Understanding the adsorption characteristics of yeast cell wall preparations associated with mycotoxin binding

Introduction

Mycotoxins are secondary products of fungal metabolism that may be produced in contaminated feeds during production and storage. These metabolites are generally associated with a group of ubiquitous fungi belonging to the Fusarium, Aspergillus, Penicillium and Claviceps species that grow on forages and grains in the field and during storage (Ledoux and Rottinghaus, 1999). It has been estimated that at least 300 fungal metabolites are potentially toxic for man and animals, and that as much as 25% of the world’s cereal grains are contaminated with measurable levels of mycotoxins (Devegowda et al., 1998). Fungal growth in silages, stored forages and byproducts can result in toxin formation. As a result, mycotoxins can be found in many types of feed materials and are commonly encountered in livestock production systems. As a group, mycotoxins are chemically diverse and have a broad range of physiological activities in animals. These toxins are typically present at low concentrations, even in highly contaminated feeds.

However, many are produced at concentrations known to have significant effects on animal health and may have a major impact on animal production. The negative influence on the growth and health of livestock makes them a major problem in many production systems.

There are a broad array of fungal metabolites that can be toxic (Figure 1). Among these, aflatoxins are the most common in the tropical and subtropical regions where warm environmental temperatures allow for the growth of the Aspergillus species during storage. These toxins can cause liver damage, decrease reproductive performance, cause tumor formation and suppress immune functions.

In contrast, fusarium toxins are more commonly encountered in temperate regions, where temperatures between 45º and 75ºF allow the fungus to infect not only stored feeds, but also the growing corn plants before harvest.


Figure 1. Diverse structure of some common mycotoxins.




The fungi in the Fusarium species produce more than 100 metabolites that are potentially toxic in animals. These include trichothecenes (deoxynivalenol, T-2 toxin, HT-toxin and diacetoxyscirpenol), zearalenone, fumonisins, moniliformin and fusaric acid. It is unlikely that any one of these toxins would be produced individually in great quantities. As a result, toxicosis associated with these fungi is often a complex process involving a number of toxins (Smith et al., 2000). The fusarium toxin, deoxynivalenol (DON), is known for its ability to inhibit protein synthesis and has been shown to incrementally decrease intake in pigs at concentrations above 2 ppm (Newman, 2000). It can also influence reproductive performance and is associated with immunosuppression (Johnson et al., 1997). T-2 and related toxins can cause irritation and hemorrhages in the gastrointestinal tract. In severe cases, T-2 causes oral lesions in poultry and swine. Zearalenone is known for its estrogenic activities and its ability to impair reproductive performance. At low concentrations it influences the development of reproductive organs and causes rectal and vaginal prolapses. Fumonisin is known for its ability to impair immune function and cause kidney and liver damage. As a result, it can have a significant impact on animal performance and the incidence of disease in many species.

Other fungal-associated toxins have been identified in forages. These are often not traditionally classified with the mycotoxins associated with grains and prepared animal feeds, but are the result of synergistic metabolic activities that develop from endophytic fungal infections in forages. These toxins are commonly associated with grazing animals maintained on tall fescue and perennial ryegrass. They also include the ergot alkaloids found in sorghum (Deo et al., 1999).


Controlling mycotoxicoses

A number of approaches have been used to control the adverse effects of mycotoxins in animal production systems. Many of the strategies that prevent the formation of mycotoxins in feeds have merit and provide an initial line of defense against toxin-induced health problems. The objective of these strategies is to prevent the growth of fungi and the formation of toxin by altering feed management practices. Practices that keep feed moisture levels low or that prevent damage of grains during processing can be effective in decreasing the prevalence of mycotoxin-producing fungi in the feeds, but may not always eliminate toxin formation.

Strategies that use microbial or thermal inactivation of toxins, physical separation of contaminated feedstuffs, irradiation, ammoniation and ozone degradation have all been examined as tools for destroying or modifying mycotoxins (CAST, 1989; McKenzie et al., 1998). However, these strategies tend to be costly or time consuming and have not been found to be of practical use. A number of strategies are based on nutritional manipulation that can help overcome the toxic effects of ingested toxins. These strategies are based on the fact that many subtle effects of mycotoxins can be overcome by maintaining animals in a healthy, disease-free environment.

