Understanding Mycotoxin Mitigation and Developing New Strategies for Risk Remediation through the Use of Yeast Cell Wall Extract (Mycosorb®)

An increasing amount of work is focusing on integrated agricultural strategies to provide control measures to ensure that food, feed and the environment are mycotoxin-free.However, the mycotoxins represent an unavoidable risk that necessitate analytical tools for their determination as a selective “surveillance radar” to the removal of the material that is at risk. To provide an adequate protective method or solution for mycotoxin mitigation, the correct determination of their presence is needed thereby calling for the improvement of sampling methods and mycotoxin measuring. The principal challenge to the development of analytical procedures is the important diversity of those molecules, around 500 known today, each of them exhibiting differences in their toxicity and economical implication. Furthermore, the contamination is greatly dependent on the environmental context that may offer the adequate conditions to the growth of moulds and to the triggering of mycotoxin synthesis. Then, suitable techniques need to be implemented to allow the determination of those contaminants from multiple matrices that can inherently impact the extractability of the toxins, as of the referenced “masked” mycotoxins.

Mycotoxin, biological fate and overall detection

Because of the detrimental  impact of mycotoxins, the level of some of them have been strictly regulated in food and feed samples, in the range μg/kg (ppb) to mg/kg (ppm) range for the majority of them. The very high chemical diversity of the fungal metabolites can result in a multitude of chemical structures that can be expected from an extract thus rendering their search difficult and prone to errors. In this context, sampling still represents the majority of the issue related to the correct evaluation of the mycotoxin content of a feed commodity distributed to an animal since these contaminations are often occurring in very specific areas, often defined as “hot spots” of contamination, where the specific environmental and stress conditions (temperature, moisture, aw, pH…), invading and colonizing fungal species and the triggering of the mycotoxinogenic metabolic pathways will be reunited and conductive to the production of mycotoxins. This, added to the wide variety of fungi and vegetal matrices, truly represents the limitation of the implementation of corrective measures; critical control points that will help mitigate the level of crop contamination at the field level, or during storage and processing; the further mycotoxin production and the spoilage of feed and food commodities by toxinogenic strains of mould susceptible to synthesize mycotoxins. If the only observation for visible signs of fungal contamination or fungal spore count as first evaluation tool for feed and food poorly correlates with the mycotoxin presence, chemotaxonomy can be used as a first approach in high throughput screenings to assess the identity and metabolites and through the knowledge of mold species identity. However the complexity of this approach is challenging, especially because of the inherent variation of mycotoxin synthesis pattern in the environment. More complex techniques can be used as an indirect screening method for the recognition of toxigenic vs. atoxigenic strains of fungi by detecting specific volatile metabolites and generally through the use of gas-chromatography coupled to mass spectrometry detection or the development more recently of an electronic nose. But of major importance, the correct evaluation of the mycotoxin level is strictly linked to the selection of representative samples that is achieved only by the increase of the sample size and number. It has been estimated that a correct sample size could be between 1.25 to 10.0 kg according to the feed matrix, 50 to 100 samples and subsequently homogenized sub-sample of 62.5 to 500 g. The sample size should be homogeneous between samples with variation in weight of not more than ± 5% (Whitaker, 2003).

No matter what, a portion of the good lots will be eventually rejected by the sampling plan and conversely a portion of bad lots will be accepted by the sampling plan (seller's/buyer's risk or false negatives). The magnitude of these risks, but in a lesser extent, is also directly related to the magnitude of the variability associated with the mycotoxin test procedure. This approach is even complicated because of the differences in regulation among trading countries. The errors associated with sampling can be as high as 80%,sub-sampling10% and error during sample preparation analysis less than 10%.

