**10. Bioaccessibility**

B1 consumption may produce a significant decrease of serum leptin levels (Abdel-Wahhab, Ahmed, Hagazi, 2006). Leptin concentration is usually associated with the high levels of cor‐ tisol and interleukin-6, which act together to influence the feeding response (Barber, McMil‐ lan, Wallace, Ross, Preston, 2004). *Lactobacillus reuteri* might have contributed to reduce the aflatoxin B1 absorption in bacteria-treated rats and thus diminish its effect on leptin levels in

The volume of the stomach is considered an important parameter for oral dosing in experi‐ mental animals. For rats, maximum oral dosage volume recommended is 10 mL kg–1 of body weight; for a 200 g rat this would mean a dosing volume of 2 mL (McConnell, Basit, Mur‐ dan, 2008). Therefore, it is possible that the volume supplied (every third day) by oral gav‐ age of aflatoxin and/or bacteria over the experiment, had partially met the basic water needs of the rats, which may explain the observed reduction in water consumption at the end of

A world-wide-accepted method for protecting animals against mycotoxicosis is the use of adsorbent materials. An effective adsorbent is one that tightly binds the mycotoxin in contaminated feed without dissociating in the gastrointestinal tract of the animal. The toxin–adsorbent complex passes then through the gastrointestinal tract without absorp‐ tion and is eliminated via the faeces. In other words, the bioavailability of the mycotox‐ in is reduced as less mycotoxin is absorbed because it is bound to the adsorbent, i.e. lower bioaccessibility. Therefore, these adsorbents can be used to evaluate the use of the in vitro digestion model as indicator for the in vivo bioavailability. The following mate‐ rials, representative for different classes of adsorbents, have been selected: an aluminosi‐ licate (HSCAS), which is a common anticaking additive in animal feeds to reduce mycotoxicosis in animals; activated charcoal, which is used in humans and animals as an antidote against poisoning; cholestyramine is an anion exchange resin and binds bile acids in the gastrointestinal tract and it has been used for over 20 years in the clinic for

The effect of chlorophyllin on intestinal transport of aflatoxin B1 was studied by measure‐ ment of the transport of aflatoxin B1 with the intestinal Caco-2 cells. The rate at which com‐ pounds are transported across the Caco-2 cells, which is expressed as a permeability

Transport of 5ng/mL aflatoxin B1 across Caco-2 cells revealed that after 4h, 25±6% aflatoxin B1 was transported across Caco-2 cells into the basolateral compartment. Addition of chloro‐ phyllin (1 mg/mL) greatly reduced (>20-fold) the transport of aflatoxin B1 to only 1±1%. From this transport, a permeability coefficient can be calculated for aflatoxin B1 of 9x10-6 cm/s in absence, and 0.4x10-6 cm/s in presence of chlorophyllin. When we compare these transport rates with the S-shaped correlation found for absorption of compounds in hu‐ mans, the permeability coefficient of aflatoxin B1 alone (9x10-6 cm/s) corresponds with high absorption in humans whereas the permeability coefficient of aflatoxin B1 in presence of chlorophyllin (0.4x10-6 cm/s) indicates an intermediate absorption. Thus, these data are in ac‐ cordance with the human intervention study on chlorophyllin and aflatoxin B1, where a 50%

coefficient, is correlated with absorption in humans (Artursson, Karlsson, 1991).

blood serum.

the experimental period (21 days).

354 Aflatoxins - Recent Advances and Future Prospects

reduction of lowdensit y lipoproteins and cholesterol.

Bioaccessibility has been defined as the fraction of a compound that is released from its ma‐ trix in the gastrointestinal tract and thus becomes available for intestinal absorption i.e. en‐ ters the blood stream (Benito, Miller, 1998). Bioaccessibility includes the entire sequence of events that take place during the digestive transformation of food into material that can be assimilated by the body, the absorption/assimilation into the cells of the intestinal epitheli‐ um, and lastly, the presystemic metabolism (both intestinal and hepatic). Bioaccessibility analyses can be approached using general experimental techniques (there are systematic techniques common to all types of foods) that can be adapted to all types of claims regard‐ ing nutritional content.

