v1/d6ce18a74bda7981add90ef0.jpeg"},{"id":99497861,"identity":"9f1b846d-d23b-4db4-9fd8-44ec9bfd5002","added_by":"auto","created_at":"2026-01-05 07:00:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49097,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot of FB\u003csub\u003e1 \u003c/sub\u003e\u003cem\u003ein vitro\u003c/em\u003e adsorption performance at pH 5 (citrate buffer 0.1 mol/L + 10% methanol) by five different algae-based formulations (0.1% w/v). Values are means +/- SD (standard deviation) of technical replicates (n), green color plots represent green algae-based products, red color plots are for red algae-based products. A1-A3: products based on green algae; A4-A5: products based on red algae; AC: activated charcoal.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8407612/v1/79d4ed2258aaa19b102d40b0.jpeg"},{"id":105755068,"identity":"77a14615-8d24-4918-9897-41ded19da07e","added_by":"auto","created_at":"2026-03-30 16:24:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1023513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8407612/v1/34959100-ffc0-4be2-82ce-8d9dd0b038a1.pdf"},{"id":99497879,"identity":"090c5e64-84a2-455b-a5a8-4a344da7fbac","added_by":"auto","created_at":"2026-01-05 07:00:57","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":902619,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8407612/v1/a4f46559be33d481f0e7d783.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"In vitro adsorption of Fumonisin B1 by multiple algae-modified clay formulations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMycotoxins are toxic secondary metabolites produced by filamentous fungi, including species within the \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e genera. These microorganisms are known to infect cereals at multiple stages of production, ranging from field to storage; and result in a persistent risk of mycotoxin contamination throughout the entire production chain (Daou et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Considering that cereals are major components of regular livestock diets, animals become vulnerable to the ingestion of contaminated grains. This exposure may lead to reduced performance, such as lower feed intake, milk yield, and growth efficiency and/or fertility (Vila-Donat et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Kihal et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the most commonly detected toxins in feed are Aflatoxins (AFs), Ochratoxin A (OTA), Trichothecenes, Zearalenone (ZEN) and Fumonisins (FBs) (Awuchi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Considering FBs, Fumonisin B\u003csub\u003e1\u003c/sub\u003e (FB\u003csub\u003e1\u003c/sub\u003e) is the predominant compound encountered, especially in maize and its by-products (Beccaccioli et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pamphile and Azevedo \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Therefore, the occurrence of FB\u003csub\u003e1\u003c/sub\u003e in animal feed has been documented worldwide, with reports from Europe, the Americas, Asia and Africa showing contamination levels ranging from 24.5% to 100% (Gao et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe presence of FB\u003csub\u003e1\u003c/sub\u003e in products intended for animal consumption is concerning, as it has been linked with equine leukoencephalomalacia and porcine pulmonary edema. Additionally, its ingestion can also result in the reduction of feed intake, lower egg production, hepatic necrosis, and thymic cortical atrophy in poultry (Awuchi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Schrenk 2022; Yang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Effects on ruminants are briefly described, with previous studies showing lower milk yield after FB\u003csub\u003e1\u003c/sub\u003e ingestion by dairy cattle; whereas adult beef cattle seem to be more resistant, exhibiting only mild hepatic necrosis (Diaz et al. 2000; Osweiler et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Smith \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, economic pressures, particularly in the livestock industry, are motivating producers to seek effective solutions to prevent the adverse effects of mycotoxins on animal health and productivity. However, if grain contamination has already occurred, preventative measures are no longer feasible. Consequently, the implementation of mitigation strategies intended for the removal, degradation or inactivation of toxins becomes crucial. Therefore, integrated approaches that combine multiple strategies are essential to guarantee safe mycotoxin levels in animal feed (Shi et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDescribed methods include chemical, biological and physical treatments of grain. Chemical agents, such as ammonia, hydrogen peroxide, and organic acids, are highly effective in reducing mycotoxin levels. Nevertheless, the use of such reagents can make raw materials inedible for animals and contributes to environmental pollution. Biological strategies involve the use of enzyme-producing microorganisms to degrade or bio-transform toxins, which presents challenges for in-field application due to environmental interactions. Lastly, physical methods include sorting and separation, floating and segregation by density, irradiation, ultrasound treatment, dehulling, milling and adsorption (Luo et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdsorption involves the use of an adsorbing agent (AA), which forms mycotoxin\u0026ndash;adsorbent complexes through direct binding with the toxins. This process reduces mycotoxin bioaccessibility and, therefore, intestinal absorption; as the formed complexes are excreted via feces. As a consequence, this also leads to a decrease in mycotoxin uptake and in its spread to target organs (Boudergue et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). In contrast with other mitigation strategies, which aim to diminish mycotoxin levels during feed production, AAs are used as feed additives. Additionally, the main advantages of such binders are their cost, safety and ease of administration through inclusion in feed (Peng et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong employed AAs are organic (yeast cell wall, yeast cell wall beta-D-glucan fraction, oat and alfalfa fibers) and inorganic (bentonites, montmorillonites, zeolite and activated carbon) compounds (Kihal et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In general, inorganic binders are highly efficient in adsorbing AFs, due to the high polarity and small molecule size of these toxins. However, such products are relatively inefficient in adsorbing other mycotoxins, especially \u003cem\u003eFusarium\u003c/em\u003e toxins, such as Deoxynivalenol (DON) and ZEN. In this context, the use of organic binders is recommended (Vila-Donat et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering the FB\u003csub\u003e1\u003c/sub\u003e molecule size and its structural conformation, adsorption may be a challenging issue, especially with inorganic products. To address this limitation, adsorption can be increased through the incorporation of biological components, such as algae extracts, to inorganic binders. This modification enhances the interlayer spaces of clays, increasing surface