Purification of Bacillus amyloliquefaciens lipopeptides for postharvest disease control

South Africa is the largest exporter of fresh fruit (by volume) in the southern hemisphere and as such, the quality of fruit must be maintained throughout the global supply chain.Several fungal pathogens contribute to significant fruit loss and wastage due to disease during postharvest storage.The use of chemical fungicides as a postharvest disease control strategy has become limiting due to negative environmental concerns (toxicity, non-specificity and non-biodegradability) while the efficient use of bacterial cells/spores as biocontrol agents is restrictive as the viability is dependent on suitable postharvest environments.An alternative and novel biocontrol strategy proposed in this study is to use antifungal lipopeptides, namely fengycin and iturin, produced by Bacillus amyloliquefaciens DSM 23117, as second-generation biocontrol agents for postharvest disease control.However, to be sufficiently effective, concentration and purification of the lipopeptides is necessary due to the low yields produced and the presence of metabolic products such as lipid and protein impurities that are not desired in the final product.The aim of the study was to develop an appropriate downstream concentration and purification programme for antifungal lipopeptides using acid precipitation, solvent extraction and macroporous adsorption as downstream unit operations.To concentrate the lipopeptides, while effecting some degree of purification, acid precipitation studies were conducted by acidification of the cell-free supernatant to pH values 1 -4 to determine the effect of pH on the recovery and purity of fengycin and iturin.Reverse-phase high performance liquid chromatography (RP-HPLC) quantification of acid precipitate showed high recoveries of 78% and 62% for fengycin and iturin respectively at pH 2 with an optimal fengycin purity of 64% obtained at pH 3.Acid precipitation of the cell-free supernatant at pH 3 would be suitable as the initial concentration step, due to the percentage purity of fengycin and iturin.To improve the purity of fengycin and iturin above that obtained from acid precipitation, solvent extraction was the next unit operation in the purification programme.Organic solvents of varying polarity indices were screened for their efficiency in extracting fengycin and iturin.Following screening experiments, a three-stage methanol extraction and a three-stage diethyl ether-methanol extraction were conducted to further improve the purity.Lipopeptides in solvent extracts were quantified by thin layer chromatography (TLC) which showed methanol to be the best solvent which extracted 100% of lipopeptides with a purity of 74% for fengycin.A final purity of 89% and 16% for fengycin and surfactin respectively was obtained after the 3-stage methanol extraction, while final purities of 74% and 13% were obtained for fengycin and surfactin respectively, with the diethyl ether-methanol extraction.The extraction efficiency of fengycin by the organic solvents was found to be related the functional groups of the solvents, in addition to the polarity.