# Natural Clays as Potential Amino Acids Carriers for Animal Nutrition Application
## 4 Discussion
Due to the buffer capacity of both the amino acids, in view of its zwitterionic nature, and the carrier, which is able to adsorb H\({}^{+}\) and OH\({}^{-}\), no pH effect on the capture process is evident, and the same equilibrium values are reached between pH 1 and 8. This absence of pH effect is confirmed by the IR findings, where the spectral features of the samples at different pH are actually very similar. Therefore, pH does not affect either the nature of the interaction nor methionine structure. Methionine is adsorbed in its natural form, i.e., neutral and unmodified, and the interaction with the carrier is manifest. This assumption is confirmed by both FTIR and TG-DTG measurements. Indeed, in the FTIR spectra of the MET-ADS spectrum, bands belonging to methionine, despite very weak, are always present and their broadening can be due to methionine interaction with the carrier. Methionine interacts with the carrier in its zwitterionic form. Moreover, the reduced relative intensity of the ammonium bands at 1585 cm\({}^{-1}\), together with the disappearing of the band at 1655 cm\({}^{-1}\), suggests that MET-carrier interaction mainly affects the -NH\({}_{3}\)\({}^{+}\) group. The slight shift to higher frequencies of the bands of the CH\({}_{3}\) group and of the CS group suggests a limited perturbation of the side chain after interaction, also. Upon multiple contacting experiments, an increased methionine-to-clay band intensity ratio is observed, in line with an increased methionine content in the sample. In addition, in the case of the multiple contacting steps process, methionine is present in its zwitterionic form, while the side chain of the amino acid is detected almost unchanged. The comparison with spectra upon a single-step contacting process seems to indicate that, in the multiple-step one, the molecule is even less affected by interaction with the matrix. It can be assumed that, possibly, more than one layers of methionine are deposited, loosening the interaction with clay surface, and therefore leading to features close to the pristine methionine compound. The presence of free or weakly bonded methionine on the MET-ADS samples is also supported by TG-DTG analysis, where the sharp weight loss in the range 220-240 \({}^{\circ}\)C corresponds to pure methionine degradation (Figure 7). The extent of the phenomenon increases upon increasing methionine content, in line with FTIR indication that suggests the presence of more than one layer of methionine characterized by a very weak interaction with clay surface. However, TG data also evidenced a weight loss at 500-550 \({}^{\circ}\)C that corresponds to the decomposition of methionine strongly interacting with the carrier, being intercalated in the bentonite and/or trapped in the zeolite cages. This thermal phenomenon is only present in the MET-ADS samples, and not in the mechanical mixture, thus supporting the presence of methionine molecules characterized by different interaction degree with the carrier. FTIR data on MET ADS C6 thermal evolution also shows gas phase CO\({}_{\rm x}\) formation in the high-temperature range, in agreement with this picture. MET-ADS samples also demonstrated release of methionine after in vitro digestion simulation. Thisapproach provided a suitable strategy to produce and characterize the molecules that can be generated during _in vivo_ digestion. Thus, the final hydrolysate obtained represents the pool of molecules (e.g., amino acids and peptides) resembling those generated during the physiological digestion of MET-ADS. A weak release (<5%) of methionine from MET-ADS was observed at the end of the procedure. It is evident that the bioaccessibility of methionine from MET-ADS is low, at least lower than the reasonable requirement for feed application. Such a behavior is not thought to be due to the preparation procedure, but mainly related to the carrier structure and phase composition. Indeed, the large zeolite content of Adsorbene makes methionine prone to strong interactions with the molecules.