In vitro assessment of commercial multi-mycotoxin binders to reduce the bioavailability of emerging mycotoxins in livestock

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9685567/ 

In vitro assessment of commercial multi-mycotoxin binders to reduce the bioavailability of emerging mycotoxins in livestock

https://doi.org/10.1016/j.emcon.2023.100256Get rights and content
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Abstract

The changing climate and agricultural practices continue to drive the increased prevalence and levels of both regulated and emerging mycotoxins in feed and food crops. This poses a considerable challenge for the agricultural sector in developing and developed countries as there is growing evidence that the emerging contaminants are capable of inducing a wide range of adverse health effects in livestock. This study evaluated and compared the capacity of commercially available mycotoxin binders to reduce the bioavailability of eight important emerging mycotoxins commonly found in livestock feeds. A novel in vitro model simulating the gastrointestinal track (GIT) of a monogastric animal was developed. Then, the efficiency of ten commercial binders with multi-mycotoxin binding claims were evaluated using the developed in vitro GIT model. Whist all the ten products demonstrated the capacity to simultaneously decrease the levels of more than one emerging mycotoxins, only one product (a mixed silicate) was able to considerably reduce the concentrations of all eight emerging mycotoxins simultaneously under the simulated in vitro conditions. This study highlights, for the first time, a significant gap in emerging mycotoxin mitigation through the deployment of binding agents and identifies the need for the development of novel mitigation strategies for this important group of mycotoxins.

Keywords

Emerging mycotoxins
Livestock
Mycotoxin binders
In vitro model
Mitigation

1. Introduction

Globally, annual compound feed production averages around one billion tonnes, with over US $400 billion turnover [1]. This is expected to increase rapidly as the production of animal proteins is projected to grow by around 1.7% per year, with meat production expected to rise by nearly 70% and aquaculture by 90% [2]. Animal feeds form a central component of the agri-food supply chain, not only to maintain the health, welfare, and performance of livestock animals but also for the quality and safety of animal derived food products [3]. However, feed safety is frequently compromised by natural and processed contaminants [4]. Mycotoxins are one of the major natural contaminants that pose a feed safety challenge both in the developed and developing world [5,6]). Mycotoxins are toxic secondary metabolites produced under favourable environmental conditions by fungal species belonging to the Aspergillus, Penicillium, Alternaria and Fusarium genera. Hundreds of these metabolites have been reported to contaminate agricultural commodities, with deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FBs), aflatoxins (AFs), ochratoxins (OTs), and T-2/HT-2 the most frequently detected and which also have regulatory and/or guidance levels [7]. Moreover, emerging mycotoxins (EMs), commonly termed ‘mycotoxins that are neither routinely determined nor legislatively regulated’, are of grave concern. They are also produced mostly by Aspergillus, Alternaria and Fusarium species [8]. Thus, the majority of agricultural commodities worldwide are co-contaminated with both emerging and regulated mycotoxins.

Recent advances in liquid chromatography coupled with mass spectrometry (LC-MS/MS) methods have enabled the simultaneous detection of emerging and regulated mycotoxins in feed crops [[9], [10], [11], [12]]. Moreover, recent surveys on the occurrence of mycotoxins in agricultural commodities revealed a continuous increase in the prevalence and concentrations of EMs in feed and feed ingredients (cereals and soybeans) worldwide [[10], [11], [12], [13], [14]]). The most prevalent EMs include, beauvericin (BEA), alternariol (ALT), enniatins (ENNs), moniliformin (MON), sterigmatocystin (STG), nivalenol (NIV), patulin (PAT), diacetoxyscirpenol (DAS), tentoxin, citrinin, fusaric acid, tenuazonic acid and fusaric acid (FUS) [13].