area and resulting in more binding sites for mycotoxins (Cai et al. 2024; Oguz et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rasheed et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlgal extracts are appealing options for AAs, as they are well-known for their biosorption/bioremediation of heavy metals (Cheng et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These organisms\u0026rsquo; cell walls contain a wide range of proteins and polysaccharides, such as β-D-glucans, that are potential binding sites for mycotoxins. Additionally, other algal compounds could also be responsible for adsorption capacity, such as chlorophyll and chlorophyllin; which may also form complexes with mycotoxins, reducing their bioaccessibility (Simonich et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the inclusion of algae as feed additives may not only help prevent the adverse effects of mycotoxins but also promotes improved animal health (Perali et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Algae display high bioactive compound content, which can act as prebiotics and therefore enhance animal immunity. In addition, production performance (i.e. weight gain, feed intake, and feed conversion rate) may also be increased, since algae are rich in proteins, vitamins, and minerals (Fraga-Corral et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Makkar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yadavalli et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEstimating the adsorbent efficiency of different products is indispensable, since it depends on the type of adsorbent, its physico-chemical properties, and the target mycotoxins. \u003cem\u003eIn vivo\u003c/em\u003e testing of mycotoxin binding efficacy of a large number of adsorbents is challenging, due to the complexity and cost; making \u003cem\u003ein vitro\u003c/em\u003e analyses powerful tools for assessing and ranking the efficacy of various AAs. Nevertheless, experimental conditions for \u003cem\u003ein vitro\u003c/em\u003e tests reported in the literature may vary widely, making it difficult to establish reliable protocols (Boudergue et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Faucet-Marquis, 2014). Considering the information cited above, the aim of this study is to develop an \u003cem\u003ein vitro\u003c/em\u003e protocol for FB1 adsorption to evaluate and compare the binding efficacy of products composed of inorganic clay and algae extracts, in order to identify the most effective formulation for FB\u003csub\u003e1\u003c/sub\u003e adsorption in livestock feed.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cem\u003eMycotoxin binders\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFive AA formulations were provided by Olmix (Br\u0026eacute;han, France) for the development of this study. All products consisted of inorganic clay mixed with algae extracts, including red and green algae. They were each labeled with a code (A1 to A5) and their characteristics are summarized in Table 1. For the development and validation of the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003emethod, only A2 was employed; with the other formulations used only for efficacy testing. Additionally, activated charcoal (AC) was used as a positive control for adsorption tests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e[Please, insert Table 1 file here]\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChemicals and reagents\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFB\u003csub\u003e1\u003c/sub\u003e external standards were acquired from Sigma-Aldrich (Fallavier, France), with stock solutions prepared in acetonitrile/water (50/50; v/v) for long-term storage at -20 \u0026ordm;C and further dilution (calibration curves and experiments). The solvents used for the FB\u003csub\u003e1\u003c/sub\u003e external standard dilution, mobile phase and buffer preparation for subsequent analysis by high-performance liquid chromatography coupled with a triple quadrupole mass spectrometry (LC-MS/MS), were HPLC grade and purchased from J.T. Baker (Phillipsburg, USA). In addition, validation of the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003emethod was performed using citrate buffer (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e7\u0026nbsp;\u003c/sub\u003e\u0026bull; H\u003csub\u003e2\u003c/sub\u003eO and C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003e \u0026bull; 2H\u003csub\u003e2\u003c/sub\u003eO; 0,1 M) containing 10% methanol, adjusted to pH 3, 5 and 7.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChromatographic conditions and method validation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLC-MS/MS for mycotoxin quantification was performed using the Agilent 1290 Infinity LC-System (Agilent Technologies, Santa Clara, USA) coupled to a 6460 triple quadrupole (Agilent Technologies, Santa Clara, USA) mass spectrometer, at the Food Toxicology Laboratory, Food Engineering School, State University of Campinas (UNICAMP).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Zorbax SB-C8 column (3.0 mm x 100 mm; 1.8 \u0026micro;m) was used for chromatographic separation at 25 \u0026ordm;C. The analyses were performed in isocratic mode, using 30% of 0.1% (v/v) formic acid in water (mobile phase A) and 70% of 0.1% (v/v) formic acid in methanol (mobile phase B), with a flow rate of 0.35 mL/min and injection volume of 3 \u0026micro;L.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMass spectrometry conditions were: Electrospray Source Ionization in the positive mode (ESI+) with gas temperature at 300 \u0026deg;C, gas flow at 10 L/min, nebulizer at 35 psi, sheath gas flow at 10 L/min, sheath gas temperature at 350 \u0026deg;C, capillary voltage at 3.0 kV and nozzle voltage at 0.5 kV. Ultrapure nitrogen was used in the nebulizer and as collision gas.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor FB\u003csub\u003e1\u003c/sub\u003e, m/z 722 (precursor), m/z 353 (quantifier transition) and m/z 334 (qualifier transition) were monitored. The fragmentor applied was 185 V and collision energies were 40 eV for both quantifiers and qualifier transitions. Data acquisition and analyses were performed using MassHunter software workstation version 7.00 (Agilent Technologies, Santa Clara, USA) in selected reaction monitoring (SMR) mode, using a dwell time of 100 ms per channel.\u003c/p\u003e\n\u003cp\u003eThe linearity of FB\u003csub\u003e1\u003c/sub\u003e was obtained based on calibration curves in the matrices (Figure S1; citrate buffer 0.1 M with 10% methanol at pH 3, 5, and 7). FB\u003csub\u003e1\u003c/sub\u003e standard was prepared at seven concentrations (0.5, 1, 2.5, 5, 7.5, 10, and 12.5 \u0026micro;g/mL) in triplicates, and subsequently analyzed under the conditions previously described (Sun et al. 2018). All linearities (R\u003csup\u003e2\u003c/sup\u003e) obtained were \u0026ge; 0.99 at pHs 3, 5, and 7 (Figure S1). \u0026nbsp;Recovery was evaluated in triplicates on the same day as the analysis, with results ranging from 95% to 110% for citrate buffer at pH 3, 5, and 7 (EC 2006). The limits of detection (LODs) and the limits of quantification (LOQs) for each matrix are provided in Table S1; these were calculated through the minimum concentration detected with a 3x and a 10x signal-noise ratio, respectively.