# Natural Clays as Potential Amino Acids Carriers for Animal Nutrition Application
## 4 Discussion  
As demonstrated in the Results section, the experimental approach here adopted allowed for the effective capture of methionine on mineral substrates, namely Adsorbene, the bentonite-zeolite-based mixed material. By a single intercalation step, ADS is able to capture up to 0.18 gMET/gADS, via a mechanism that is mainly related to methionine trapping inside the pores of the material. Adsorption mechanism is favored by the material morphology, characterized by the high surface area of 93 m\({}^{2}\)/g, and it is confirmed by the occurrence of a very fast reaction, where equilibrium is reached within 15 min. This picture is consistent with the phase composition of ADS that predominantly consists of zeolites rather than expandable clay components. Indeed, ADS morphology, composition, and behavior are very close, if not the same, to those of BIO, the selected reference for pure zeolite material (Table 1). The hypothesis that methionine is mainly captured by the zeolite component is also supported by the fact that both ADS and BIO, although slightly different in composition, are able to adsorb the same amount of methionine, and, once captured, the MET is not removed during washing. This occur due to a stronger interaction MET-carrier that can be attributed to the zeolite smaller porosity (dimeter = 3-4 nm) of the zeolite component, and to a capture mechanism other than intercalation. Intercalation mechanism has been reported for montmorillonite-based clay, such as VEG, where organic molecules can be allocated in neutral form, inside the clay interlayer which is expandable [42], without ion exchange [24]. Such a capture mechanism results in a progressive site saturation, in view of a limitation of the interlayer expansion, as observed in the case of VEG, but not in the cases of ADS and BIO (Figure 4), where no saturation was observed. ADS and BIO can adsorb, for each single step, a lower amount of MET; however, although in greater quantities, the methionine adsorbed by VEG is only partially strongly interacting with the carrier, and \begin{table}
\begin{tabular}{c c c} \hline \hline
**Sample** & **Sample Condition** &
\begin{tabular}{c} **Percentage Recovered after** \\ **in vitro Digestion (\%)** \\ \end{tabular} \\ \hline
**CTR** & ADS alone pH 7 (no MET) & n.d. \\
**MET-ADS 2.5 mM** & ADS + 2.5 mM MET & 4.92 \(\pm\) 0.34 \\
**MET 2.5 mM** & 2.5 mM MET & 99.64 \(\pm\) 11.98 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Methionine percentage quantified after in vitro digestion. Results are presented as percentage of the methionine initial dosage recovered (%) after in vitro digestion. almost 55% is weakly, or not, bonded with the carrier. The interaction is so weak that part of the adsorbate can be washed away by demineralized water. The system MET-VEG could be considered as a mechanical mixture of free methionine and MET-intercalated VEG. A situation different than a mechanical mixture was confirmed for the MET-VEG system, where the TG-DTG analysis evidenced that the MET-containing samples cannot be a simple mixture of the raw methionine and the raw Adsorbene (Figure 7). Indeed, the thermal decomposition of the organoclay samples is a complex multistep phenomenon, largely different than that of the single raw components. Thus, the presence of some MET-ADS interaction stronger than a simple surface interaction must be active. Once more, adsorption, via pore trapping, appears to be the mechanism of choice to explain MET-VEG interaction. To increase the methionine content, multiple adsorption steps can be performed; indeed, the methionine capture is a linear process, so that, by repeating the single step process six times on the same sample, the final adsorbed methionine was six-fold the one after a single step (Figure 5). Once the operating conditions are fixed, the adsorption process is fully reproducible, and the final methionine content in the sample can be managed by the number of steps.