In terms of health effects, several in vitro and in vivo studies have demonstrated that these emerging hazardous compounds, particularly BEA, ENNs, MON, NIV and ALT, are capable of eliciting a wide range of adverse health effects in farm animals, such as reproductive disorders, neurotoxicity, intestinal toxicity, hepatotoxicity, and genotoxicity [[15], [16], [17], [18], [19]]. Furthermore, there are currently no established maximum permitted levels in Europe for the residues of these compounds in feed ingredients intended for animal consumption. Thus, livestock are constantly exposed to a wide range of doses and mixtures that may profoundly impact their health, welfare, and performance. Beside the toxic effects of EMs on animal health and productivity, exposure of farm animals to contaminated feed can lead to increased emission of greenhouse gases due to feed waste [11].

Regarding mitigation, while several feed additives or mycotoxin binders have been developed and used to mitigate the toxicity of regulated mycotoxins (DON, ZEN, FBs, T-2/HT-2, OTs, and AFs) in livestock animals, there are currently no mitigation strategies for EMs. Given their increased prevalence and concentrations in animal feed, there is a need to find suitable approaches to minimize livestock exposure. As many of the EMs have similar molecular structures or functional groups with regulated mycotoxins (for instance, T-2 toxin and DAS; AFs and STG), we hypothesized that some of the commercial binders currently used for the mitigation of regulated mycotoxins in animal feeds will also have the capacity to bind and reduce the bioavailability of EMs in farm animals. Thus, we developed and validated a sensitive LC-MS/MS assay and novel in vitro gastrointestinal (GIT) model simulating the GIT of monogastric animals (pig and poultry). Both assays were then used to evaluate and compare the efficacy of ten commercially available mycotoxin binders to reduce the levels of eight EMs that are mostly prevalent in animal feeds worldwide – ENNs (ENNA, ENN A1, ENN B, ENN B1), NIV, DAS, BEA, and STG.

2. Materials and method

2.1. Chemicals and reagents

Hydrochloric acid (HCl), citric acid, pancreatin, pepsin, formic acid, bile salt, pepsin, pancreatin, sodium chloride (NaCl), potassium chloride (KCl), sodium bicarbonate (NaHCO3), LC-MS/MS grade methanol and acetonitrile were supplied by Sigma-Aldrich (Gillingham, UK). EMs — ENN (A, A1, B and B1), NIV, DAS, and STG —crystalline solids were obtained from Romer Labs GmbH (Tulln, Austria). Ultra-pure water was obtained from a Milli-Q Gradient A10 water purification device (Millipore, France). Polytetrafluoroethylene (PTFE) filters and LC-MS/MS glass vials were purchased from Waters. All chemicals used were of analytical grade.

2.2. Mycotoxin binders

Ten commercially available products claiming multi-mycotoxin adsorption or binding were obtained directly from the respective manufacturers in Europe and America. Due to non-disclosure agreements with the manufacturers, the ten products were coded with numbers to protect the confidentiality of the products and the identity of the manufacturers. However, the details of each product, including mode of actions and composition (as provided by the manufacturers) are illustrated in Table 1.

Table 1. Composition and mode of actions (as stated on the product labels) of ten commercial mycotoxin binders.

ProductCompositionMode of action
1Yeast cell wallBinding/Detoxification
2Mixed silicatesBinding/Detoxification
3Yeast extractAdsorption/Binding
4Yeast extractAdsorption/Binding
5Yeast cell wallDetoxification
6AluminosilicatesAdsorption
7AluminosilicatesBinding
8AluminosilicatesBinding
9BentoniteBinding
10Yeast cell wallBinding

2.3. In vitro gastrointestinal model

An artificially contaminated feed sample was made by spiking 10 g of finely ground poultry feed with 1 mL of multi-mycotoxin stock solution to reach a concentration of 0.5 μg/kg for ENN (A, A1, B and B1), NIV, BEA, DAS, and STG. The spiked feed samples were incubated overnight to allow the evaporation of methanol and water. A validated LC-MS/MS method was used to check the homogeneity of each batch [11]. Subsequently, an in vitro automated gastro-intestinal model simulating the GIT conditions of a monogastric animal in terms of compartment, duration, pH, and enzymes, was used to assess the efficacy of the commercial products to reduce the levels of EMs in feed.

