Prevention of Bacterial Contamination of a Silica Matrix Containing Entrapped β-Galactosidase through the Action of Covalently Bound Lysozymes

Abstract

β-galactosidase was successfully encapsulated within an amino-functionalised silica matrix using a "fish-in-net" approach and molecular imprinting technique followed by covalent binding of lysozyme via a glutaraldehyde-based method. Transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy were used to characterise the silica matrix hosting the two enzymes. Both encapsulated β-galactosidase and bound lysozyme exhibited high enzymatic activities and outstanding operational stability in model reactions. Moreover, enzyme activities of the co-immobilised enzymes did not obviously change relative to enzymes immobilised separately. In antibacterial tests, bound lysozyme exhibited 95.5% and 89.6% growth inhibition of Staphylococcus aureus ATCC (American type culture collection) 653 and Escherichia coli ATCC 1122, respectively. In milk treated with co-immobilised enzymes, favourable results were obtained regarding reduction of cell viability and high lactose hydrolysis rate. In addition, when both co-immobilised enzymes were employed to treat milk, high operational and storage stabilities were observed. The results demonstrate that the use of co-immobilised enzymes holds promise as an industrial strategy for producing low lactose milk to benefit people with lactose intolerance.

Keywords: covalent binding; encapsulation; lysozyme; β-galactosidase.

Figure 1

The strategy for co-immobilisation of the two enzymes relative to the silica substrate.

Figure 2

Transmission electron microscope (TEM) image (a) and X-ray pattern (b) of the silica matrix containing both enzymes.

Figure 3

Scanning electron microscope (SEM) images of NH₂-Silica (a) and co-immobilised enzymes (b).

Figure 4

Fourier transform infrared (FTIR) spectrum of NH₂-Silica (a); the co-immobilised enzymes (b); lysozyme (c); and β-galactosidase (d).

Figure 5

Antibacterial assay on Petri dishes: Staphylococcus aureus ATCC (American type culture collection) 653 incubated with the co-immobilised enzymes (a); Staphylococcus aureus ATCC 653 incubated with NH₂-Silica (b); Escherichia coli ATCC 1122 incubated with the co-immobilised enzymes (c); Escherichia coli ATCC 1122 incubated with NH₂-Silica (d).