Focusing on multi-residues of mycotoxin

In order to have a better grasp on the dual issue related to the analytical challenges of detecting mycotoxins and the multi-contamination issue, our Research Department has diligently worked on a method using hyphenated techniques of mass spectrometry that could provide accurate quantification for up to 48 mycotoxins at once in raw material commonly used in animal nutrition such as cereal grains (corn, wheat, barley, oat…); processed cereal by-products(Distiller Dry Grains with solubles); complex feed matrices such as silages and finally complete feed or total mixed rations. This analytical approach also took into consideration the cost effectiveness of the methodology used, depending on the analytical instrumentation and the use of external standards of mycotoxins as well as stable isotopes as internal standards (Jackson et al., 2012; Jackson and Yiannikouris, 2012). Measurement of mycotoxins using mass spectrometry techniques represents a real analytical challenge given their diverse extraction efficiencies and signal suppression/enhancement properties in matrixes encountered in the feed and food industry. These differences are largely due to the structural diversity of mycotoxins. Currently, methods have been developed to normalize LC-MS/MS data by comparing the instrument response to an isotopically enriched version (isotopologue) of a particular toxin (Cramer et al., 2007; Haubl et al., 2006). The method presented herein sets the basis for a novel approach using non-analogous mycotoxins to normalize classes of mycotoxins which have similar extractability and which experience similar matrix suppression/enhancement effect to the isotopologue during analysis. The method developped uses four isotopologues to normalize the MS signal of known concentrations of 10 mycotoxins from a certified feed material by serving as surrogates for their extraction, detection, and quantification by Ultra-Pressure Liquid Chromatography coupled to ElectroSpray Ionization tandem Mass Spectrometry(UPLC-ESI-MS/MS). To our knowledge, our work is the first example that uses isotopically labeled internal standards to normalizenon-analogous mycotoxins in LC- MS/MS for quantification purposes. The complexity of mycotoxin detection can be easily understood because of the numerous approaches that were used more or less effectively to analyze for their presence, acknowledged by the number of techniques available. The pitfalls in their use and application are also broad due to the inherent properties of mycotoxins, their chemical diversity, and their metabolism into other molecular species. The complexity of the interaction with matrices represents another limitation. In this respect mycotoxin can be found as conjugated metabolite to one or several glycosides or glucuronides residues, masking the mycotoxin from being detected as such even using the referenced analytical procedure. Constant evolution of the analytical devices and the extraction procedures will help mitigate the errors as well as better understand, predict and spot the mycotoxin occurrence in field situation. The importance of developing accurate methods for mycotoxin quantification is part of our actual research prerogative in order to develop appropriate ways of controlling this inherent exposure. This precise analytical determination of mycotoxins in feed samples usingLC-MS/MS clearly demonstrated the impact of larger mycotoxin spectrum than believed, which naturally contaminates feed commodities. This better insight to the global contamination also shows that a more holistic approach is necessary to tackle unsuspectedmulti-contamination pattern that can be revealed only when appropriate testing with appropriate methodology is performed.

Survey and Risk Assessment

Using this technological advancement, a survey and interpretation of the analysis done in 2012 on 889 samples were performed. Conclusion to this study established clearly the prevalence of measurable levels of mycotoxins in 100% of the sample analyzed. In average, 8 mycotoxins were present at measurable level in every sample analyzed, with a span of 2 to 22 mycotoxins found per sample, and with close to 80% of the samples being contaminated by 3 to 11 mycotoxins present concomitantly. Overall, 99.9% of the samples analyzed were contaminated with measurable levels of mycotoxins from the 48 analyte investigated. Fumonisins were the most represented population, which could also be attributed to a higher visibility since often involving ppm concentrations compared to ppb concentration for most of the other toxins, followed by combined trichothecenes and interestingly by Penicillium toxins. This result was the first in its kind. Finally, the threshold of toxin in the samples were evaluated showing that trichothecenes of the B group could contaminate 50% samples atchronic-high levels (close to 1000 ppb) and that more than 20% of the samples were contaminated at more than 1000 ppb.

