**6. Aflatoxin binders and strategies to reduce toxicity to farm animals**

Adsorbents are necessary and important and may have great impact on improving animal production and health, providing greater security to consumers of animal products, due to the reduction and/or removal of mycotoxins in these products.

Considering that aflatoxins were the first discovered mycotoxins, there are many data avail‐ able searching for binders and other methods to reduce toxicity in animals. However, due to methodologies used for evaluation, there is certain degree of variation in results.

The most common additives used in animal diets are aluminosilicates, produced syntheti‐ cally or extracted from clay mines. There are also other alternatives to reduce aflatoxin toxic‐ ity, as presented.

#### **a.** *Clay derived sorbents:*

This type of binder is basically composed by single or blended type of clay. The most com‐ mon clay is hydrated sodium calcium aluminosilicate (HSCAS). However, there are other sort of clays which can be used as toxin binders, like sodium or calcium bentonites and zeo‐ lites. Not often, any particular varying sort of clay can be used as well. It has to be consid‐ ered that such materials can be synthesized industrially or obtained from mines around the world. In the case of natural sources (mines) it has to be considered that each source may present specific particularities in terms of composition, which can impact the binding ca‐ pacity, and even clays obtained from the same place, can vary from batch to batch, that has to be well controlled throughout quality control.

There is much information available in the literature comparing those different clays [49]. These authors for example, compared zeolite, bentonite and HSCAS for AFB1 binding capacity using *in vitro* method, simulating gastrointestinal fluids. This method seems to be the most fre‐ quent technology adopted for such assays, including a double condition of pH (3.0 and 7.0). Those researchers find that zeolite and bentonite aflatoxin binding capacity varied according to the pH used for the assay, and both clays were less effective than HSCAS (Figure 2).

age, processing and packaging should be accomplished in order to prevent that contaminat‐

*Mycotoxins Corn and byproducts\**

**Table 8.** Maximum tolerated levels for mycotoxins according to Resolution RDC 07/2011 [47].\*The maximum tolerated levels refer to results obtained by methodologies that comply with the performance criteria established by

**6. Aflatoxin binders and strategies to reduce toxicity to farm animals**

Adsorbents are necessary and important and may have great impact on improving animal production and health, providing greater security to consumers of animal products, due to

Considering that aflatoxins were the first discovered mycotoxins, there are many data avail‐ able searching for binders and other methods to reduce toxicity in animals. However, due to

The most common additives used in animal diets are aluminosilicates, produced syntheti‐ cally or extracted from clay mines. There are also other alternatives to reduce aflatoxin toxic‐

This type of binder is basically composed by single or blended type of clay. The most com‐ mon clay is hydrated sodium calcium aluminosilicate (HSCAS). However, there are other sort of clays which can be used as toxin binders, like sodium or calcium bentonites and zeo‐ lites. Not often, any particular varying sort of clay can be used as well. It has to be consid‐ ered that such materials can be synthesized industrially or obtained from mines around the world. In the case of natural sources (mines) it has to be considered that each source may present specific particularities in terms of composition, which can impact the binding ca‐ pacity, and even clays obtained from the same place, can vary from batch to batch, that has

There is much information available in the literature comparing those different clays [49]. These authors for example, compared zeolite, bentonite and HSCAS for AFB1 binding capacity using *in vitro* method, simulating gastrointestinal fluids. This method seems to be the most fre‐ quent technology adopted for such assays, including a double condition of pH (3.0 and 7.0).

methodologies used for evaluation, there is certain degree of variation in results.

Aflatoxins B1, B2, G1 and G2, ppb 20 Deoxynivalenol, ppb 3000 Fumonisin B1+B2, ppb 5000 Zearalenone, ppb 400

the reduction and/or removal of mycotoxins in these products.

ed food is sold or consumed.

184 Aflatoxins - Recent Advances and Future Prospects

Codex Alimentarius.

ity, as presented.

**a.** *Clay derived sorbents:*

to be well controlled throughout quality control.

**Figure 2.** Adsorption percentage of AFB1 (8 µg/ml) to sorbents (0.5% w/v) in simulated gastrointestinal fluid at pH 3 and pH 7 [49].

It is important to establish the correct inclusion rate to animal diets in order to optimize the binding response. In Figure 3, it can be observed how those three types of clays perform un‐ der the same pH (7.0) when increasing doses are included.

It has been shown that montmorillonite (0.5%) added to the diet containing 5 ppm of afla‐ toxin has proven its effectiveness in preventing the effects of aflatoxicosis in broilers [50].