\u003c/p\u003e\n\u003cp\u003eFurthermore, precision was assessed at three FB\u003csub\u003e1\u003c/sub\u003e concentration levels (2.5\u0026nbsp;\u0026micro;g/mL, 5 \u0026micro;g/mL and 7.5 \u0026micro;g/mL) by analyzing five replicates of each level in citrate buffer pH 5, without the presence of adsorbent. Intra-day precision was assessed by analyzing all replicates on the same day under the same conditions. Inter-day precision was determined by repeating the procedure on three different days. The results were expressed as the coefficient of variation (CV%) for each level (Table S2). All CV% values were within the acceptable limits of \u0026le; 20%, in accordance with SANTE/11312/2021 (EC 2021) (Table S2).\u003c/p\u003e\n\u003cp\u003eThe selectivity of the method was assessed by analyzing blank and fortified samples (2.5 \u0026micro;g/mL of FB\u003csub\u003e1\u003c/sub\u003e) in citrate buffer (pH 3, 5, and 7). The fortified samples exhibited well-defined peaks in the qualifier transition (m/z 334) with consistent retention times ranging from 1.928 to 1.955 minutes (Figure S2).\u0026nbsp;No interfering peaks were observed in the blank samples at the corresponding retention times, demonstrating the selectivity of the method for FB\u003csub\u003e1\u003c/sub\u003e analysis (Figure S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEffect of pH and FB\u003csub\u003e1\u003c/sub\u003e concentration on adsorption capacity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003epH\u003c/p\u003e\n\u003cp\u003eThree different pH levels (3, 5 and 7) were evaluated, to determine the optimal level to proceed with the trials. For this purpose, three FB\u003csub\u003e1\u003c/sub\u003e working solutions (5 \u0026micro;g/mL) were prepared in citrate buffer with 10% methanol at pH 3, 5 and 7, separately. Next, 5 mL of each solution was pipetted into 50 mL polypropylene tubes containing either: 0.1% (w/v) of A2, according to manufacturer instructions; 0.1% (w/v) of activated charcoal (AC; AC positive control); or no adsorbent (control without adsorbent); in triplicates (Faucet-Marquis et al. 2014)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSamples were then incubated (30 minutes; 37\u0026ordm;C; 8 x g) and centrifuged (15 minutes; 8586 x g). Subsequently, 1 mL of supernatant from each sample was retrieved, filtered by syringe filter (0.22 \u0026micro;m) and analyzed with HPLC-MS/MS (item 2.3.). \u0026nbsp;After chromatographic analyses, the adsorption percentage of each test was calculated based on equation 1:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cimg width=\"157\" height=\"32\" src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1767594229.png\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (1)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eC\u003csub\u003eads\u003c/sub\u003e\u003c/em\u003e is the adsorbed mycotoxin yield (\u0026micro;g/mL) and \u003cem\u003eC\u003csub\u003e0\u003c/sub\u003e\u003c/em\u003e is the concentration of FB\u003csub\u003e1\u003c/sub\u003e in the controls without adsorbent (\u0026micro;g/mL) (Joannis-Cassan et al. 2011).\u003c/p\u003e\n\u003cp\u003eFB\u003csub\u003e1\u003c/sub\u003e concentration\u003c/p\u003e\n\u003cp\u003eFour FB\u003csub\u003e1\u003c/sub\u003e concentrations (1 \u0026micro;g/mL, 2.5 \u0026micro;g/mL, 5 \u0026micro;g/mL, and 10 \u0026micro;g/mL) were prepared in 5mL of pH 5 citrate buffer (10% methanol) as solvent. Considering the evaluated product should demonstrate a high affinity and capacity to adsorb mycotoxins at its low inclusion rate in livestock diets, 0.1% (w/v) of product A2 was employed. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe tests were performed in triplicates, using 0.1% (w/v) of AC as positive control (AC positive control) and mycotoxin working solution without adsorbent as\u0026nbsp;control without adsorbent. Next, the tubes were incubated (30 minutes; 37 \u0026ordm;C; at 8 x g) and centrifuged (15 minutes; at 8586 x g). After, approximately 1 mL of the supernatant was withdrawn, filtered by syringe filter (0.22 \u0026micro;m), and taken for HPLC-MS/MS analysis using the conditions described above (item 2.3.); and adsorption percentage was calculated based on Formula 1.\u003c/p\u003e\n\u003cp\u003eIsotherm curve\u003c/p\u003e\n\u003cp\u003eConsidering the previously determined optimal pH (item 2.4.1.), 5 mL of increasing amounts of FB\u003csub\u003e1\u003c/sub\u003e (0.5, 1, 2.5, 5, and 7.5 \u0026micro;g/mL) were incubated in 50 mL polypropylene tubes with 0.1% (w/v) of adsorbent (A2), 0.1% (w/v) of AC (AC positive control) or without adsorbent (control without adsorbent). All tests were performed in triplicates and incubation conditions were 37 \u0026ordm;C, with rotation 8 x g for 30 min. Next, all samples were centrifuged (15 minutes; 8586 x g) and 1 mL of their supernatants were withdrawn separately, filtered by syringe filter (0.22 \u0026micro;m), and taken for HPLC-MS/MS analysis; as formerly described (item 2.3.).\u003c/p\u003e\n\u003cp\u003eThis step was performed to determine the ideal intermediate concentration of FB\u003csub\u003e1\u003c/sub\u003e, to evaluate the adsorption capacity of different algae-based products. For this purpose, the adsorption percentage of each concentration was calculated based on Formula 1 (item 2.4.1). Moreover, an isotherm curve was also elaborated by plotting the variations in the residual FB\u003csub\u003e1\u003c/sub\u003e concentration after adsorption in \u0026micro;g/mL (\u003cem\u003eC\u003csub\u003eeq\u003c/sub\u003e\u003c/em\u003e) against the quantity of adsorbed FB\u003csub\u003e1\u003c/sub\u003e per gram of adsorbent (\u003cem\u003eQ\u003csub\u003eeq\u003c/sub\u003e\u003c/em\u003e), calculated using Formula 2:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003cimg width=\"108\" height=\"32\" src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img176759422916.png\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (2)\u003c/p\u003e\n\u003cp\u003ewhere\u0026nbsp;\u003cem\u003eC\u003csub\u003e0\u003c/sub\u003e\u003c/em\u003e is the concentration of FB1 in the blank controls (\u0026micro;g/mL),\u0026nbsp;\u003cem\u003em\u003c/em\u003e is the mass of adsorbent in grams and \u003cem\u003eV\u003c/em\u003e is the final volume of the solution in liters.\u003c/p\u003e\n\u003cp\u003eAdditionally, the data obtained for the isotherm curve was fitted into two different models, Freundlich and Hill (Joannis-Cassan et al. 2011). In this regard, the first assumes the sorption occurs on a heterogeneous surface and is described by Formula 3:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cimg width=\"98\" height=\"33\" src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1767594228.png\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (3)\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eK\u003csub\u003eF\u003c/sub\u003e\u0026nbsp;\u003c/em\u003eis a constant representing the capacity of the adsorbent to bind to the mycotoxin (milligram \u003csup\u003e1 -\u0026nbsp;\u003c/sup\u003e(\u003csup\u003e1 ∕ \u003cem\u003en\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003csub\u003eF\u003c/sub\u003e\u003c/em\u003e)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u0026times; liter \u003csup\u003e1 ∕\u003cem\u003e\u0026nbsp;n\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003csub\u003eF\u003c/sub\u003e\u003c/em\u003e per gram) and \u003cem\u003en\u003csub\u003eF\u003c/sub\u003e\u003c/em\u003e is a constant concerning the affinity of the adsorbent (Joannis-Cassan et al. 2011).