Mitigation Strategies

Early recognition that no technology existed to totally eliminate mycotoxins from feed ingredients started the development of scientific projects for practical applied solution, such as the evaluation of potential adsorbents that could help mitigate mycotoxin bioavailability in animal feeding strategies. Considering this, adsorption of mycotoxins prior to their entry into the blood circulation of animals represents the major practical and effective technique to combat mycotoxins. If clays (at large) have been characterized for their affinity to aflatoxin B1 (AFB1) when added at appropriate levels to the contaminated animal feed, they failed to demonstrate good binding to other mycotoxins at practical levels of inclusion. Thus, the search shifted toward finding an organic compound capable of efficient adsorption of a wider spectrum of toxins that coexist in mixed feeds. A low inclusion rate and biodegradability were also critical in order to maximize formulation space for nutritional use and to minimize the environmental burden. In the past 20 years, research emphasized on the use of modified Saccharomyces cerevisiae cell wall extract in mycotoxin detoxification strategies. Previous in vitro/in silico research has shown that the carbohydrate fraction of glucan polymers in the yeast cell wall was responsible for the sequestration efficacy toward aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol, and patulin (Yiannikouris et al., 2004a; Yiannikouris et al., 2006; Yiannikouris et al., 2004b, c; Yiannikouris et al., 2004d; Yiannikouris et al., 2003). The interaction was promoted further when higher glucan levels were present in yeast cell wall in combination with a complex dynamic structure organization made of triple helix and fibers. In parallel, in vivo investigations focused on the organic adsorbent strategy applied to artificially contaminated feed ingredients in numerous animal species, studying performance, hematology, metabolism, and immunological parameters… Data has shown that the product was able to mitigate or prevent the adverse effect in acute or chronic cases of mycotoxicosis. In recent years, a broad range of specific, adverse mycotoxin effects have been investigated based on the measurement of biomarkers of immune cell modulation, reproductive performance, oxidative stress, and blood parameters. In avians, biomarkers affected by mycotoxin challenge and partially but significantly restored by the addition of Saccharomyces cerevisiae cell wall extracts follow: Urea nitrogen, antibody titers, immune cells modulation, serum proteins and serum urea, organ weights, histopathology when natural Fusarium toxins in combination with AFB1, and in some cases FB1, were administered to the animal (Aravind et al., 2003; Chowdhury and Smith, 2004; Chowdhury et al., 2005a, b; Girgis et al., 2010a; Girgis et al., 2010b; Girgis et al., 2008; Girish and Devegowda, 2006; Girish et al., 2008a; Girish and Smith, 2008; Girish et al., 2010; Girish et al., 2008b; Karaman et al., 2005; Mogadem and Azizpour, 2011; Raju and Devegowda, 2000; Raju and Devegowda, 2002; Reddy et al., 2004; Swamy et al., 2002a; Swamy et al., 2004a; Swamy et al., 2004b; Swamy et al., 2002c; Wang et al., 2006); antioxidants (Aravind et al., 2003; Chowdhury and Smith, 2004; Chowdhury et al., 2005a, b; Girgis et al., 2010a; Girgis et al., 2010b; Girgis et al., 2008; Girish and Devegowda, 2006; Girish et al., 2008a; Girish and Smith, 2008; Girish et al., 2010; Girish et al., 2008b; Karaman et al., 2005; Mogadem and Azizpour, 2011; Raju and Devegowda, 2000; Raju and Devegowda, 2002; Reddy et al., 2004; Swamy et al., 2002a; Swamy et al., 2004a; Swamy et al., 2004b; Swamy et al., 2002c; Wang et al., 2006);the antioxidant status of egg yolk, Se-GSH-Px activity in liver, and selenium status after the consumption of T2 or aurofusarin (Dvorska et al., 2007; Dvorska and Surai, 2001; Dvorska and Surai, 2004; Dvorska et al., 2003). In ruminants, the biomarkers influenced by the addition of Saccharomyces cerevisiae cell wall extracts following a mycotoxin challenge implicated: AFM1 excretion in milk after AFB1 consumption (Diaz et al., 2004; Firmin et al., 2011; Moran et al., 2013); biochemical, proteomic and genomic plasma markers in response to natural contamination with DON, mycophenolic acid and other Penicillium toxins, and ZEA in feed; increase in prolactin after the ingestion of ergot alkaloids (Merrill et al., 2007). In pigs restored biomarkers involved antibody titers, immune cells modulation, serum protein and serum urea when natural Fusarium toxins in combination were administered to the animal (Diaz-Llano and Smith, 2007, 2006; Diaz- Llano et al., 2010; Swamy et al., 2004b; Swamy et al., 2002b; Swamy et al., 2003). Finally, in rodents, a full absorption, desorption, metabolization and excretion study showed a reduction in the absorption and an increase in the fecal excretion of AFB1, and to a lesser extend OTA, when Saccharomyces cerevisiae cell wall extracts was administered in the diet (Firmin et al., 2010).