**Figure 3.** Amount of aflatoxin B1 adsorbed on sorbents at different concentrations of the adsorbents in simulated in‐ testinal fluid at pH 7 [50].

Based on the data presented in Figures 2 and 3, it is clear that assays condition (especially pH) and toxin:sorbent dosing rate are extremely important. These conditions should be con‐ sidered when product performance reports are compared. However, when evaluating [51] nine different toxin binders (4 activated charcoals, 3 sodium bentonites, 1 calcium bentonite and 1 esterified glucomannan) all products presented adsorption above 95% of AFB1, re‐ gardless of the pH used (3.0, 7.0, 10.0 and the original pH of each product).

vated charcoal has been used to prevent animal intoxication by several compounds, in‐ cluding mycotoxins. Plant extracts with specific mode of action, like liver protection, have

Aflatoxins Importance on Animal Nutrition http://dx.doi.org/10.5772/51952 187

Milk thistle (*Silybum marianum*), which is a medicinal herb found in Pakistan, has been used to treat liver diseases. This herb was tested in poultry feed contaminated with AFB1 (80 ppb for the first week and 520 ppb from the second until the fifth) at a dose of 1%. The results indicated that milk thistle is effective as hepatoprotectant and growth promoter in the pres‐ ence of AFB1 in the feed [54]. Protection against the negative effects of aflatoxin on perform‐ ance of broiler chickens was observed when broilers were fed during 4 weeks with 0.05%

Another report concluded that dietary citric acid supplementation can be used as an addi‐ tive to degrade aflatoxins in the ration as well as to promote growth performance in young broiler chickens. Results showed that aflatoxins in the diet, at a concentration of 39 ppb were

Other alternatives to degrade aflatoxins have been tested, like the use of microorganism. Bacteria (*Nocardia corynebacteroides*, NC) showed ability to degrade AFB1 [57]. In a trial per‐ formed with broiler fed AFB1 (800-1,200 ppb) NC was safe to the birds and showed protec‐ tion to the animal, indicating that it can be used as a tool to detoxify feed contaminated with

Humic acid, generated during matter decomposition, has binding capacity for many mole‐ cules. The use of oxihumate was evaluated as AFB1 binder, *in vitro* and *in vivo* [35]. Oxihu‐ mate showed a high *in vitro* affinity for AFB1. *In vivo* trial showed that oxihumate decreased adverse effects caused by AFB1 on broiler body weight and also protective effect against liv‐ er damage, stomach and heart hyperplasia, acting positively preserving standard blood pa‐ rameters. Enzyme degradation of aflatoxin has been tested as well. Data suggest that

There are many contradictory data available in the main scientific journals. One and prob‐ ably the main reason for that is the way the trials have been performed, differing in terms of toxin levels, and environmental condition of the trials. Under a real field condition, the challenges the animals suffer are far stronger then under experimental situation. Toxin binder, especially clays, may affect the cation binding capacity of feeds and consequently influencing water intake and feed consumption. Also, the effects of none, medium (1 g kg-1 feed) and high (2.5 g kg-1 feed) inclusion levels of HSCAS was evaluated in broiler mycotoxin free diets [60]. The data suggest that increasing HSCAS to diets may modify performance, internal organ weights, gastrointestinal and biochemical parameters. How‐ ever, other authors [61] did not see effect as the consequence of toxin binders (EGM) pres‐

been used as well to reduce the toxicity of some mycotoxins, specially aflatoxin.

ETE (ethanolic turmeric extract, *Curcuma longa*) plus 3 ppm aflatoxins [55].

almost degraded (92%) by the acidification procedure (up to 50 g kg-1) [56].

lactoperoxidase can be used to hydrolyze aflatoxin [59].

ence on broiler body weight and feed efficiency.

AFB1 at high levels [58].

**d.** *Other aspects:*

Other methods can be used to evaluate toxin adsorbents, as *in vivo* trials. In this case, the most frequent inconsistency when comparing research reports is related to the source used to obtain aflatoxin (synthetic crystalline vs natural aflatoxin obtained by fermentation) and also the aflatoxin level used in the specific assay. When comparing analytical reports with field accepted levels of aflatoxins, there is much difference. One reason for that is the differ‐ ence between the experimental conditions (well controlled) where animals are not submit‐ ted to stress situation in comparison to the real farm condition.