The use of antioxidants to control tissue damage associated with mycotoxins has been investigated by a number of research groups (Lin et al., 1994; Atroshi et al., 1995; Grosse et al., 1997; Ibeh and Saxena, 1998). It is clear from these studies that antioxidants can act as mycotoxin antagonists and help maintain the tissue integrity of animals exposed to certain types of mycotoxins. Other nutritional approaches have used specific phenolic compounds to detoxify mycotoxins (Aboobaker et al., 1994); and compounds like aspartame (Baudrimont et al., 1997), piperine (Reen et al., 1997), coumarin (Goeger et al., 1998), chlorophyll derivatives (Dashwood et al., 1998) and serotonin antagonists (Prelusky et al., 1997) have been used as chemoprotectants against the negative effects of mycotoxins. However,
most studies of these potential strategies have been performed in basic biochemical test systems, and it is not clear how the protective effects of such strategies can be used to define nutritional approaches for control of mycotoxin-associated toxicosis. As a result, such strategies have not become widespread practical applications in animal production systems.

Another approach for attenuating the effects of mycotoxins is based on the use of specific materials that adsorb mycotoxins in animal feeds. These have become some of the most practical methods for controlling mycotoxins in feeds. They are based on the ability of the adsorbents to ‘tie up’ or ‘bind’ the toxins. This allows the toxins to pass through an animal’s digestive tract without being absorbed. Both inorganic and biological adsorbents have been examined and used to control the bioavailability of mycotoxins. Inorganic clay-based adsorbents and activated charcoal have been shown to adsorb specific mycotoxins and are attractive as feed supplements because they are relatively inert from a nutritional standpoint. These include hydrated sodium calcium aluminosilicates (HSCAS), zeolites, bentonites, specific clays and activated charcoals prepared from different sources (Piva et al., 1995). For the most part, these strategies are known for their ability to bind aflatoxins, but adsorption to other types of mycotoxins has also been examined (Table 1).


Table 1. Some inert adsorbent and binding compounds used for decreasing the bioavailability of mycotoxins in animal feeds.




There has been a great deal of interest in using natural biological products to reduce the bioavailability of mycotoxins in animal production systems. One available strategy for attenuating the effects of some groups of mycotoxins uses the unique adsorptive capacity of the carbohydrate complexes in the yeast cell wall. The potential usefulness of these types of materials was first demonstrated in poultry in the early 1990s. Initially used as a nutritional aid and a growth promoter, a commercially available viable yeast culture preparation based on Saccharomyces cerevisiae strain 1026 (Yea-Sacc), was found to improve hatchability (McDaniel, 1991) and broiler body weights (Stanley et al., 1993). Investigators attributed the yeast culture preparation’s ability to alter growth patterns of poultry to its ability to bind toxins found in the diets used in these studies. In controlled studies, viable yeast cultures added to broiler diets containing aflatoxin resulted in significant improvement in weight gain and enhanced immune response (Devegowda et al., 1995). Additionally, in vitro studies clearly established up to 90% adsorption of aflatoxin to yeast cells in a dose-dependent fashion (Devegowda et al., 1994). This work was the basis for a set of new strategies that use yeast-derived products to overcome problems associated with mycotoxin contaminated feeds.

Studies with whole yeast cells have led to a closer evaluation of specific components in yeast cultures and their ability to bind mycotoxins. Modification in manufacturing techniques have allowed for the production of a specific modified yeast cell wall preparation with the ability to bind a range of mycotoxins (Table 2). The yeast cell wall-derived glucomannan product, Mycosorb, has also been repeatedly shown to reduce the toxic effects of mycotoxin-contaminated grains in poultry (Smith et al., 2000), leaving little doubt about the nature of the active components. Data from these and similar studies suggest that organic adsorbents prepared from the yeast cell wall of specific strains of S. cerevisiae may play a critical role in strategies for controlling the toxicity of mycotoxins in poultry feeds.