The GIT model consisted of four compartments connected in series with peristaltic valve pumps to simulate 1) stomach/proventriculus, 2) duodenum/jejunum and 3) ileum. The pHs in the gastric and small intestine compartments were monitored with pH sensors attached to the compartments and adjusted using NaHCO3 and HCl. The average pH of stomach/proventriculus, duodenum/jejunum and ileum were, respectively, 3, 5 and 7. The gastric secretions comprised of pepsin (0.25 g/L) derived from porcine gastric mucosa, NaCl 2 g/L, and NaHCO3 0.50 g/L. Duodenal secretions consisted of 4% pancreatin solution NaHCO3 0.50 g/L, KCl 0.20 g/L, NaCl, 2 g/L, and 0.4% bile salt. The temperature was kept constant throughout the experiment at 39 °C. Approximately 10 g of feed contaminated with eight EMs and 200 mg of each product (0.2%) was used for each batch of digestion in quintuplicate, with total incubation time of 6 h. Blanks, positive and negative controls contained only ileal digesta (ILD), only EMs and only mycotoxin binders, respectively. After digestion, approximately 5 mL of the ILD was withdrawn and mixed with 5 mL of 2% formic acid in acetonitrile, followed by a rigorous vortex and centrifugation at 10,000×g for 30 min. Subsequently, 1 mL of the supernatant was filtered through 0.2 μm PTFE filter and transferred to a glass vial for LC-MS/MS analysis.

2.4. LC-MS/MS conditions

Qualitative and quantitative analyses of the eight EMs, ENN (A, A1, B and B1), NIV, DAS, BEA, and STG, were carried out on an ExionLC™ AD ultra-high-performance liquid chromatography system (Framingham, MA, USA) coupled to a SCIEX 5500+ QTrap triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA) equipped with a Turbo V™ electrospray ionisation source. Chromatographic separation was performed using a Phenomenex C18-column (100 × 4.6 mm, 5 μm) maintained at 30 °C. The mobile phase was composed of mobile phase A — methanol/water/acetic acid 10:89:1 (v/v/v) and mobile phase B — methanol/water/acetic acid 97:2:1 (v/v/v), both containing 5 mM ammonium acetate buffer. Mycotoxins were eluted following a gradient elution program as follows: 0 min 1% B, held for 1 min at 1% B, 5 min 65% B, 7 min 80% B, 8.5 min 80% B, 9 min 95% B, 10 min 95% B and 12 min 1% B. Mobile phase flow rate was maintained at 0.7 mL/min, with sample injection volume set at 5 μL. The total runtime was 12 min. The mass spectrometry was operated in both positive and negative electrospray ionisation mode, with data acquisition carried out in scheduled multiple reaction monitoring mode. The capillary voltage and source temperatures were set at 4.5 kV and −4.5 kV for ESI+ and ESI- respectively, with the temperature set at 600 °C. With regard to gas parameters, ion source gas 1, ion source gas 2, collision gas, and curtain gas were set at 60, 60, 9 and 35 psi, respectively. Two MRM characteristic transitions (1 precursor ion, 2 product ions) were monitored for each analyte. The selected MRM transitions and their respective analyte-dependent operating conditions, including collision energy, declustering potential and collision cell exit potential are listed in Table 2. Analyst® Software and SCIEX OS-Q Software were used for acquiring and processing data, respectively.

Table 2. Triple quadruple mass spectrometry (MS/MS) parameters for the determination of emerging mycotoxins.

MycotoxinsPrecursor ion (m/z)Product ion (m/z)PolarityIonDP (V)CE (V)CXP (V)
Enniatin A699.3682.4Positive[M+H]+1002724
699.3210.21003922
Enniatin A1685.3668.5Positive[M+H]+1002722
685.3210.11003910
Enniatin B657.3640.3Positive[M+H]+1002712
657.31961003910
Enniatin B1671.3654.4Positive[M+H]+62222
671.3196.164122
Sterigmatocystin325310.1Positive[M+H]+1213516
325281.61214114
Nivalenol371.1281.1Negative[M + CH3COO]-−75−22−15
371.159.1−75−42−7
Beauvericin801.3784.3Positive[M + NH4]+1412714
801.3244.11414312
Diacetoxyscirpenol384.2307.2Positive[M+H]+81179
384.2105.181617