Another additional evaluation that should be performed is the presence of aflatoxin in spe‐ cific organs, like liver. Low level of AFB1 (50 ppb) on broiler performance was studied on biochemical parameters and aflatoxin presence in liver tissue, when monensin and sodium bentonite were added to the feed, from 18 up to 46 days of age. The authors concluded that monensin and AFB1 compete for adsorption sites on sodium bentonites, indicating a non-se‐ lective adsorption capacity of this particular binder. The researchers comment as well that different substances, such as coccidiostats, vitamins, minerals, aminoacids or other dietary components, could affect the ability of the adsorbent to bind low levels of aflatoxin. In addi‐ tion, significant levels of AFB1 in livers indicate that this determination is important not only for diagnosis of aflatoxicosis in broilers, but also for quality control of avian products [52].

#### **b.** *Organic sorbents:*

The most well-known natural toxin binders are yeast based products. Glucans are yeast cell wall constituents. Those compounds have been submitted to esterification process generat‐ ing a new additive with toxin binding capacity, called esterified glucomannan (EGM). Effi‐ cacy of EGM was tested against mycotoxins naturally present in broiler feed [53], being 0.05% EGM efficient to counteract the adverse effects of mycotoxins (Table 9).


\* Aflatoxin 168 ppb, ochratoxin 8.4 ppb, zearalenone 54 ppb and T2-Toxin 32 ppb [53].

**Table 9.** Efficacy of esterified glucomannans (EGM) on broiler live weight (LW), feed intake (FI) and feed conversion ratio (FCR) fed with a mycotoxin contaminated diet, from one up to 35 days.

#### **c.** *Other strategies:*

The use of mechanisms that improve animal health and physiology can be helpful. One example is the use of probiotics which have been used to ameliorate mycotoxicosis. Acti‐ vated charcoal has been used to prevent animal intoxication by several compounds, in‐ cluding mycotoxins. Plant extracts with specific mode of action, like liver protection, have been used as well to reduce the toxicity of some mycotoxins, specially aflatoxin.

Milk thistle (*Silybum marianum*), which is a medicinal herb found in Pakistan, has been used to treat liver diseases. This herb was tested in poultry feed contaminated with AFB1 (80 ppb for the first week and 520 ppb from the second until the fifth) at a dose of 1%. The results indicated that milk thistle is effective as hepatoprotectant and growth promoter in the pres‐ ence of AFB1 in the feed [54]. Protection against the negative effects of aflatoxin on perform‐ ance of broiler chickens was observed when broilers were fed during 4 weeks with 0.05% ETE (ethanolic turmeric extract, *Curcuma longa*) plus 3 ppm aflatoxins [55].

Another report concluded that dietary citric acid supplementation can be used as an addi‐ tive to degrade aflatoxins in the ration as well as to promote growth performance in young broiler chickens. Results showed that aflatoxins in the diet, at a concentration of 39 ppb were almost degraded (92%) by the acidification procedure (up to 50 g kg-1) [56].

Other alternatives to degrade aflatoxins have been tested, like the use of microorganism. Bacteria (*Nocardia corynebacteroides*, NC) showed ability to degrade AFB1 [57]. In a trial per‐ formed with broiler fed AFB1 (800-1,200 ppb) NC was safe to the birds and showed protec‐ tion to the animal, indicating that it can be used as a tool to detoxify feed contaminated with AFB1 at high levels [58].

Humic acid, generated during matter decomposition, has binding capacity for many mole‐ cules. The use of oxihumate was evaluated as AFB1 binder, *in vitro* and *in vivo* [35]. Oxihu‐ mate showed a high *in vitro* affinity for AFB1. *In vivo* trial showed that oxihumate decreased adverse effects caused by AFB1 on broiler body weight and also protective effect against liv‐ er damage, stomach and heart hyperplasia, acting positively preserving standard blood pa‐ rameters. Enzyme degradation of aflatoxin has been tested as well. Data suggest that lactoperoxidase can be used to hydrolyze aflatoxin [59].

#### **d.** *Other aspects:*

and 1 esterified glucomannan) all products presented adsorption above 95% of AFB1, re‐

Other methods can be used to evaluate toxin adsorbents, as *in vivo* trials. In this case, the most frequent inconsistency when comparing research reports is related to the source used to obtain aflatoxin (synthetic crystalline vs natural aflatoxin obtained by fermentation) and also the aflatoxin level used in the specific assay. When comparing analytical reports with field accepted levels of aflatoxins, there is much difference. One reason for that is the differ‐ ence between the experimental conditions (well controlled) where animals are not submit‐

Another additional evaluation that should be performed is the presence of aflatoxin in spe‐ cific organs, like liver. Low level of AFB1 (50 ppb) on broiler performance was studied on biochemical parameters and aflatoxin presence in liver tissue, when monensin and sodium bentonite were added to the feed, from 18 up to 46 days of age. The authors concluded that monensin and AFB1 compete for adsorption sites on sodium bentonites, indicating a non-se‐ lective adsorption capacity of this particular binder. The researchers comment as well that different substances, such as coccidiostats, vitamins, minerals, aminoacids or other dietary components, could affect the ability of the adsorbent to bind low levels of aflatoxin. In addi‐ tion, significant levels of AFB1 in livers indicate that this determination is important not only for diagnosis of aflatoxicosis in broilers, but also for quality control of avian products [52].

