Immobilization of Nisin on Natural Montmorillonite

CAROLINA IBARGUREN; PABLO M NARANJO; M CARINA AUDISIO; EDGARDO SHAM; E MÓNICA FARFÁN TORRES; FRANK A. MÜLLER

Kosice

Conferencia; International Scientific Conference on Bacteriocins and Antimicrobial Peptides; 2012

The aim of this work is the development of immobilized nisin systems using natural clay as a support structure to produce food grade materials capable of releasing antimicrobial agents. Montmorillonite (MMT), a natural abundant inexpensive clay approved by the FDA as a food additive, was used as support material. This hydrated sheet alumina-silicate is characterized by a high cation exchange capacity (CEC) and a large surface area (1). The immobilized antimicrobial agents were pure nisin and its commercial form Nisaplin® (Danisco, UK). Two series of antibacterial compounds, one for each type of nisin, were prepared using the monosodium clay fraction (≤2 m) of a natural MMT from Río Negro (Argentina). This fraction was obtained by saturation with NaCl, rinse with distilled water and centrifugation (12000 rpm, 10 min). Different amounts of the solid were put into contact with pure nisin (5 mg/mL) and Nisaplin® (100 mg/mL) aqueous solutions (2 h, room temperature) in order to achieve nisin loadings (NL) equivalent to a 0.06-1.21 and 0.03-0.6 fold of the MMT CEC (84.7 meq/100g), respectively. The residual antimicrobial activity of the supernatants recovered by centrifugation (12000 rpm, 10 min) after the contact with the solid was determined by the serial 1:2 dilution method using Enterococcus faecium C1 as indicator strain (2). The peptide adsorption of the resulting solid samples was analysed, prior lyophilisation, by X-ray diffraction (XRD) (Brucker AXS D8 Discover diffractomer, CuK radiation (40 mV, 40 mA), 3°≤2≤70°) and Fourier Transform Infrared Attenuated Total Reflectance spectroscopy (FTIR-ATR) (Brucker Alpha FTIR Spectrum, Pt single reflection diamond, 4000-375 cm-1). The residual antimicrobial activity of the supernatants recovered after contact with the clay decreased with the increase of solid content, suggesting the adsorption of the peptide. The XRD patterns showed a split of the d(001) spacing signal characteristic for MMT (3) into two  values, suggesting an increase in the MMT interlayer distance after contact with the peptide solution due to a ?partial? intercalation of the nisin molecules in the solid matrix. The FTIR spectra showed a slight shift of the band at 995 cm-1 corresponding to Si(Al)-O stretching vibrations (4) to higher wavelengths which reflect interactions of the peptide with MMT tetrahedral layers upon intercalation. Moreover, the amide I (1638 cm-1, stretching vibrations of the C=O bond) and amide II (1520 cm-1 bending vibrations of the N-H bond) bands of nisin (5) intensified with the increase of NL values. Additionally, the amide bands for the peptide adsorbed on MMT from Nisaplin® solution were more intense in terms of the signal of the non-adsorbed commercial nisin, suggesting that preferably nisin fraction is adsorbed, which conducted to the purification of the peptide. The results concerning antimicrobial activity, FTIR and XRD measurements showed evidences of nisin adsorption on montmorillonite. These preliminary results are encouraging for the development of nisin-based bioprotectors immobilized on natural montmorillonite and for the elucidation of the interaction between the peptide and the support matrix.