2.5. Method performance

The developed LC-MS/MS method was validated for specificity, limit of quantification (LOQ), linearity, recovery, and matrix effect (SSE) using the ILD. The linearity of the assay was evaluated by spiking ILD with eight different levels of EMs, ranging from 0.5 to 500 ng/mL. For the determination of LOQ, ILD spiked with EMs at a concentration of 10 ng/mL was serially diluted by up to tenfold. The LOQ was recorded as the lowest concentration at which each EM could be quantified in ILD. Matrix-induced ion suppression/enhancement (SSE) was determined by comparing the response of ILD samples spiked with eight different concentrations of EMs with pure solvent standards at similar concentrations. SSE was calculated as the ratio of calibration curve slope for matrix-matched standards and pure solvent standards multiplied by 100. The extraction recovery of each EM from ILD was calculated as the ratio between toxin concentration after extraction and spiked concentration before sample extraction, expressed as a percentage.

2.6. Quantification of mycotoxins and statistical analysis

The percentage adsorption or binding of each EM by each commercial product was calculated as follows: Adsorption (%) = (CEM – CSPT)/CEM × 100; where CEM is the mycotoxin concentration in positive control with no binder (ng/mL); and CSPT, the amount of mycotoxin in the supernatant of sample (ng/mL). The means of treatments showing significant differences in one and two-way ANOVA were compared using Tukey's honestly significance difference multiple-comparisons post-test. All statements of significance are based on the 0.05 level of probability.

3. Results and discussion

Exposure of livestock to a wide range of types and concentrations of mycotoxins can result in various adverse health effects, with the symptoms and their severity depending on the type and level of mycotoxin, interactions with other mycotoxins, the exposure time and the age, sex, and health status of the animal species [11,20]. The contamination of feed with mycotoxins can increase production costs due to increased veterinary care costs and reduced livestock productivity. Feed mycotoxin contamination can also increase environmental emissions due to wastage when animals refuse feed, meaning that additional feed must be produced to maintain livestock energy requirements [11].

A variety of mycotoxin decontamination methods including thermal processing, chemical treatment, irradiation, and supplementation of feed with additives have been developed and widely used by farmers and feed manufacturers [20,21]. However, the supplementation of feed with mycotoxin binders or feed additives is the most prevalent strategy for reducing livestock exposure to mycotoxins, primarily for economic reasons [22,23]. Several mycotoxin binders are available on the global market for farmers and animal nutrition companies to mitigate against regulated mycotoxins (AFs, OTs, FBs, ZEN, T-2/HT-2, and DON) [22,23]. However, there are currently no strategies available to reduce livestock exposure to EMs. This is the first study to examine the potential of commercially available products to minimize livestock exposure to EMs.

3.1. In vitro gastrointestinal model

Animal feeding trials are the most accurate approach for determining the efficacy of a wide range of products to mitigate against mycotoxin-induced toxic effects in farm animals [22]. Feeding trials, however, are very expensive and time-consuming. Therefore, a properly designed gastrointestinal model simulating the GIT conditions of the target animal specie is a very useful tool for the rapid screening and identification of substances that may have mycotoxin sequestering potential. The in vitro GIT model used for this study was developed to mirror the GIT conditions associated with monogastric animals, including poultry and pigs. As illustrated in Fig. 1, the in vitro model consisted of proventriculus/stomach, duodenum/jejunum, and ileum, with peristaltic valve pumps that allow the movement of controlled amounts of chyme. The model is also equipped with temperature and pH sensors to reflect the body temperature of monogastric species, and the pH associated with various GIT compartment.