\u003c/p\u003e\n\u003cp\u003eAs for the Hill model, it is usually employed to describe the adsorption of diverging compounds onto a heterogeneous substrate (Joannis-Cassan et al. 2011), using Formula 4:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003cimg width=\"103\" height=\"42\" src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img176759422932.png\" alt=\"image\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (4)\u003c/p\u003e\n\u003cp\u003ewhere Q\u003csub\u003eHmax\u003c/sub\u003e is the maximal mycotoxin adsorption corresponding to the site saturation\u003cem\u003e\u0026nbsp;\u003c/em\u003e(mg/g), \u003cem\u003eK\u003csub\u003eD\u003c/sub\u003e\u0026nbsp;\u003c/em\u003esymbolizes the Hill constant (mg/L) and \u003cem\u003en\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e corresponds to the cooperativity coefficient of the interaction among product and toxin (Joannis-Cassan et al. 2011).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEvaluation of adsorption capacity of five algae-based products\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe ideal conditions (pH and FB\u003csub\u003e1\u003c/sub\u003e concentration) obtained from the last steps (item 2.4) were applied, to compare FB\u003csub\u003e1\u003c/sub\u003e adsorption capacities of five different algae-based products (Table 1). Tests were performed using 0.1% (w/v) of each product (A1-A5), 0.1% (w/v) of AC (AC positive control) and no adsorbent (control without adsorbent) in triplicates. FB\u003csub\u003e1\u003c/sub\u003e working solutions (2.5 \u0026micro;g/mL) were prepared in citrate buffer, pH 5 (0.1 M) with 10% methanol, and pipetted (5 mL) into 50 mL polypropylene tubes containing either the test products, AC (AC positive control), or no product (control without adsorbent). All tubes were incubated (30 minutes; 37 \u0026ordm;C; 8 x g) and centrifuged (15 minutes; 8586 x g), followed by the removal of 1 mL of each supernatant. Samples were then filtered by syringe filter (0.22 \u0026micro;m) and taken for HPLC-MS/MS analysis according to item 2.3. Adsorption efficacy was calculated based on Formula 1 (item 2.4.1).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMean and standard deviation calculations of assay replicates were performed in Microsoft Excel 2016. The equilibrium point isotherm with two model fits (Freundlich and Hill model) was elaborated by plotting the experimental data and other resources in R software (R Core Team 2025). Lastly, unpaired t-test results for the adsorption of red algae and green algae-based products were obtained from GraphPad Prism software version 8, with no correction for multiple comparisons (GraphPad, 2018, v. 8.0.1).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eEffect of pH and FB\u003csub\u003e1\u003c/sub\u003e concentration on adsorption capacity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEffect of pH\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pH level is an influential parameter for \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eadsorption tests (Faucet-Marquis et al. 2014). Thus, three different assays were performed: at pH level 3, 5, and 7. All of the AC positive control samples showed adsorption levels above 90%, as expected (Table 2). Conversely, the A2 product adsorption performance varied greatly according to the pH.\u003c/p\u003e\n\u003cp\u003eAt a lower pH level (3), FB\u003csub\u003e1\u003c/sub\u003e adsorption was the highest (88.06%\u0026plusmn;0.011) and the closest to the AC positive control samples. When pH was increased to 5, the adsorption was reduced almost by half to 48.06%\u0026plusmn;0.032. Lastly, at pH 7, FB\u003csub\u003e1\u003c/sub\u003e adsorption was the closest to 0% (2.8%\u0026plusmn;0.018), and showed a very high coefficient of variation, ruling it out as an alternative. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBetween pH levels 3 and 5, the former presented a lower coefficient of variation and standard deviation. However, its adsorption level is too high and similar to AC, which could hinder comparisons among products. Alternatively, pH 5 presented an ideal and intermediate adsorption rate, with an adequate coefficient of variation and higher recovery, when compared to pH 3. In this context, all further assays were carried out at pH 5.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e[Please, insert Table 2 file here]\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEffect of FB\u003csub\u003e1\u003c/sub\u003e concentration\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of FB\u003csub\u003e1\u003c/sub\u003e concentration on the adsorption capacity, 1 \u0026micro;g/mL, 2.5 \u0026micro;g/mL, 5 \u0026micro;g/mL, and 10 \u0026micro;g/mL of FB\u003csub\u003e1\u003c/sub\u003e were tested, considering 0.1% (w/v) of the product A2 and citrate buffer at pH 5. All the recovery tests were acceptable, ranging between 97% and 109% (EC, 2006). AC positive control assays exhibited the highest adsorption rates (Table 3). Moreover, FB\u003csub\u003e1\u003c/sub\u003e adsorption by A2 ranged from 33%\u0026plusmn;0.01 (1 \u0026micro;g/mL), to 45%\u0026plusmn;0.04 (5 \u0026micro;g/mL). In this context, higher adsorption was observed for 2.5 \u0026micro;g/mL of FB\u003csub\u003e1\u003c/sub\u003e (Table 3), with no statistical difference when compared to FB\u003csub\u003e1\u003c/sub\u003e at 5 \u0026micro;g/mL. Nevertheless, when FB\u003csub\u003e1\u003c/sub\u003e concentration was 10 \u0026micro;g/mL, mean adsorption dropped by 10%, indicating that higher FB\u003csub\u003e1\u003c/sub\u003e concentrations might cause saturation in the binding sites of the product (i.e. 10 mg of FB\u003csub\u003e1\u003c/sub\u003e:1 g of the product).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e[Please, insert Table 3 file here]\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEquilibrium point and isotherm curve\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter selecting pH 5 for the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eadsorption test, an isotherm curve was elaborated with different FB1 concentrations (0.5, 1, 2.5, 5, and 7.5 \u0026micro;g/mL). As a result, the experimental data were used to build an equilibrium isotherm curve (Figure 1), which shows an exponential relationship among \u003cem\u003eC\u003csub\u003eeq\u003c/sub\u003e\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Q\u003csub\u003eeq\u003c/sub\u003e\u003c/em\u003e and non-saturation of the adsorbent\u0026rsquo;s binding sites.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsidering the two models applied for curve fitting, the Freundlich model demonstrates agreement with the experimental data; predominantly at lower FB\u003csub\u003e1\u003c/sub\u003e concentrations. In contrast, the Hill model displays a better fit at higher mycotoxin levels. Thus, the Freundlich model seems to be the most suitable for the acquired experimental data due to its significant n factor; which reflects the non-linear nature of the adsorption. Additionally, the Hill model is mostly related to the binding of multiple species and presents many uncertainties in the conditions of the present study; contributing to its unsuitability to the experimental data (Joannis-Cassan et al. 2011).