Table 2. Demonstrated relative efficiency of adsorption of Mycosorb for several mycotoxins measured in an in vitro binding assay.




In many respects, the organic adsorbent derived from yeast overcomes the inherent drawbacks associated with using some of the inorganic adsorbents that have traditionally been used to address intoxication problems. Claybased adsorbents are typically used at high concentrations (>1.0% of the diet) in animal feeds. This can decrease the nutrient density of the diet and provide an excess adsorbent capacity that may decrease the bioavailability of important micronutrients. In contrast, the high adsorptive capacity of the yeast cell wall-derived binders make it possible for them to provide beneficial activities at much lower concentrations (<0.1% of the diet). Since these preparations are designed to adsorb to mycotoxins at low concentrations, their inclusion would have minimal effect on the nutrient density of the diets. In addition, these adsorbents would not be expected to influence the concentrations of micronutrients that are typically present in the diet at concentrations greater than those of contaminating mycotoxins.


In vitro measures of mycotoxin binding

In order to understand the best way to use specific mycotoxin adsorbents, it is important to understand the basic mechanisms of interactions with specific toxins. While in vitro adsorption assays may not always be indicative of the in vivo responses to specific mycotoxins, these types of laboratory studies can be used to define approximate dose requirements for an adsorbent and define a strategic mechanism for using a particular adsorbent in an animal system.

A number of techniques have been used to measure in vitro mycotoxin binding or adsorption. The simplest of these tests measure adsorption of purified toxin preparations in an aqueous medium. In these systems, a known amount of mycotoxin is reacted with a known amount of adsorbent in water. The amount of toxin remaining in the liquid after separation of the mycotoxinbinder complex is then quantitatively determined and the amount of adsorbed toxin is estimated by difference. Because of the relative insolubility of the mycotoxins, these tests are generally run at very low mycotoxin concentrations. The ability of the assay system to accurately measure adsorption is limited by the detection limits of the mycotoxin assay system. The increased sensitivity of high performance liquid chromatographic techniques has made routine evaluation of many mycotoxin adsorbents more practical for many laboratories. These types of assay systems have been used to quantitatively study the binding of adsorbents to aflatoxins (Ledoux and Rottinghaus, 1999).

Other test systems used to evaluate mycotoxin binding are based on measuring adsorption in a two-step process. These systems attempt to measure the strength of the mycotoxin binding by initially measuring the quantity of toxin bound in an aqueous system, and then measuring the desorption of the mycotoxin after exposure to a second solvent system. The adsorption efficiency compares the initial binding (weak binding) with the binding after desorption (strong binding). Such tests for strong mycotoxin binding have been used to measure the relative efficacy of the yeast cell wall-derived glucomannan (Table 2). As might be expected from the diverse structural differences in mycotoxins, the yeast cell wall material displays variations in adsorption efficiencies, depending on the mycotoxin. While such tests are useful in demonstrating in vitro binding, they do not provide much information for comparing different types of adsorbents or an understanding of how a specific adsorbent can be used in a practical feed management program. Since an ideal adsorbent would be most effective at the relatively low mycotoxin levels found in the gastrointestinal tract, these in vitro tests at relatively high mycotoxin concentrations give a poor representation of the adsorbent’s true usefulness.

Considerable information on the adsorption of mycotoxins to specific binders can be obtained by using adsorption isotherms (Ramos and Hernandez, 1996; Grant and Phillips, 1998). These systems recognize that mycotoxin adsorption in biological systems is a reversible process that can be characterized as a chemical equilibrium. As a result, adsorption is a concentration-dependent process influenced by mycotoxin concentration, the amount of adsorbent, and the relative affinity of the adsorbent for the mycotoxin. Adsorption isotherms plot the amount of mycotoxin adsorbed per unit weight of adsorbent versus the mycotoxin concentration in solution at equilibrium and constant temperature (Figure 2). These tests can be used to compare the relative binding capacity and affinity of adsorbents. They clearly can be used to demonstrate differences in clay-based adsorbents and those derived from yeast cell wall preparations.