The most well-known natural toxin binders are yeast based products. Glucans are yeast cell wall constituents. Those compounds have been submitted to esterification process generat‐ ing a new additive with toxin binding capacity, called esterified glucomannan (EGM). Effi‐ cacy of EGM was tested against mycotoxins naturally present in broiler feed [53], being

*Mycotoxin\* EGM (%) LW (g) FI (g) FCR (g g-1)* ---- ---- 1,391.2 b 3,017.6 b 2.17 b ---- 0.05 1,441.4 c 2,994.0 b 2.07 a +++ ---- 1,258.8 a 2,803.4 a 2.22 c +++ 0.05 1,381.0 b 2,952.6 b 2.15 b SEM 7.25 20.01 0.015

**Table 9.** Efficacy of esterified glucomannans (EGM) on broiler live weight (LW), feed intake (FI) and feed conversion

The use of mechanisms that improve animal health and physiology can be helpful. One example is the use of probiotics which have been used to ameliorate mycotoxicosis. Acti‐

0.05% EGM efficient to counteract the adverse effects of mycotoxins (Table 9).

\* Aflatoxin 168 ppb, ochratoxin 8.4 ppb, zearalenone 54 ppb and T2-Toxin 32 ppb [53].

ratio (FCR) fed with a mycotoxin contaminated diet, from one up to 35 days.

gardless of the pH used (3.0, 7.0, 10.0 and the original pH of each product).

ted to stress situation in comparison to the real farm condition.

186 Aflatoxins - Recent Advances and Future Prospects

**b.** *Organic sorbents:*

**c.** *Other strategies:*

There are many contradictory data available in the main scientific journals. One and prob‐ ably the main reason for that is the way the trials have been performed, differing in terms of toxin levels, and environmental condition of the trials. Under a real field condition, the challenges the animals suffer are far stronger then under experimental situation. Toxin binder, especially clays, may affect the cation binding capacity of feeds and consequently influencing water intake and feed consumption. Also, the effects of none, medium (1 g kg-1 feed) and high (2.5 g kg-1 feed) inclusion levels of HSCAS was evaluated in broiler mycotoxin free diets [60]. The data suggest that increasing HSCAS to diets may modify performance, internal organ weights, gastrointestinal and biochemical parameters. How‐ ever, other authors [61] did not see effect as the consequence of toxin binders (EGM) pres‐ ence on broiler body weight and feed efficiency.


Additionally, different regions across the world have been dealing with mycotoxin subject in different ways. In US for instance, no toxin binders are officially registered, as a conse‐ quence of the control quality assumed for feedstuffs. In EU, since 2009, toxin sequestrants have been considered as a sort of feed additive, and a scientific group of specialists, namely European Food Safety Authority [62] have been working on re-evaluation of the efficacy and biological effects of detoxifying agents in animals. In Tables 10 and 11, a summary of

Aflatoxins Importance on Animal Nutrition http://dx.doi.org/10.5772/51952 189

**Detoxifying agents AFB1 DON-NIV Fumonisin OTA T2 Toxin ZEA**

Montmorilonite - -

Zeolite + +

Yeast glucomannans + +/- - +

Alfafa **+**

**-**

Cells highlighted in dark gray indicate that the product has shown positive effects in counteracting deleterious effects

In other regions, like South America, due to the climate and grain production conditions, mycotoxin has been a significant challenge along the past decades. This particular situation has been forcing the development of research groups which are involved with commercial sequestrants evaluation. As a consequence, the maximum acceptable aflatoxin limit has been established for different raw materials and feed, as well the specificity and inclusion levels of toxin binders in animal feed. However there are many different criteria for toxin adsorb‐

+: positive effect of the mycotoxin-detoxifying agent; -: negative effect of the mycotoxin-detoxifying agent; +/-: positive effect of the mycotoxin-detoxifying agent on some parameters, no effect on other parameters.

the outcome of that technical group is presented.