*In vivo*, as soon as a compound is released from its matrix in the chyme, the compound can be transported across the intestinal epithelium into the body thereby keeping the compound concentration low in the chyme.

may be underestimated when saturation of the compound occurs in the chyme. Thus, one

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 357

The bioaccessibility of aflatoxin B1 in chime has been determined from nine peanut slurries ranging from 0.6 to 14 µg/kg aflatoxin B1 (contamination level in peanuts 1.5-36 µg/kg). Afla‐ toxin B1 was almost completely mobilised from the peanut slurries during digestion evi‐ dencing a mean bioaccessibility of 94%. The concentration of aflatoxin B1 in chyme of the two highest contaminated peanut slurries was higher than those in the calibration curve and a smaller volume of chyme was used for analysis (Versantvoort, Oomen, Van de Kamp,

The amount of aflatoxin B1 released from the peanut slurries into the chyme is dose pro‐ portional to the contamination level in the peanut slurries. These results showed no sat‐ uration of the chyme with aflatoxin B1. The amount of food in the digestion model was varied in order to study whether release of the contaminant from its food matrix was linearly, i.e. whether bioaccessibility was independent from the amount of food in the model. Application of 0.5 g and 4.5 g peanut slurry in the in vitro digestion model cor‐ responds to the consumption of approximately 10 and 100 g peanuts, respectively. The bioaccessibility of some bioactive compounds can be influenced by the food composition as observed by Versantvoort et al. (Versantvoort, Oomen, Van de Kamp, Rompelberg, Sips, 2005) that studied the effects of different food components on the bioaccessibility of aflatoxin B1 from peanut slurry considering an average meal. Bioaccessibilities of afla‐ toxin B1 (108±11%) from 6 g food-mix (4.5g standard meal + 0.5g peanut slurry + 1g buckwheat) were compared to the bioaccessibility of aflatoxin B1 from 0.5g correspond‐ ing peanut slurry (83±18%) showing that the bioaccessibility of aflatoxin B1 did not vary

Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample prepara‐ tion, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level. In this regard, specific plans have been developed and tested rigorously for some commodities such as corn, peanuts, and tree nuts; sampling plans for some other com‐ modities have been modeled after them. A common feature of all sampling plans is that the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample. Methods of sampling and analysis for the official control of mycotoxins, including aflatoxins, are laid down in Commission Regulation No 401/2006. This ensures that the same sampling criteria in‐ tended for the control of mycotoxin content in food are applied to the same products by the competent authorities throughout the EU and that certain performance criteria, such as recovery and precision, are fulfilled. In 2008, the Codex Alimentarius set a maximum

factor potentially affecting bioaccessibility is the level of contamination.

**11. Advanced analysis of aflatoxins in biological fluids**

**11.1. Sampling and sample preparation**

Rompelberg, Sips, 2005).

significantly.

Different analytical approaches can be applied to measure bioaccessibility of nutrients and bioactive compounds: *in vivo* and *in vitro* studies both present strengths and drawbacks. Within *in vivo* studies, balance studies and tissue concentration are two strategies that allow determination of the absorbed amount of nutrients, bioactive compounds, or their metabo‐ lites. Balance studies determine the absorbed amount by measuring the difference between the fed and excreted amounts of the nutrient or bioactive compound. Tissue concentration consists of monitoring the increase in plasma/serum concentration of the nutrient or bioac‐ tive compound. These approaches have been applied these approaches have been used with both animals and humans to determine absorption of carbohydrates, minerals, vitamins, phytochemicals, and different compounds (Benito, Miller, 1998; Hallberg, 1991). *In vivo* hu‐ man studies are the criterion standard approach to determine bioaccessibility of food nu‐ trients or bioactive compounds, although some experimental approaches are ethically and technically unaffordable.