Figure 2. Isothermal adsorption of aflatoxin B1 with a yeast cell wall-derived adsorbent (Mycosorb) and a clay-based adsorbent at 37°C in an aqueous solution.




Grant and Phillips (1998) were able to use multiple isotherm equations to determine not only full capacity and affinity, but also average capacity, enthalpy of binding, heterogeneity coefficient, multiple site distribution coefficients, and multi-site capacity of HSCAS for aflatoxin B1. Similarly, we have used these techniques to compare the adsorption of aflatoxin B1 to the yeast cell wall preparation, Mycosorb, and to a clay-based adsorbent (Figure 2). In these studies, major differences were noted in the overall adsorption capacity of the two commercial mycotoxin clay-based adsorbents and that of Mycosorb at high mycotoxin concentrations. A measure of the efficiency of the mycotoxin binder over a wide range of specific mycotoxin concentrations can also be used to describe and compare the role of specific adsorbents at low mycotoxin concentrations. Mycosorb was a more effective adsorbent at low mycotoxin concentrations than were either of the clay-based binders tested (Figure 3). This reflects the higher affinity of Mycosorb for aflatoxin and suggests that this material would be more effective at the low mycotoxin concentrations found in the gastrointestinal tract. The yeast cell wall-derived adsorbent also tended to bind a greater proportion of mycotoxin at relatively high mycotoxin concentrations. This suggests that these types of adsorbents have a greater overall capacity to bind the mycotoxin and can be used at lower inclusion rates. The combination of high affinity for aflatoxins and lower inclusion rates makes Mycosorb an attractive supplement for controlling aflatoxicosis. These observations are consistent with the efficacy of the product in controlling aflatoxin uptake in dairy cattle (Whitlow et al., 2000).


Figure 3. Efficiency of aflatoxin B1 adsorption to three different binding agents.




Characteristics of aflatoxin binding

In vitro studies have also helped to characterize the interactions between the yeast cell wall-derived adsorbents and specific mycotoxins. Serial elution of the aflatoxins adsorbed to the glucomannan clearly demonstrates that the adsorption of the mycotoxin is reversible, and that aflatoxins are not modified during the adsorption process. This is important, since it clearly establishes the basic kinetic mechanisms associated with adsorption and lack of an extensive chemical modification during adsorption. Aflatoxin adsorption is also influenced by the pH (relative acidity) in the aqueous environment. Maximum binding occurs at a pH of approximately 4.0. In addition, adsorption is also influenced by the relative phosphate concentrations in an aqueous environment (Figure 4). Maximum binding was observed in a phosphate buffer that contained 0.5 M phosphate. Both pH and phosphate concentration optima are consistent with those found in the gastrointestinal tract and suggest that the conditions in the gastrointestinal tract would enhance adsorption and not decrease the mycotoxin-adsorbent interactions.

Figure 4. Effects of phosphate concentrations on the binding efficiency of a yeast cell wall-derived glucomannan preparation (Mycosorb).




Improving mycotoxin binding using yeast cell wall derivative Recent studies have examined the effects of chemical modifications of yeast cell wall glucans on the basic adsorption of a wide variety of mycotoxins. Specific chemical modification of the surfaces associated with the basic glucan structure has made it possible to tailor derivatives with activities that are mycotoxin-specific. One such derivative is specific for zearalenone (Figure 5). This particular derivative has a greater capacity to adsorb zearalenone across a broad concentration range and has a greater affinity for zearalenone than the original glucomannan preparation. These chemical modifications can be made without influencing the particulate nature of the adsorbent and allow for the development of mycotoxin-specific activities based on the structure of the toxins. Since the surface area of the modified yeast cell wall is not significantly different from the original yeast cell wall preparations, it appears that adsorption in these systems is not always correlated with the surface area, but is in some cases related to the chemical nature of the adsorbent surface. These studies suggest that the basic glucan structure derived from crude yeast cell wall preparations may provide a basic structural unit for more advanced mycotoxin adsorbents in the future.


Figure 5. Efficiency of zearalenone adsorption with Mycosorb and a yeast cell wallglucan derivative.