HSCAS + -

Sodium bentonite + -

Ammonium carbonate -

Polyvinylpolypyrrolidone -

Apple pommace **+**

Content of large intestine of hens **+** *Eubacterium* **+**

Combination of Eubacterium BBSH 797 with dried yeast and clays

Charcoal +/-

of mycotoxins, while light gray color depicts that the product was not effective.

**Table 11.** Mycotoxin detoxifying agents tested *in vivo* in pigs. Adapted from ref. [62].

Calcium bentonite +

Sepiolite + Palygorskite +

1BHT = butylhydroxytoluene; 2PVPP = Polyvinylpolypyrrolidone; 3Mycotoxins AFB1 = aflatoxin B1, CPA = cyclopiazonic acid; DAS = diacetoxyscirpenol, DON = deoxynivalenol; NIV = nivalenol, OTA = ochratoxin A, ZEA = zearalenone. Cells highlighted in dark gray indicate that the product has shown positive effects in counteracting deleterious effects of mycotoxins, while light gray color depicts that the product was not effective.

+: positive effect of the mycotoxin-detoxifying agent;


+/-: positive effect of the mycotoxin-detoxifying agent on some parameters, no effect on other parameters.

**Table 10.** Mycotoxin detoxifying agents tested *in vivo* in poultry. Adapted from ref. [62].

Additionally, different regions across the world have been dealing with mycotoxin subject in different ways. In US for instance, no toxin binders are officially registered, as a conse‐ quence of the control quality assumed for feedstuffs. In EU, since 2009, toxin sequestrants have been considered as a sort of feed additive, and a scientific group of specialists, namely European Food Safety Authority [62] have been working on re-evaluation of the efficacy and biological effects of detoxifying agents in animals. In Tables 10 and 11, a summary of the outcome of that technical group is presented.

**Detoxifying agents Mycotoxin3**

+/-

+

Xylanase -

PVPP2 +/- -

+: positive effect of the mycotoxin-detoxifying agent; - : negative effect of the mycotoxin-detoxifying agent;

of mycotoxins, while light gray color depicts that the product was not effective.

**Table 10.** Mycotoxin detoxifying agents tested *in vivo* in poultry. Adapted from ref. [62].

Clinoptilolite +/- -

188 Aflatoxins - Recent Advances and Future Prospects

Acidic phyllosilicate -

BHT1 +

Yeast glucomannans +

Live yeast culture residue + *Nocardia corynebacteroides* +/-

*Saccharomyces cerevisiae* + Ammonia + Calcium propionate +/-

Cell wall *Saccharomyces*

Yeast *Trichosporon mycotoxinivorans*

*cerevisiae*

Modified nanomontmorilonite + Mg K aluminosilicate +/- Sodium bentonite + Ca montmorillonite +

Synthetic crystalline aluminosilicate

**AFB1 CPA DAS DON Fusaric**

HSCAS + - - -

Zeolite + - - -

Superactivated charcoal +/- +/-

Esterified glucomannans + +/- + + - +

1BHT = butylhydroxytoluene; 2PVPP = Polyvinylpolypyrrolidone; 3Mycotoxins AFB1 = aflatoxin B1, CPA = cyclopiazonic acid; DAS = diacetoxyscirpenol, DON = deoxynivalenol; NIV = nivalenol, OTA = ochratoxin A, ZEA = zearalenone. Cells highlighted in dark gray indicate that the product has shown positive effects in counteracting deleterious effects

+/-: positive effect of the mycotoxin-detoxifying agent on some parameters, no effect on other parameters.

*Eubacterium* + + +

Diatomaceous earth + Charcoal -

**acid**

**NIV OTA T2**

**Toxin**

+ -

**ZEA**


Cells highlighted in dark gray indicate that the product has shown positive effects in counteracting deleterious effects of mycotoxins, while light gray color depicts that the product was not effective.

+: positive effect of the mycotoxin-detoxifying agent; -: negative effect of the mycotoxin-detoxifying agent;

+/-: positive effect of the mycotoxin-detoxifying agent on some parameters, no effect on other parameters.

**Table 11.** Mycotoxin detoxifying agents tested *in vivo* in pigs. Adapted from ref. [62].

In other regions, like South America, due to the climate and grain production conditions, mycotoxin has been a significant challenge along the past decades. This particular situation has been forcing the development of research groups which are involved with commercial sequestrants evaluation. As a consequence, the maximum acceptable aflatoxin limit has been established for different raw materials and feed, as well the specificity and inclusion levels of toxin binders in animal feed. However there are many different criteria for toxin adsorb‐ ents registration in different countries, some demanding an extend documentation about product efficacy and others are less restrictive.

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