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe highest adsorption percentage was observed at 7.5 \u0026micro;g/mL (61%\u0026plusmn;0.03), followed by 5 \u0026micro;g/mL (40%\u0026plusmn;0.01), 1 \u0026micro;g/mL (32%\u0026plusmn;0.01), 2.5 \u0026micro;g/mL (30%\u0026plusmn;0.04) and 0.5 \u0026micro;g/mL (29%\u0026plusmn;0.01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite observing higher adsorption rates for FB\u003csub\u003e1\u003c/sub\u003e at concentrations of 7.5 and 5 \u0026micro;g/mL during this experiment, the recovery tests approached the upper limit of 110% for concentrations of 0.5, 1, 5, and 7.5 \u0026micro;g/mL. In contrast, a recovery rate of 97% was achieved at 2.5 \u0026micro;g/mL. Consequently, this concentration was selected for the comparison of the five different algae-based products. In addition, while conducting the previous experiment, the FB\u003csub\u003e1\u003c/sub\u003e concentration of 2.5 \u0026micro;g/mL achieved a mean adsorption of 44%\u0026plusmn;0.02; therefore, further corroborating this choice for future product comparisons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e[Please insert Figure 1 file here]\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAdsorption capacity of five algae-based products\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo compare five different algae-based formulations, \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eadsorption tests that employed the ideal conditions from previous analyses (pH 5; 2.5 \u0026micro;g/mL FB1) were performed, and the results are shown in Figure 2. Disregarding the AC positive control, the greatest adsorption performance was achieved by A2 (48%\u0026plusmn;0.05), followed by A3 (42%\u0026plusmn;0.04), A1 (38.5%\u0026plusmn;0.03), A4 (38%\u0026plusmn;0.03) and A5 (34%\u0026plusmn;0.05). Additionally, the Q\u003csub\u003eeq\u003c/sub\u003e of the tested products showed the same pattern as the adsorption percentage, where A2 exhibited the highest values (1.022 \u0026micro;g of FB\u003csub\u003e1\u003c/sub\u003e adsorbed per mg of product) and A5 the lowest (0.718 \u0026micro;g of FB1 adsorbed per mg of product).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThree green algae-based and two red algae-based formulas were tested. In this regard, a significant difference (p \u0026lt; 0.05) was found between the adsorption performance of products containing green algae (mean adsorption = 43.06 \u0026plusmn;0.06) and red algae (mean adsorption = 36.25\u0026plusmn;0.05); suggesting that green algae-based formulations may be more efficient in adsorbing FB1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e[Please insert Figure 2 file here]\u003c/em\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e adsorption tests are generally used as a screening method for the efficacy assessment of mycotoxin binders. Some of the main advantages of performing such assays are their lower cost and quicker results, when compared to \u003cem\u003ein vivo\u003c/em\u003e analyses. However, most of the studies on \u003cem\u003ein vitro\u003c/em\u003e adsorption trials do not present standardized and repeatable conditions, especially regarding the pH levels and mycotoxin concentration (Faucet-Marquis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kihal et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This is particularly important, as these assays represent the gold standard used by most companies developing adsorbents for livestock feed, since they mimic the animal gastrointestinal system. During digestion, mycotoxins ingested with feed can be bound by the adsorbent, reducing their absorption and subsequent biotransformation in the liver. Consequently, lower toxin levels reach systemic circulation, decreasing their transfer through the food chain into animal-derived products such as milk, eggs or meat (Avantaggiato et al. 2006; Kihal et al. 2023). Thus, validating a robust adsorption protocol is essential to ensure both animal and food safety.\u003c/p\u003e \u003cp\u003eThe adsorption capacity of mineral products, such as bentonites, is directly linked to their physico-chemical characteristics, especially their interlayer spaces\u0026rsquo; conformation/charge and cation exchange capacity. The latter is highly influenced by the pKa of the adsorbent (point zero charge) and the medium\u0026rsquo;s pH level. In this regard, when the pH level of the medium is lower than the pKa of the adsorbent, it results in a loss of charge and lower cation exchange capacity. Conversely, when the medium\u0026rsquo;s pH level is higher than the bentonite\u0026rsquo;s pKa, it becomes more electronegative and adsorption capacity is increased (Du et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kihal et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering that FB\u003csub\u003e1\u003c/sub\u003e is a highly polar compound, minor pH alterations can result in structural modifications as well as protonation/deprotonation of carbonyl groups. In acidic conditions, the FB\u003csub\u003e1\u003c/sub\u003e molecule is protonated, while in neutral-alkaline pH it exists as an anion (Momany and Dombrink-Kurtzman \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Šegvić and Pepeljnjak \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The results of the present study, show that adsorption was higher at pH 3, which is not consistent with the physico-chemical characteristics of FB\u003csub\u003e1\u003c/sub\u003e and bentonites. At this pH range, the cation exchange capacity and electronegativity of the AA should be low; which considerably reduces the binding performance when only bentonite is utilized (Barrientos-Vel\u0026aacute;zquez et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results obtained from the current study suggest that the adsorption of FB\u003csub\u003e1\u003c/sub\u003e by the algae-based product complex occurs mainly due to the presence of algae in the interlayers of bentonites. Algae mycotoxin adsorption mechanisms are not yet well understood. However, it is known that algal cell walls contain polymers, such as β-D-glucans, xylans, galactanes and mannans. These components are likely key contributors to the adsorption capacity of algae, as they can bind to mycotoxins through Van der Walls interactions or hydrogen bonds (Cheng et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fraga-Corral et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jouany et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Yiannikouris et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Such interactions are not based on charge exchange but rather depend on the structural stability of the polysaccharide molecules. In the case of β-D-glucans and other glucose-based polymers, this stability is pH-dependent. Under acidic conditions (pH 3), the structural rigidity of these polysaccharides increases, which improves their ability to bind to mycotoxins. Conversely, at neutral pH levels (5\u0026ndash;7), the molecules are less stable and can suffer conformational changes, which might hinder mycotoxin adsorption (Faucet-Marquis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yiannikouris et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Yiannikouris 2006).