IMMOBILIZATION OF NISIN ON NATURAL MONTMORILLONITE

C. IBARGUREN; P. M. NARANJO; E. L. SHAM; M. C. AUDISIO; E. M. FARFÁN TORRES; F. MÜLLER

Kocise

Congreso; nternational Scientific Conference on Bacteriocins and Antimicrobial Proteins – BAMP2012; 2012

Institute of Animal Physiology of the Slovak Academy of Sciences, Slovakia

The main focus of this work is the development of immobilized nisin systems using natural clay as a support structure to produce food grade material capable of releasing antimicrobial agents. Montmorillonite (MMT), a natural abundant inexpensive solid approved by the FDA as a food additive, was used as support material. This hydrated alumina-silicate layered clay is characterized by a moderate negative surface charge (cation exchange capacity, CEC) and a high surface area. The immobilized antimicrobial agent was pure nisin and its commercial form Nisaplin® (Danisco, UK). Two series of antibacterial compounds, one for pure nisin and one for Nisaplin®, were prepared using the sodium fraction ≤2 m obtained by saturation with NaCl, distilled water rinse and centrifugation (12000 rpm for 10 min) of a natural MMT from Río Negro (Argentina). Different fractions of the solid were put in contact at room temperature with pure nisin (5 mg/mL) and Nisaplin® (2.5 mg/mL) aqueous solutions in order to achieve nisin loadings (NL) equivalent to between 0.3-6.0 and 0.2-3.0 times of the MMT CEC, for nisin and Nisaplin® respectively. The residual antimicrobial activity in the supernatants recovered by centrifugation (12000 rpm for 10 min) after the contact with the solid was determined by the serial 1:2 dilution method using Enterococcus faecium C1 as indicator strain. The peptide adsorption was analysed in the resulting solid samples, prior liophilization, 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, 4 cm-1 resolution, 24 scans). The analysis of the antimicrobial activity on the supernatants recovered post-contact with the clay evidenced the adsorption of the peptide as the residual antimicrobial activity decreased with the increase of NL values. The XRD patterns showed a split of the d(001) spacing signal characteristic for MMT into two  values, suggesting an increase in the MMT interlayer distance after contact with the peptide solution due to a “partial” intercalation in the solid matrix of the nisin molecules. The FTIR spectra showed a slight shift of the band at 995 cm-1 corresponding to Si(Al)-O stretching vibrations to higher wavelengths which reflect interactions of the peptide with MMT tetrahedral layers upon intercalation. Also, 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 intensified with the increase of NL values. The amide bands for the peptide adsorbed on MMT from Nisaplin® solution were clearly resolved respect to the not adsorbed commercial bacteriocin signal, suggesting a purification of the peptide once immobilized on the clay. The antimicrobial activity, FTIR and XRD measurements performed showed evidences of nisin adsorption on montmorillonite. These preliminary results are encouraging for the development of nisin-based bioprotectors supported on natural montmorillonite and for the elucidation of the interaction between the peptide and the support matrix.