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Effect of lactoferrin digest and ozone treatments on Penicillium spp. isolated from bottled water Liceaga Gesualdo, Andrea


Recently, consumers have complained about visible fungi contamination in bottled water; this has caused the industry a series of rejected products. These fungi seem to be resistant to the common sterilization processes, such as ozonation, used for bottled water. In this study, the main objective was to investigate the effect of an antimicrobial peptide and ozone treatments on a Penicillium spp. isolated from bottled water. Foreign bodies were isolated from bottled water and plated on potato dextrose agar. A loopfull from a visible colony was examined by phase contrast microscope for identification based on its morphology. Fungi isolated from the bottled water were identified as Penicillium sp. using three specialized media. A stock suspension was obtained by washing the spores after 7 days incubation at 25°C on PDA. Lactoferrin (5% w/v), isolated from cheese whey, was digested at 37°C for 4 hrs using porcine pepsin (3% w/w) at pH 2.3. Digestion was terminated by increasing the temperature to 80°C for 15 min and the pH to 7.0. The digest was centrifuged and the supernatant filtered through a 0.45 μ cellulose acetate filter. Digestion was confirmed by SDS-PAGE electrophoresis. Lactoferrin digest (LFD), containing lactoferricin, was used without further purification. The antimicrobial assay was performed using a 96 well microplate containing peptone yeast glucose medium (PYG) and known concentrations of LFD. Each well was inoculated with the spore suspension (7xl0³ mL⁻¹ spores); bovine serum albumin digest was used as negative control. Culture optical density was monitored at 595 nm over time. LFD at concentrations of 0.06 and 0.3 mg/mL inhibited spore germination and mycelial fusion for up to 9 and 21 days at 30°C, respectively. Ozonation experiments were carried out at 0.1-0.4 ppm ozone in 100 mL of distilled deionized water with or without LFD (0.03, 0.06 0.30 mg/mL). Two experimental set ups were used; one consisted of adding ozonated water (65 mL) to 35 mL of spore suspension (3xl0⁶ mL⁻¹ spores). Samples (1 mL) of ozonated spores were retrieved at 0, 4, 8 and 12 min and plated on potato dextrose trypan blue agar using the hydrophobic grid membrane filtration (HGMF) method. The second set up consisted of directly ozonating 100 mL inoculated water (0.9 mL spore suspension with 99.1 mL d-d water). Samples (1 mL) were retrieved every 30 seconds for a period of 3 minutes and plated as described above. Ozone concentrations in the range of 0.1-0.4 ppm stimulated spore germination, mycelial fusion and growth. LFD when added, led to slower development of mould colonies on the HGMF filters.

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