\u003c/p\u003e \u003cp\u003eTherefore, the low FB\u003csub\u003e1\u003c/sub\u003e adsorption observed at pH 5\u0026ndash;7 likely reflects the reduced activity of the algae component, due to pH-sensitive structural changes. Simultaneously, the bentonite may not have attained sufficient surface electronegativity to effectively contribute to adsorption at this pH interval. Altogether, this suggests that pH 5\u0026ndash;7 likely represents an intermediate zone, where neither components perform optimally, leading to the observed decrease in overall adsorption efficiency. Moreover, our results also support the evidence that the modification of inorganic mineral products (i.e. bentonites) by adding organic extracts may increase their adsorption capacity; since previous reports indicate FB\u003csub\u003e1\u003c/sub\u003e adsorption by clays alone is moderate to low (Elliott et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this respect, Rasheed et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), through the addition of orange peel extracts to pure bentonite, obtained efficient \u003cem\u003ein vitro\u003c/em\u003e adsorption of Aflatoxin B1 (AFB\u003csub\u003e1\u003c/sub\u003e), FB\u003csub\u003e1\u003c/sub\u003e, and OTA. In their study, FB\u003csub\u003e1\u003c/sub\u003e adsorption exceeded 80% in buffered solutions at pH 2.5 and pH 7, as well as in simulated gastric fluids (acidic conditions). However, when the reaction was performed in simulated intestinal fluids (near-neutral conditions), the performances was considerably reduced to under 20% (Rasheed et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These observations are consistent with our results, in which higher adsorption efficiency was also observed at acidic pH 3. Structurally, the organic components were found within the interlayers of the clay, like the algae-based products, generating additional binding sites for mycotoxins. The authors suggested that FB\u003csub\u003e1\u003c/sub\u003e might have formed peripheral interactive forces, especially Van der Waals interactions with the organic fraction of the product (Rasheed et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, Oguz et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) evaluated the adsorption efficacy of clays, plant extracts, and glucomannans, individually and in combination, using \u003cem\u003ein vitro\u003c/em\u003e methods. For FB\u003csub\u003e1\u003c/sub\u003e, the authors reported moderate binding levels for clays and glucomannans alone. However, when plant extracts and glucomannans were added to a mixture of clays, there was a 20% increase in the adsorption capacity of the product, particularly under acidic conditions (pH 3), where FB\u003csub\u003e1\u003c/sub\u003e adsorption reached 54.61%. (Oguz et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These findings corroborate the results of the current study, in which FB\u003csub\u003e1\u003c/sub\u003e adsorption by algae-modified clay was greatest at pH 3.\u003c/p\u003e \u003cp\u003eAs for \u003cem\u003ein vivo\u003c/em\u003e studies, Tsiouris et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), by adding yeast cell walls and silymarin to clays, observed the reduction of adverse effects related to AFB\u003csub\u003e1\u003c/sub\u003e and OTA in broiler chicks. Treated animals also showed healthier intestinal conditions after ingesting the formulated product, which could hinder the development of gut pathogens, such as \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eClostridium perfringens\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e spp. and \u003cem\u003eCampylobacter\u003c/em\u003e spp. (Tsiouris et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, El-Nekeety et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) also formulated an organically modified clay and, as a result, the product was able to efficiently adsorb FB\u003csub\u003e1\u003c/sub\u003e and ZEN, reducing their toxic effects in rats.\u003c/p\u003e \u003cp\u003eAn isotherm curve was constructed to characterize FB\u003csub\u003e1\u003c/sub\u003e adsorption by one algae-based formulation, to select an optimal concentration for testing different products (Boudergue et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Joannis-Cassan et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The shape of the curve resembled those observed in other studies involving FBs and organically modified clays (Baglieri et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). According to Baglieri et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and Lemke et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), such a profile indicates the binding of the mycotoxin to specific sites. Furthermore, the data revealed that binding site saturation was not achieved at the tested concentrations; however, our preliminary results suggest that an FB\u003csub\u003e1\u003c/sub\u003e concentration of 10 \u0026micro;g/mL combined with 0.1% (w/v) of adsorbent may saturate the binding sites of the product.\u003c/p\u003e \u003cp\u003eAdditionally, two different models were applied for isotherm curve fitting: the Freundlich model and the Hill model. The Freundlich model is applicable to non-linear multilayer adsorption, showing the exponential distribution of the active binding sites and their energies. In contrast, the Hill model describes cooperative interactions in biological systems and is mainly related to the binding of different species. Therefore, the Freundlich model appears to better fit the conditions and experimental data of the current study when compared to the Hill model (Al-Ghouti and Da\u0026rsquo;ana \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rajahmundry et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter determining the FB\u003csub\u003e1\u003c/sub\u003e concentration to be 2.5 \u0026micro;g/mL from the isotherm curve, five different algae-based products were tested. The results showed adsorption percentages from 48% (A2) to 34% (A5). Moreover, green algae-based products exhibited significantly higher adsorption compared to those derived from red algae. This contrast may be attributed to the distinct compositions of the red and green algae cell walls (Romera et al. 2008). Red algae cell walls contain sulfated polysaccharides composed of galactanes serving as potential adsorption sites, whereas green algae have glycoprotein-rich walls with diverse functional groups (amino, carboxyl, sulfate, hydroxyl) acting as binding domains (Romera et al. 2008). At present, there are no studies comparing mycotoxin adsorption by multiple products containing different types of algae. However, both algal extracts and live algae have been widely used for heavy metal biosorption, with results showing that green algae generally display higher adsorption capacity than red algae, consistent with our findings (Boukarma et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Romera et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering the use of only algae or algae-based products for mycotoxin adsorption, Perali