Mycotoxin Multi-Residues Adsorption with Mycosorb® and Risk Remediation

Since it is clear that mycotoxins are always co-occurringin feed given the previous results obtained on the distribution of mycotoxins in animal feed samples, it is essential to adapt strategically the use of mitigation strategy to accommodate this broader spectrum challenge and investigate the use of a broader spectrum solution such as the use of adsorbent of greater spectrum of activity. It is thus worthwhile to also move from single mycotoxin configuration for adsorption evaluation to more complex systems that, even in buffer condition, truly account for the complexity of the natural mycotoxin profile and thus for the efficacy of an adsorbent more realistically. Of course, increasing the number of mycotoxins present in an experimental environment means a fortiori a dramatic increase in the possibilities in terms of mycotoxin distribution patterns. In this context, a novel approach has been investigated as part of our research strategy. The concept relies on the advancements that were made analytically in terms of bundled detection of mycotoxins and the characterization of an adsorption efficacy through in vitro adsorption experiments. Mycotoxin concentrations were chosen according to their regulatory limits and guideline values proposed by the European Commission (Regulation, 2006) and expert report committee (French Food Safety Agency, Risk assessment for mycotoxins in human and animal food chains, December 2006). The levels used in this study could be subjected to change but represent a challenging mycotoxin distribution but still complying with the regulatory limits and guidelines or practical limits. Also, from the results accumulated in 2012 using the analysis, it clearly appears that in average and based on the analysis of around 1000 feed samples, 8 mycotoxin are contaminating in average a given sample at measurable levels. The concentrations chosen are also all above the limit of quantitation and limit of detection with asignal-to-noise value above 10. These concentrations selected for each individual mycotoxin arbitrarily matched the threshold corresponding to the limit between chronic and acute intoxication and also for the ones subjected to regulation, the regulatory limit of acceptance of mycotoxin in feed. A holistic approach was investigating by operating the adsorption evaluation on a pattern of 14 different mycotoxins in buffer conditions using the common adsorption test procedures. The sorbent was then tested for its efficiency in mycotoxin adsorption and was allowed to interact with the toxin for 90 min at 37°C under orbital agitation (150 rpm) in ammonium citrate buffer pH 4.0. A control sample was used that was made out of the mixture of mycotoxins only and that was submitted to the same experimental conditions. After incubation, the samples were centrifuged at 10,000 rpm for 15 min and the supernatants were collected. A volume of 400 µL of supernatant was collected in silanized amber glass autosampler vials. Samples were further diluted 1:10 with loading buffer before injection. The amount of toxin adsorbed is evaluated according to the concentration of toxins remaining in the supernatant. The mean adsorption for individual mycotoxin with each formula was calculated and the standard deviation reported. All sorption tests were carried out in three replicates per sample. The following mycotoxins were used: AFB1, ergotamine D-tartrate(ERGOTAM), OTA, FB1, DON, fusaric acid, T2, DAS, ZEA, roquefortine C (ROQC), sterigmatocystin (STERIG), cyclopiazonic acid (CPA). Toxins received as dry powders were reconstituted in acetonitrile. Standard solutions of mycotoxins were used to build calibration curve for every mycotoxin quantified. Evaluation of each the sorbent efficacy was determined according to the interaction between sorbent and the multiple-mycotoxincontaminated mixture prepared. The original method developed for the analysis of 48 mycotoxins was used to analyze the sample by means of UPLC-MS/MS(Jackson et al., 2012; Jackson and Yiannikouris, 2012). Analysis was performed on a Waters AcquityUPLC-Xevo-TQD system (Waters Corp., Milford, MA, USA) utilizing a BEH C18 analytical column (1.7 µm particle size, 2.1 mm × 100 mm, Waters Corp., Milford, MA, USA) maintained at 40.0°C. A binary mobile phase was used consisting of water (eluent A) or methanol containing 0.1% formic acid (eluent B). The flow rate was set to 0.40 mL/min and the total analysis time was 15 min using a gradient elution. Mycotoxin identity confirmation was performed by the monitoring of at least 2 transition ions per analyte. The UPLC system utilized a binary solvent system consisting of Optima grade water and methanol/formic acid mixture. Calibration curves were built individually using 5 concentration levels (8ppb to 5ppm), injected in triplicate into theUPLC-MS/MS system for each mycotoxin analyzed. The peak area of the mycotoxins was plotted against the concentration using a 1/x calibration curve. The adsorption was above 80% for AFB1, FB1, ZEA, ERGOTAM, ROQC, STERIG, above 70% for OTA, CPA and around40-50% for DON, T2, DAS and fusaric acid. The adsorption was efficient enough to get below the regulatory values or the recommended safe levels of contamination.