Digestion and absorption involve several different steps, and each one could cause an effect on the nutrient or bioactive compound so that a detailed picture is not obtained with the bal‐ ance and bioassay studies. *In vitro* studies have been developed to simulate the physiologic conditions and the sequence of events that occur during digestion in the human gastrointes‐ tinal tract. In a first step, an in vitro gastrointestinal method is applied to the food, mirroring the physiochemical conditions that take place during human digestion, considering the three areas of the human digestive system (mouth, stomach, and intestine).

The main features of the in vitro gastrointestinal methods are temperature, shaking or agita‐ tion, and the chemical and enzymatic composition of saliva, gastric juice, duodenal juice, and bile juice (Wittsiepe, Schrey, Hack, Selenka, Wilhelm, 2001). When physical processes that oc‐ cur in vivo are not reproduced (shear, mixing, hydration, changes in conditions over time, peri‐ stalsis), the in vitro gastrointestinal model is defined as a static or biochemical model. The dynamic models mimic the *in vivo* physical processes so that they take into account new varia‐ bles, such as changes on viscosity of the digesta, particle size reduction, diffusion, and parti‐ tioning of nutrients. Several examples of *in vitro* gastrointestinal static and dynamic models have been described (Rotard, Christmann, Knoth, Mailahn, 1995; Arcand, Mainville, Farn‐ worth, 2007). During the application of the in vitro gastrointestinal method, food nutrients or bioactive compounds can be monitored to determine whether they are affected by digestion conditions (pH, enzymes) or if interactions with other food components (fiber, sucrose polyest‐ er, fat replacers) take place, which could affect efficiency of digestion. The final processed ma‐ terial of the experimental procedure is a digesta or intestinal preparation.

To analyze the lipophilic content that has been effectively incorporated to mixed micelles, the micellar fraction can be isolated from that processed material by the application of an ultracentrifugation protocol (Hernell, Staggers, Carey, 1999). In the digestion model, the compounds are not removed from the chyme during digestion and therefore, bioaccesibility may be underestimated when saturation of the compound occurs in the chyme. Thus, one factor potentially affecting bioaccessibility is the level of contamination.

The bioaccessibility of aflatoxin B1 in chime has been determined from nine peanut slurries ranging from 0.6 to 14 µg/kg aflatoxin B1 (contamination level in peanuts 1.5-36 µg/kg). Afla‐ toxin B1 was almost completely mobilised from the peanut slurries during digestion evi‐ dencing a mean bioaccessibility of 94%. The concentration of aflatoxin B1 in chyme of the two highest contaminated peanut slurries was higher than those in the calibration curve and a smaller volume of chyme was used for analysis (Versantvoort, Oomen, Van de Kamp, Rompelberg, Sips, 2005).

The amount of aflatoxin B1 released from the peanut slurries into the chyme is dose pro‐ portional to the contamination level in the peanut slurries. These results showed no sat‐ uration of the chyme with aflatoxin B1. The amount of food in the digestion model was varied in order to study whether release of the contaminant from its food matrix was linearly, i.e. whether bioaccessibility was independent from the amount of food in the model. Application of 0.5 g and 4.5 g peanut slurry in the in vitro digestion model cor‐ responds to the consumption of approximately 10 and 100 g peanuts, respectively. The bioaccessibility of some bioactive compounds can be influenced by the food composition as observed by Versantvoort et al. (Versantvoort, Oomen, Van de Kamp, Rompelberg, Sips, 2005) that studied the effects of different food components on the bioaccessibility of aflatoxin B1 from peanut slurry considering an average meal. Bioaccessibilities of afla‐ toxin B1 (108±11%) from 6 g food-mix (4.5g standard meal + 0.5g peanut slurry + 1g buckwheat) were compared to the bioaccessibility of aflatoxin B1 from 0.5g correspond‐ ing peanut slurry (83±18%) showing that the bioaccessibility of aflatoxin B1 did not vary significantly.
