Recognition of Membrane Sterols by Polyene Antifungals Amphotericin B and Natamycin, A (13)C MAS NMR Study.

The molecular action of polyene macrolides with antifungal activity, amphotericin B and natamycin, involves recognition of sterols in membranes. Physicochemical and functional studies have contributed details to understanding the interactions between amphotericin B and ergosterol and, to a lesser extent, with cholesterol. Fewer molecular details are available on interactions between natamycin with sterols. We use solid state (13)C MAS NMR to characterize the impact of amphotericin B and natamycin on mixed lipid membranes of DOPC/cholesterol or DOPC/ergosterol. In cholesterol-containing membranes, amphotericin B addition resulted in marked increase in both DOPC and cholesterol (13)C MAS NMR linewidth, reflecting membrane insertion and cooperative perturbation of the bilayer. By contrast, natamycin affects little either DOPC or cholesterol linewidth but attenuates cholesterol resonance intensity preferentially for sterol core with lesser impact on the chain. Ergosterol resonances, attenuated by amphotericin B, reveal specific interactions in the sterol core and chain base. Natamycin addition selectively augmented ergosterol resonances from sterol core ring one and, at the same time, from the end of the chain. This puts forward an interaction model similar to the head-to-tail model for amphotericin B/ergosterol pairing but with docking on opposite sterol faces. Low toxicity of natamycin is attributed to selective, non-cooperative sterol engagement compared to cooperative membrane perturbation by amphotericin B.

A 90-day oral toxicity study of nisin A, an anti-microbial peptide derived from Lactococcus lactis subsp. lactis, in F344 rats.

This study was designed to evaluate and characterize any adverse effect of nisin A, when administered to both sexes of F344/DuCrlCrlj rats (10 males and 10 females in each group) at dietary levels of 0%, 0.2%, 1.0% and 5.0% for 90 days. Animals given NaCl at a dietary level of 3.712% (equivalent to the NaCl content in 5.0% nisin A diet) served as a reference material treated group. There were no deaths, and the treatment had no toxicologically significant effects on clinical signs, body weights, food consumption, ophthalmology, hematology, or gross pathology. Statistically significant increases of water consumption, urine volume, and urinary sodium and chlorine, and decreases of urinary potassium and serum sodium, along with increases of absolute and relative kidney weight, and incidences of minimal squamous cell hyperplasia of limiting ridge in the forestomach, were found in nisin A-treated groups. It was considered that these changes were related to NaCl, since they were also noted in rats given diet containing the reference substance. Thus, no toxicologically significant changes were apparent in both sexes of F344/DuCrlCrlj rats fed diet containing 0%, 0.2%, 1.0% and 5.0% nisin A for 90 days. Therefore, the no-observed-adverse-effect level (NOAEL) for nisin A was concluded to be a dietary level of 5.0% (2996 mg/kg/day for males and 3187 mg/kg/day for females).

Investigation of the effectiveness of Ascopyrone P as a food preservative.

Ascopyrone P (APP), a novel antibacterial from fungi, was evaluated as a food preservative. Efficacy was generally assessed by comparing the time taken for test strains to grow to 10(6) CFU/g in food +/- APP. In chilled chicken soup, 2000 mg kg-1 APP prevented Bacillus cereus, Listeria monocytogenes, Pseudomonas fluorescens, Salmonella and Escherichia coli reaching this threshold for >60 days. Good activity was also observed at 500-1000 mg kg-1 but not against L. monocytogenes. No activity was observed against Saccharomyces cerevisiae. Activity was reduced at 20 degrees C, although 2000 mg kg-1 was still effective against B. cereus and P. fluorescens. APP was less effective in chilled cooked meat systems and ineffective in raw meat. In a cooked meat system at 8 degrees C, bacteriostatic effect was generally observed at 2000 mg kg-1 against Salmonella typhimurium, E. coli and P. fluorescens but not against L. monocytogenes or Lactobacillus sake. Activity against Gram-negative enteric bacteria was enhanced by low temperature. In milk, 2000 mg l-1 was effective against P. fluorescens at chilled but not ambient temperature. APP was ineffective against yeasts and the mould Byssochlamys in apple juice. A minimum of 2000 mg kg-1 APP would appear to be necessary for antibacterial efficacy in food, although low-temperature storage may help. Observed variations in sensitivity may be related to APP stability, which decreases >pH 5.5. Toxicology testing is needed before consideration of APP for food use.

Effective use of nisin to control Bacillus and Clostridium spoilage of a pasteurized mashed potato product.

Heat-resistant spore-forming bacteria such as Bacillus and Clostridium can survive and grow in cooked potato products. This situation represents both a public health problem and an economic problem. The natural food preservative nisin is used in heat-treated foods to prevent the growth of such bacteria. A cocktail of Clostridium sporogenes and Clostridium tyrobutyricum spores was inoculated into cooked mashed potatoes, which were vacuum packed, pasteurized, and incubated at 8 and 25 degrees C. The shelf life of the mashed potatoes at 25 degrees C was extended by at least 58 days with the addition 6.25 microg of nisin per g. At 8 degrees C, in control samples not containing nisin, the natural contaminant Bacillus grew, but the inoculated Clostridium strains did not until the temperature was raised to 20 degrees C after 39 days. No bacterial growth occurred in nisin-containing samples. The shelf life of the mashed potatoes was extended by at least 30 days with 6.25 microg of nisin per g. In trials involving a cocktail of Bacillus cereus and Bacillus subtilis strains, 6.25 microg of nisin per g extended the shelf life of mashed potato samples that were not vacuum packed by at least 27 days at 8 degrees C. At 25 degrees C, 25 microg of nisin per g extended shelf life by a similar period. Shelf life extension was also observed at lower nisin levels. Microbiological analysis of the mashed potato ingredients showed that a high spore level was associated with the onion powder. It is emphasized that the preservative and the ingredients must be well mixed to ensure good nisin efficacy. Nisin remained at effective levels after pasteurization, and good retention was observed throughout the shelf life of the mashed potatoes.