Fig. 1
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Fig. 1

3.2. LC-MS/MS assay

As the ILD contains a complex mixture of feed, gastric and intestinal fluids, we also developed and validated a sensitive LC-MS/MS assay to accurately determine trace levels of eight EMD in ILD. The performance characteristics evaluated include linearity, LOQ, matrix effect, recovery. As shown in Table 3, the coefficients of determination (R2) for selected mycotoxins in the matrix ranged from 0.9911 to 0.9996. The retention times of the analyte in the sample extract were checked to correspond to that of the calibration standards and were within a tolerance of ±0.1 min. Also, the ion ratios were within 20% for all the target analytes. The recoveries obtained for all the emerging toxins were acceptable and ranged from 86% to 98%. LOQ values were between 2 μg/kg and 10 μg/kg, indicating the assay is very sensitive considering the complexity of our matrix (ileal digesta). The performance of the LC-MS/MS assay is presented in Table 3. Fig. 2 shows the chromatograms obtained for all the eight EMs in a spiked ILD matrix.

Table 3. Method performance characteristics determined for emerging mycotoxins, including retention time, recovery, repeatability, linearity, limit of detection (LOD) and limit of quantification (LOQ).

MycotoxinsRetention timeExtraction recovery (%)RSD (%)Linearity (R2)LOQ (ng/mL)
Enniatin A10.09820.99742
Enniatin A19.99750.99442
Enniatin B9.59530.99912
Enniatin B19.79740.99932
Sterigmatocystin8.39230.99925
Nivalenol2.28660.994310
Beauvericin9.89040.99965
Diacetoxyscirpenol7.48780.991110

RSD: relative standard deviation.

Fig. 2
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Fig. 2

3.3. Efficacy of commercial binders on emerging mycotoxins

All the ten commercial mycotoxin binders evaluated were found to simultaneously reduce the levels of ENNs (A, A1, B, and B1), BEA, and STG under the in vitro conditions. Their binding capacities on ENNs, BEA, and STG ranged from 79% to 85% (Fig. 3). Only product 8 (aluminosilicates) had a poor binding capacity on BEA (13%). For DAS, products 2, 3 and 5 were the only commercial products found to have a significant binding/detoxifying activity on DAS, with an adsorption rate of 83%, 44% and 46%, respectively (p < 0.05). The remaining products (1, 4, 6, 7, 8, 9) had little to no effect on the level of DAS (<20%) (p > 0.05). With regard to NIV, all the products evaluated had a very poor activity on NIV, with the exception of product 2 (30%).

Fig. 3
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Fig. 3

The majority of commercially available mycotoxin binders are derived from clay minerals and yeast extracts [22]. These substances prevent the absorption of mycotoxins from the GIT of farm animals by interacting and adsorbing toxins to their surface to form a mycotoxin-binder complex [22,24]. The bound mycotoxins are later excreted along with the binder/product in animal faeces. In this study, 50% of the commercial products evaluated are clay-based products, while the remaining 50% are derived from yeast cells (Table 1).

Certain clay minerals possess negatively charged surface area and high cation exchange capacity (CEC), which enable them to adsorb mycotoxins to their interlayer spaces. The mechanisms of action for binding mycotoxins include chemisorption and ion exchange [24]. The physicochemical properties of these clay minerals including their CEC, particle size, charge, expandability, and pH differ significantly, and largely dependent on the geographical origin [25,26]. Thus, even clay minerals from the same origin can have different mycotoxin-binding capacity.

The cell wall of yeast (Saccharomyces cerevisiae) is composed of lipid, protein, and polysaccharides, with glucans and mannans being the two main constituents of the polysaccharide fraction. The mechanism of mycotoxin adsorption is mainly through hydrogen bonding and ionic or hydrophobic interactions [27,28]. Many researchers have shown that β-D-glucans are the principal yeast cell wall component responsible for binding mycotoxins [28,29]. Furthermore, the reticular organisation of the β-D-glucans and the distribution between β-(1,6)-D-glucans and β-(1,3)-D-glucans play vital roles in determining their capacity to adsorb mycotoxins [28,29]. The yeast strain, growth conditions, pH and the availability of binding sites influence the mycotoxin-binding efficiency of yeast extracts.