Conclusion and Perspectives

Research developed during the last 20 years gave a comprehensive insight into the functionality of modified Saccharomyces cerevisiae cell wall extract (Mycosorb®) with indisputable series of data to explain its mode of action of in adsorbing several mycotoxins in vitro and in vivo. These in vitro researches were carried out while respecting several essential physiological parameters modeling the animal organism. The dual in vitro – in vivo facet is essential in acknowledging the efficacy of a product and should be mandatorily implemented as part of the demonstration of product efficacy. The general organization of yeast cell wall composition is governed by the genome of the yeast but also by the production technical characteristics and nutritional strategy used to generate the yeast biomass. This can explain why all yeasts and yeast cell walls can present in nature various composition even for a same strain. Therefore, the careful selection of efficient yeast strains is required. In vivo investigations have focused on the impact of organic adsorbent strategies with artificially contaminated feed ingredients or on feeding blends of grains naturally contaminated with mycotoxins in numerous animal species and studying performance, hematology, metabolism, and immunological parameters. Data has shown that modified Saccharomyces cerevisiae cell wall extracts were able to mitigate or prevent the adverse effect in acute or chronic cases of mycotoxicosis. A further step in our investigations evaluated their sequestration activity toward AFB1 and ZEA ex-vivo. In this respect and in addition to some of the recent results obtained on the toxinokinetics of 3H-labelled mycotoxins in rats after oral administration (Firmin et al., 2010),series of experiment were conducted to evaluate the gastrointestinal uptake of the mycotoxins and the consequent of sequestering activity associated with the product. In vivo experiments with radioactively labeled AFB1 have shown that the product effectively inhibits the absorption of AFB1 from the gastrointestinal tract of rats. As the main region of nutrient adsorption, the small intestine is likely an important site for its uptake. In live animal, absorption as such is difficult to study, as mycotoxin levels in blood plasma or other tissues of animals are always a sum of different processes: absorption, metabolism, and excretion. To inhibit intestinal absorption, an adsorbent should be able to compete for the binding of the toxin even in the presence of gastrointestinal epithelium which may offer a lot of binding sites for the lipophilic mycotoxins. Experiment performed in a Ussing chamber system clearly demonstrated that the product was able to strongly suppress AFB1 transport across the intestinal tissue, respectively -66and -72%(p<0.001 at least) against control. In the case of ZEA, the sequestration efficacy reached 67% whereas barely reaching 30% with HSCAS demonstrating the advantages of the organic sequestrant strategy. Furthermore, accumulation of free toxins within the intestinal tissue was also less pronounced in the case of our adsorbent (Yiannikouris et al., 2013).

The knowledge and understanding of the yeast cell wall, the genetics of the yeast, and how different components within the yeast cell wall can be modified and adapted well enough to positively influence the efficacy of an organic adsorbent, gives to the particular Saccharomyces cerevisiae yeast cell wall extract investigated its unique characteristics toward the binding of several different types of mycotoxins.

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This paper was presented at AMENA Congress in Puerta Vallarta, Mexico, October 2013.