et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) tested the binding efficacy of \u003cem\u003eLithothamnium calcareum\u003c/em\u003e, for the adsorption of AFB\u003csub\u003e1\u003c/sub\u003e in broiler chicks. As a result, the authors showed not only a reduction of adverse effects related to mycotoxin ingestion, but also an improved body weight, weight gain, and feed intake in the animals treated with \u003cem\u003eL. calcareum\u003c/em\u003e (Perali et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Conversely, another study involving DON and algae-modified clay, showed that the AA was not effective in adsorbing the toxin nor in avoiding the adverse effects related to DON in nursery pigs; possibly due to a low affinity of the product to this mycotoxin (Frobose et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAltogether, when preventive measures against mycotoxin contamination in animal feed are ineffective, adsorbents are recommended to reduce toxin uptake and prevent productivity losses (Boudergue et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Jouany, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Vila-Donat et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). FB\u003csub\u003e1\u003c/sub\u003e, one of the most commonly found toxins in feed, has a complex and elongated structure, which makes its adsorption challenging. Therefore, it is essential to test and evaluate novel AAs to mitigate its adverse effects and enhance livestock performance (Gao et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Oguz et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). In this context, incorporating algae into livestock diets may simultaneously contribute to mycotoxin adsorption and improve animal health and immunity (Fraga-Corral et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yadavalli et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e adsorption tests are useful tools for evaluating the adsorption capacity of multiple agents on a small scale, with lower cost and analysis time when compared to \u003cem\u003ein vivo\u003c/em\u003e assays. However, adsorption mechanisms are complex, especially when dealing with organic-based products, with conditions such as pH, saturation of the adsorbent, and type of mycotoxin influencing the product performance being important. In this regard, the evaluation of multiple test parameters in the present study resulted in an adequate \u003cem\u003ein vitro\u003c/em\u003e FB1 adsorption protocol to screen different algae-based products. The results also showed different adsorption capacities of the formulations evaluated, showing that green algae were more efficient than red algae-based products. Such findings may indicate that the different compositions of these organisms\u0026rsquo; cell walls can influence their performance in FB\u003csub\u003e1\u003c/sub\u003e adsorption. In summary, the evaluated products, particularly those containing green algae, show promising potential for reducing FB\u003csub\u003e1\u003c/sub\u003e exposure in livestock. However, further research is needed to assess their efficacy across different feed matrices and under simulated digestion conditions, as well as through in vivo studies on FB\u003csub\u003e1\u003c/sub\u003e bioavailability. This is especially relevant given that the inclusion of algae in livestock diets has already been linked to improvements in animal health.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics declarations\u003c/h2\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eData statement\u003c/h2\u003e\n\u003cp\u003eData supporting this study are available from the corresponding author (Rocha, L. O.) under request.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was financially supported by the Olmix Group (Br\u0026eacute;han, France); The S\u0026atilde;o Paulo Research Foundation (FAPESP) [grant number 2023/16635-7]; and the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) [grant number 140978/2023-2].\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization: MG, LOR; Methodology: LAGAS, MM, PACB; Validation: LAGAS, MM; Investigation: LAGAS, MM, PACB; Data curation: LAGAS, MM; Visualization: LAGAS, MM; Resources: APAB, MG, PNC, JB; Writing \u0026ndash; original draft: LAGAS; Writing \u0026ndash; review and editing: LAGAS, MM, APAB, MG, PNC, JB, LOR; Supervision: MG, LOR; Project administration: MG, LOR; Funding acquisition: MG, PNC, JB, LOR.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank the Olmix Group (Br\u0026eacute;han, France) for the partnership and funding, The S\u0026atilde;o Paulo Research Foundation (FAPESP, Project number: 2023/16635-7) and the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq, Project number: 140978/2023-2).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData supporting this study are available from the corresponding author (Rocha, L. O.) under request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Ghouti MA, Da\u0026rsquo;ana DA (2020) Guidelines for the use and interpretation of adsorption isotherm models: A review. J Hazard Mater 393:122383. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhazmat.2020.122383\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2020.122383\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvantaggiato G, Solfrizzo M, Visconti A (2005) Recent advances on the use of adsorbent materials for detoxification of \u003cem\u003eFusarium\u003c/em\u003e mycotoxins. 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J Food Prot 67:2741\u0026ndash;2746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4315/0362-028X-67.12.2741\u003c/span\u003e\u003cspan address=\"10.4315/0362-028X-67.12.2741\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Products used for FB1 adsorption in the current study.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"632\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.2975%;\"\u003e\n \u003cp\u003eCode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.7025%;\"\u003e\n \u003cp\u003eProduct description\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.2975%;\"\u003e\n \u003cp\u003eA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83.7025%;\"\u003e\n \u003cp\u003eBentonite combined with dry green algae\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.2975%;\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83.7025%;\"\u003e\n \u003cp\u003eBentonite combined with green algae extract, under development\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.2975%;\"\u003e\n \u003cp\u003eA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83.7025%;\"\u003e\n \u003cp\u003eBentonite combined with green algae extract, for commercial use\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.2975%;\"\u003e\n \u003cp\u003eA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83.7025%;\"\u003e\n \u003cp\u003eBentonite combined with red algae extract, under development\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 16.2975%;\"\u003e\n \u003cp\u003eA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83.7025%;\"\u003e\n \u003cp\u003eBentonite combined with red algae extract, for commercial use\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Effect of different pH levels on FB\u003csub\u003e1\u003c/sub\u003e (5 \u0026micro;g/mL) adsorption by the A2 algae-based product.