Conclusions

Mycotoxins and their associated health problems are common in modern livestock and poultry production systems. Adsorbents that bind mycotoxins and decrease their bioavailability show a great deal of promise as tools for use in strategies that attenuate mycotoxin-induced toxicosis. The high affinity and high adsorption capacity of yeast-derived glucomannan preparations make their use as adjuncts for controlling naturally occurring mycotoxins in feeds attractive.


References

Aboobaker, V.S., A.D. Balgi and R.K. Bhattacharya. 1994. In vitro effect of dietary factors on the molecular action of aflatoxin B1: Role of nonnutrient phenolic compounds on the catalytic activity of liver fractions. In Vivo 8:1095-1098.

Atroshi, T., A. Rizzo, I. Biese, M. Salonen, L.A. Lindberg and H. Saloniemi. 1995. Effects of feeding T-2 toxin and deoxynivalenol on DNA and GSH contents of brain and spleen of rats supplemented with vitamin E and C and selenium combination. J. Anim. Phys. Anim. Nutr. 74:157-164.

Baudrimont, I., A.M. Betbeder and E.E. Creppy. 1997. Reduction of the ochratoxin A-induced cytotoxicity in Vero cells by aspartame. Arch. Toxicol. 71:290-298.

Council for Agricultural Science and Technology (CAST). 1989. Task Force Report #16-Mycotoxins: Economic and Health Risk. Ames, IA.

Dashwood, R., T. Negishi, H. Hayatsu, V. Breinholt, J. Hendricks and G. Bailey. 1998. Chemopreventative properties of chlorophylls toward aflatoxin B1: A review of the antimutagenicity and anticarcinogenicity data in rainbow trout. Mutat. Res. 399:245-514.

Deo, P., B.J. Blaney and J.G. Dingle. 1999. Effects of mineral and organic adsorbents in meat chicken diets contaminated with sorghum ergot alkaloid. Queensland Poult. Sci. Symp. Vol. 8.

Devegowda, G., B.I.R. Aravind, K. Rajendra, M.G. Morton, A. Baburathna and C. Sudarshan. 1994. A biological approach to counteract aflatoxicosis in broiler chickens and ducklings by the use of Saccharomyces cerevisiae cultures added to feed. In: Biotechnology in the Feed Industry, Proceedings of the 10th Annual Symposium (T.P. Lyons and K.A. Jacques, eds). Nottingham University Press, Loughborough, Leics, UK. pp 235-245.

Devegowda, G., B.I.R. Aravind, M.G. Morton and K. Rajendra. 1995. A biotechnological approach to counteract aflatoxicosis in broiler chickens and ducklings by the use of Saccharomyces cerevisiae. Proc. Feed Ingredients Asia ’95, Singapore. pp 161-171.

Devegowda, G., M.V.L.N. Raju and H.V.L.N. Swamy. 1998. Mycotoxins: Novel solutions for their counteraction. Feedstuffs, December 7, 1998; 12:15.

Grant, P.G. and T.D. Phillips. 1998. Isothermal adsorption of aflatoxin B1 on HSCAS clay. J. Agric. Food Chem. 46:599-605.

Goeger, D.E., K.E. Anderson and A.W. Hsie. 1998. Coumarin chemoprotection against aflatoxin B1-induced mutation in a mammalian cell system: A species difference in mutagen activation and protection
with chick and rat liver S9. Environ. Mol. Mutgen. 30:64-74.

Grosse, Y., L. Chekir Ghedira, A. Huc, S. Obrecht Pfumio, G. Dirheimer, H. Bacha and A. Pfohl Leszkowicz. 1997. Retinaol, ascorbic acid and alpha-tocopheroal prevent DNA adduct formation in mice treated with mycotoxins ochratoxin A and zearalenone. Cancer Lett. 114:225-229.

Ibeh, I. N. and D. K. Saxena. 1998. Effect of alpha-tocopherol supplementation on the impact of aflatoxin B1 on the testes of rats. Exp. Toxicol. Pathol. 50:221-224.

