Phthalyl starch nanoparticles as prebiotics enhanced nisin production in Lactococcus lactis through the induction of mild stress in probiotics.

Abstract

Effect of internalized phthalyl starch nanoparticles (PSNs) on the antimicrobial ability of Lactococcus lactis (LL) KCTC 2013. Phthalyl starch nanoparticles were prepared by self-assembly of phthalyl starch and the amount of the hydrophobic phthalic moieties were characterized by nuclear magnetic resonance: PSN1 (DS: 14·3mol.%), PSN2 (DS: 17·8mol.%) and PSN3 (DS: 30·4mol.%). The sizes of PSN1, PSN2 and PSN3 measured by dynamic light scattering were 364·7, 248·4 and 213·4nm, respectively, and the surface charges of PSNs measured by electrophoretic light scattering were negative charges and PSNs were spherical in shape according to scanning electron microscope. It was found that when PSNs were treated with LL, the PSNs were internalized into LL through nanoparticle size-, energy- and glucose transporter-dependent mechanisms. The internalization was confirmed by confocal laser scanning microscopy and fluorescence-activated cell sorting. Nisin was isolated and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Also, more nisin was produced from PSNs-treated LL than untreated- or starch-treated LL. Co-culture assay and agar diffusion test were performed to test the antimicrobial ability. Antimicrobial ability against Gram-negative Escherichia coli k88, Salmonella gallinarum and Gram-positive Listeria monocytogenes of LL treated with PSNs was higher than that of untreated or starch-treated group. Finally, it was found that the expression level of stress response genes dnaK, dnaJ and groES was significantly higher in PSNs-treated groups compared with starch-treated group or LL alone. The internalization of PSNs into LL enhanced the production of nisin through mild intracellular stimulation, resulting in enhanced antimicrobial ability. This study shows the promising potential of PSNs as new prebiotics for increasing the production of nisin, thus demonstrating a new method for the biological production of such antimicrobial peptides.