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"553\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003eProduct\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eMean adsorption (%) \u0026plusmn; SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eMean recovery (%) \u0026plusmn; SD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 204px;\"\u003e\n \u003cp\u003e88.06\u0026plusmn; 0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003e95.63\u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 204px;\"\u003e\n \u003cp\u003e48.06\u0026plusmn;0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003e96.59\u0026plusmn;0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 204px;\"\u003e\n \u003cp\u003e2.8\u0026plusmn;0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003e110.97\u0026plusmn;0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 204px;\"\u003e\n \u003cp\u003e91.8\u0026plusmn; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003e95.63\u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 204px;\"\u003e\n \u003cp\u003e91.72\u0026plusmn;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003e96.59\u0026plusmn;0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 204px;\"\u003e\n \u003cp\u003e95.12\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003e110.97\u0026plusmn;0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Effect of FB\u003csub\u003e1\u003c/sub\u003e concentration on the adsorption of A2 algae-based product at pH 5.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003eProduct\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003eFB1 (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 215px;\"\u003e\n \u003cp\u003eMean adsorption (%) \u0026plusmn; SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 208px;\"\u003e\n \u003cp\u003eMean recovery (%) \u0026plusmn; SD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 65px;\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 215px;\"\u003e\n \u003cp\u003e33\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e109\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 215px;\"\u003e\n \u003cp\u003e44\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e97\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 215px;\"\u003e\n \u003cp\u003e45\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e98\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 215px;\"\u003e\n \u003cp\u003e36\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e103.1\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 65px;\"\u003e\n \u003cp\u003eAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 215px;\"\u003e\n \u003cp\u003e86\u0026plusmn;0.0005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e109\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 215px;\"\u003e\n \u003cp\u003e80\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e97\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 215px;\"\u003e\n \u003cp\u003e92\u0026plusmn;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e98\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 215px;\"\u003e\n \u003cp\u003e95\u0026plusmn;0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e103.1\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycotoxin-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myre","sideBox":"Learn more about [Mycotoxin Research](http://link.springer.com/journal/12549)","snPcode":"12550","submissionUrl":"https://submission.nature.com/new-submission/12550/3","title":"Mycotoxin Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Adsorption, mycotoxin, mitigation, feed, physical methods","lastPublishedDoi":"10.21203/rs.3.rs-8407612/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8407612/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMycotoxins are toxic secondary metabolites produced by fungi, and frequently encountered in cereals that compose a major part of livestock diets. Fumonisin B1 (FB\u003csub\u003e1\u003c/sub\u003e) is one of the most prevalent toxins in feed, posing a risk to animal health and productivity. Considering mycotoxin mitigation strategies, adsorbents are an advantageous alternative for reducing mycotoxin uptake by animals. In this context, the main objective of this study was to develop an \u003cem\u003ein vitro\u003c/em\u003e protocol for FB\u003csub\u003e1\u003c/sub\u003e adsorption and assess the binding efficacy of five formulated products composed of inorganic clay and algae extracts. For this purpose, algae-based formulations were provided by Olmix (Br\u0026eacute;han, France), and multiple parameters were evaluated for \u003cem\u003ein vitro\u003c/em\u003e testing, such as pH and mycotoxin concentration. After the selection of adequate conditions, the adsorption capacities of five algae-based products were compared. Results indicate that the adsorption capacity of the algae-based products is mainly linked to the presence of algae, especially green algae; which present a high polysaccharide content in their cell walls as binding sites for mycotoxins. The use of algae for mycotoxin adsorption remains underexplored, but the findings of the present work indicate that algae-based products are effective for FB\u003csub\u003e1\u003c/sub\u003e control in animal feed.\u003c/p\u003e","manuscriptTitle":"In vitro adsorption of Fumonisin B1 by multiple algae-modified clay formulations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-05 07:00:52","doi":"10.21203/rs.3.rs-8407612/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-26T09:13:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T15:12:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-15T18:23:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272821704320548061790061114941488280901","date":"2026-01-14T12:33:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237996253385423753236255975493474270417","date":"2026-01-02T19:17:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-02T12:54:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T09:30:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-23T09:30:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycotoxin Research","date":"2025-12-19T18:39:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycotoxin-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myre","sideBox":"Learn more about [Mycotoxin Research](http://link.springer.com/journal/12549)","snPcode":"12550","submissionUrl":"https://submission.nature.com/new-submission/12550/3","title":"Mycotoxin Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b5c01736-dacf-497e-8466-79a1dad87ce5","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:20:17+00:00","versionOfRecord":{"articleIdentity":"rs-8407612","link":"https://doi.org/10.1007/s12550-026-00643-3","journal":{"identity":"mycotoxin-research","isVorOnly":false,"title":"Mycotoxin Research"},"publishedOn":"2026-03-24 16:08:58","publishedOnDateReadable":"March 24th, 2026"},"versionCreatedAt":"2026-01-05 07:00:52","video":"","vorDoi":"10.1007/s12550-026-00643-3","vorDoiUrl":"https://doi.org/10.1007/s12550-026-00643-3","workflowStages":[]},"version":"v1","identity":"rs-8407612","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8407612","identity":"rs-8407612","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}