In this study, commercial products derived from yeast cell walls (1, 3, 4, 5 and 10) and clay minerals (2, 6, 7 and 9) have been found to simultaneously reduce the levels of EMs (ENN A, A1, B & B1, STG, BEA, and DAS) commonly detected in animal feed worldwide, excluding NIV. Due to the commercial nature of the tested products, we could not accurately determine their mode of action (i.e., adsorption or detoxification). However, based on the physicochemical properties of the selected EMs and the observed reduction in EMs levels, we theorize that the mode of action of the commercial binders is mainly through adsorption mechanism.

ENNs (A, A1, B and B1) are structurally related cyclic hexadepsipeptide mycotoxins, while BEA is also a cyclic hexadepsipeptide compound consisting of alternating D-hydroxy-isovaleryl-(2- hydroxy-3-methylbutanoic acid) and N-methylphenylalanine moieties (Fig. 4). STG is a precursor of AFs with a bisfuran structure. The carbonyl groups, oxygen atoms of the carbonyl groups and the tertiary amino nitrogen of the amide bonds of these compounds (ENNs, BEA and STG) contain free electron pairs that can interact with cationic compounds such as clay minerals through ion-dipole interaction and hydrogen bonding. These binding mechanisms have already been established for adsorbents with strong affinity for relatively polar compounds [6,22,24], and may explain the high adsorption rates obtained for ENNs, BEA and STG when compared with DAS and NIV, which are less polar (Fig. 4). One of the major drawbacks of many commercial mycotoxin binders is the capacity to only adsorb polar compounds due to the hydrophilicity of the products; they have little to no binding effect on weak polar and hydrophobic mycotoxins [6,22,23].

Fig. 4
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Fig. 4

The in vitro approach developed and used in the present study can rapidly identify potential products for mycotoxin mitigation and can therefore be used as tool in the selection of promising candidate products, leading to a huge reduction in the number of animals used for experiments. However, the in vitro system does not entirely reflect in vivo conditions. For instance, the impact of endogenous enzyme secretions and the role of gut microbiota were not considered. Therefore, in vivo experiments must be carried in various farm animal species to validate the in vitro data before claiming or concluding about the efficacy of a product.

3.4. Perspectives and conclusion

The term “emerging mycotoxins” is generally used to depict a group of mycotoxins that are “neither routinely determined nor legislatively regulated”. However, the term is becoming obsolete for certain toxins such as MON, PAT, ENNs, NIV, and DAS, as their levels are often determined along with regulated mycotoxins in many mycotoxin monitoring programmes. Moreover, few countries including Canada, and Israel have established regulatory guidance levels for DAS in animal feeds.

Over the past few years, a large number of surveys have reported significant increases in the levels of EMs and high co-occurrence with regulated mycotoxins in agricultural commodities. Whilst EMs have been demonstrated to only induce deleterious health effects at extremely high concentrations, recent data suggest when they co-occur with regulated mycotoxins at low to moderate doses, they can interact and cause synergistic or additive effects. Thus, there is a need for (1) cumulative risk assessments for emerging and regulated mycotoxins at low exposure levels, and (2) the development of innovative products that are low cost and environmentally friendly with broad-spectrum mycotoxin-binding activity.

In this study, we developed a sensitive LC-MS/MS assay and robust in vitro model relative to the GIT of a monogastric animal in terms of compartment, feed, gastric and intestinal fluids, temperature, pH, and transit time. The LC-MS/MS and in vitro GIT model were used to determine the capacity of commercially available mycotoxin binders to reduce the bioavailability of 8 EMs in ileal digesta – ENNs, DAS, BEA, STG and NIV. Out of the 10 commercial products evaluated, only one product (a mixed silicate) was able to significantly reduce the levels of all the EMs (p < 0.05). Further studies are needed to investigate the performance of commercial mycotoxin binders in the presence of both emerging and regulated mycotoxins.

Funding

This work was supported by Bualuang Chair Professor Fund (contract number TUBC 08/2022) and Thailand Science Research and Innovation Fundamental Fund fiscal year 2023 (Project no. 4182267).

Declaration of competing interest

All the authors have approved the content and submission of this manuscript, with no conflict of interest.

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