A Commercial Clay-Based Material as a Carrier for Targeted Lysozyme Delivery in Animal Feed

Abstract

The controlled supply of bioactive molecules is a subject of debate in animal nutrition. The release of bioactive molecules in the target organ, in this case the intestine, results in improved feed, as well as having a lower environmental impact. However, the degradation of bioactive molecules' in transit in the gastrointestinal passage is still an unresolved issue. This paper discusses the feasibility of a simple and cost-effective procedure to bypass the degradation problem. A solid/liquid adsorption procedure was applied, and the operating parameters (pH, reaction time, and LY initial concentration) were studied. Lysozyme is used in this work as a representative bioactive molecule, while Adsorbo^®, a commercial mixture of clay minerals and zeolites which meets current feed regulations, is used as the carrier. A maximum LY loading of 32 mg_LY/g_AD (LY(32)-AD) was obtained, with fixing pH in the range 7.5-8, initial LY content at 37.5 mg_LY/g_AD, and reaction time at 30 min. A full characterisation of the hybrid organoclay highlighted that LY molecules were homogeneously spread on the carrier's surface, where the LY-carrier interaction was mainly due to charge interaction. Preliminary release tests performed on the LY(32)-AD synthesised sample showed a higher releasing capacity, raising the pH from 3 to 7. In addition, a preliminary Trolox equivalent antioxidant capacity (TEAC) assay showed an antioxidant capacity for the LY of 1.47 ± 0.18 µmol TroloxEq/g with an inhibition percentage of 33.20 ± 3.94%.

Keywords: FT-IR spectroscopy; clay-based materials; feed application; lysozyme–carrier interactions mechanism; precision nutrition; target delivery; target release.

Figure 1

Lysozyme–Adsorbo^® adsorption process.

Figure 2

Methodology workflow for the preliminary lysozyme release assay.

Figure 3

Steps in the preliminary antioxidant activity assay of lysozyme.

Figure 4

LY capture: (a) as a function of reaction time, and (b) as a function of the initial LY content.

Figure 5

FT-IR skeletal spectra of (a) LY(6)-AD; (b) LY(22)-AD; (c) LY(32)-AD, upon subtraction of spectrum of pristine AD. Dashed line: FT IR skeletal spectrum of pure LY in KBr.

Figure 6

TGA (a) and DTG (b) curves of LY-AD samples at increasing LY loading (pristine AD and LY are reported for comparison, dashed lines: LY decomposition maxima). Curves were intentionally shifted to highlight differences; plotting in the original version in Figure S8 (Colours in (a) as in (b)).

Figure 7

Comparison of XRPD patterns of (a) LY(32)-AD and LY-AD-MM, AD reported for comparison, and (b) enlarged pattern of LY-AD-MM. The position of the most intense (I₁₀₀) reflections of the single phases is reported (Z: zeolite; Chab: chabazite; Phil: phillipsite; Feld: feldspar; D: dolomite).

Figure 8

Comparison of the synthetised LY(32)-AD organoclay and the mechanical mixture LY-AD-MM: (a) FT-IR spectra upon AD subtraction, and (b) DTG. In both plots: LY-AD-MM in blue, LY(32)-AD in red. Dashed line in plot (a) pure LY spectrum.

Figure 9

SEM images (magnification = 2 µm) of (a) pristine AD, (b) pristine LY, (c) LY(32)-AD the synthesised organoclay, and (d) LY-AD-MM the mechanical mixture. Yellow circles: LY aggregates.

Figure 10

SEM-EDX analysis of LY (32)-AD organoclay (a) SEM of the analysed portion, (b) EXD of the analysed portion, (c) EXD of Si, (d) EDX of Al, (e) EDX of Mg, and (f) EDX of S.

Figure 11

(a) SEM analysis of the synthesised organoclay, LY(32)-AD, (b) EDX analysis of the synthesised organoclay, LY(32)-AD, (c) SEM analysis of the mechanical mixture, LY-AD-MM, (d) EDX analysis of the mechanical mixture, LY-AD-MM. Yellow circles: lysozyme aggregates.