Johnson, P.J., S.W. Casteel, and N.T. Messer. 1997. Effect of feeding deoxynivalenol (vomitoxin)-contaminated barley to horses. J. Vet. Diagn. Investigation. 9:219-221.

Ledoux, D.R. and G.E. Rottinghaus. 1999. In vitro and in vivo testing of adsorbents for detoxifying mycotoxins in contaminated feedstuffs. In: Biotechnology in the Feed Industry. Proceedings of the 15th Annual Symposium. (T.P. Lyons and K.A. Jacques, eds), Nottingham University Press, UK. pp 369-379.

Lin, Z.H., S.G. Li, L.Y. Wu, S. Sun and Q.W. Lu. 1994. Antagonistic effect of Se on the T-2 toxin-induced changes in the ultrastructure and mitochondrial function of cultured chicken embryonic chondrocytes. J. Clinical Biochem. Nutr. 17:119-132.

McDaniel, G. 1991. Effect of Yea-Sacc1026 on reproductive performance of broiler breeder males and females. In: Biotechnology in the Feed Industry. Proceedings of the 7th Annual Symposium. Alltech Technical Publications, Nicholasville, Kentucky, USA.

McKenzie, K.S., L.F. Kubena, A.J. Denvir, T.D. Rogers, G.D. Hitchens, R. H. Bailey, R.B. Harvey, S.A. Buckley and T.D. Phillips. 1998. Aflatoxicosis in turkey poults is prevented by treatment of naturally contaminated corn with an ozone generated by electrolysis. Poultry Sci. 77:1094-1102.

Newman, K. 2000. The biochemistry behind esteried glucomannans- tirating mycotoxins out of the diet. In: Biotechnology in the Feed Industry. Proceedings of the 16th Annual Symposium. (T.P. Lyons and K.A. Jacques, eds), Nottingham University Press, UK.. pp 369-382.

Piva, A. and F. Galvano. 1999. Nutritional approaches to reduce the impact of mycotoxins. In: Biotechnology in the Feed Industry. Proceedings of the 15th Annual Symposium. (T.P. Lyons and K.A. Jacques, eds), Nottingham University Press, UK.. pp 381-399.

Piva, G., F. Galvano, A. Pietri and A. Piva. 1995. Detoxification methods of aflatoxins. A review. Nutr. Res. 5:689-715.

Prelusky, D.B., B.A. Rotter, B.K. Thompson and H.L. Trenholm. 1997. Effects of appetite stimulant cyproheptadine on deoxynivalenol-induced reductions in feed consumption and weight gain in the mouse. J. Environ. Sci. and Health. 32:429-448.

Ramos, A.J. and E. Hernandez. 1996. In vitro aflatoxin adsorption by means of a montmorillonite silicate. A study of adsorption isotherms. Animal Feed Sci. Technology 62:263-269.

Reen, R.K., F.J. Wielbel and J. Singh. 1997. Piperine inhibits aflatoxin B1-induced cytotoxicity and genotoxicity in V79 Chinese hamster cells genetically engineered to express rat cytochrome P4503B. J.
Ethnopharmacology 58:165-173.

Smith, T.K., M. Modirsanei and E.J. MacDonald. 2000. The use of binding agents and amino acids supplements for dietary treatment of Fusarium mycotoxicoses. In: Biotechnology in the Feed Industry. Proceedings of the 16th Annual Symposium. (T.P. Lyons and K.A. Jacques, eds), Nottingham University Press, UK. pp 383-390.

Stanley, V.G., R. Ojo, S. Woldesenbet and D.H. Hutchinson. 1993. The use of Saccharomyces cerevisiae to suppress the effects of aflatoxicosis in broiler chicks. Poultry. Sci. 72:1867-1872.

Whitlow, L.W., D.E. Diaz, B.A. Hopkins and W.M. Hagler. 2000. Mycotoxins and milk safety: The potential to block transfer to milk. In: Biotechnology in the Feed Industry. Proceedings of the 16th Annual
Symposium. (T.P. Lyons and K.A. Jacques, eds), Nottingham University Press, UK. pp 391-408.



* KARL A. DAWSON, JEFF EVANS and MANOJ KUDUPOJE
North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA