Methods for the In vitro examination of the antibacterial and cytotoxic activities of antimicrobial peptides

Antimicrobial peptides (AMPs) represent an interesting class of molecules with expanding properties. Nature is the primary source of AMPs since they are produced by most living organisms including prokaryotes, plants, and animals. Thanks to their hundreds of thousands of species on earth, insects are one of the most abundant and varied resources of AMPs. Among these, many families have already been well characterized while new AMPs are continuously discovered. In this chapter, the main methods for the in vitro evaluation of the biological properties of AMPs are described. In particular, to examine the antimicrobial activity, the inhibition zone assay and the techniques for the determination of the minimal inhibitory concentration and the bactericidal concentration are reported in detail. For the evaluation of the possible cytotoxic effect toward mammalian cells, the hemolytic test and the colorimetric assay based on the reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide are also described

Lipid production in batch and fed-batch cultures of <it>Rhodosporidium toruloides</it> from 5 and 6 carbon carbohydrates

<p>Abstract</p> <p>Background</p> <p>Microbial lipids are a potential source of bio- or renewable diesel and the red yeast <it>Rhodosporidium toruloides</it> is interesting not only because it can accumulate over 50% of its dry biomass as lipid, but also because it utilises both five and six carbon carbohydrates, which are present in plant biomass hydrolysates.</p> <p>Methods</p> <p><it>R. toruloides</it> was grown in batch and fed-batch cultures in 0.5 L bioreactors at pH 4 in chemically defined, nitrogen restricted (C/N 40 to 100) media containing glucose, xylose, arabinose, or all three carbohydrates as carbon source. Lipid was extracted from the biomass using chloroform-methanol, measured gravimetrically and analysed by GC.</p> <p>Results</p> <p>Lipid production was most efficient with glucose (up to 25 g lipid L⁻¹, 48 to 75% lipid in the biomass, at up to 0.21 g lipid L⁻¹ h⁻¹) as the sole carbon source, but high lipid concentrations were also produced from xylose (36 to 45% lipid in biomass). Lipid production was low (15–19% lipid in biomass) with arabinose as sole carbon source and was lower than expected (30% lipid in biomass) when glucose, xylose and arabinose were provided simultaneously. The presence of arabinose and/or xylose in the medium increased the proportion of palmitic and linoleic acid and reduced the proportion of oleic acid in the fatty acids, compared to glucose-grown cells.</p> <p>High cell densities were obtained in both batch (37 g L⁻¹, with 49% lipid in the biomass) and fed-batch (35 to 47 g L⁻¹, with 50 to 75% lipid in the biomass) cultures. The highest proportion of lipid in the biomass was observed in cultures given nitrogen during the batch phase but none with the feed. However, carbohydrate consumption was incomplete when the feed did not contain nitrogen and the highest total lipid and best substrate consumption were observed in cultures which received a constant low nitrogen supply.</p> <p>Conclusions</p> <p>Lipid production in <it>R. toruloides</it> was lower from arabinose and mixed carbohydrates than from glucose or xylose. Although high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, for lipid production from mixtures of carbohydrates fed-batch cultivation was preferable. Constant feeding was better than intermittent feeding. The feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of C5 sugars did.</p