Application of high performance liquid chromatography to the separation of microbiologically active compounds from heated sugar solutions and Lactarius necator mushrooms:Dissertation

This publication is a summary of seven research reports dealing with the application of High Performance Liquid Chromatography (HPLC) to the isolation of microbiologically active compounds in heated neutral glucose and fructose solutions and in aqueous extracts of the mushroom Lactarius necator.In both cases the starting point was a micro biologically measurable activity which was used as detection method during the isolation procedures.The formation of antimicrobial activity in neutral aqueous glucose and fructose solutions was the first object of study.Starting from the original solutions and by chromatographing them twice the compounds responsible for the antimicrobial activity could be recovered in crystalline form.The first separation step was an ordinary reverse phase chromatography, whereas the second was specific for these compounds and involved the application of a gel permeation chromatography column for reverse phase separation.As a result the compounds responsible for the antimicrobial activity were identified as various phenolic compounds.The aqueous extract of the edible mushroom Lactarius necator has been shown to exhibit strong mutagenic activity in the Ames Salmonella assay. The compound respon sible for this activity was isolated by HPLC This compound, designated here as necatorin was shown to be one of the strongest mutagens hitherto found in nature (6000 revertants/ g with the tester strain TA100).Necatorin was found to be rather stable, so that the normal methods applied in the preparation of mushrooms for consumption blanching, brining, pickling or freezing do not remove or inactivate it efficiently.Necatorin is a normal constituent of the mushroom with a rather high initial concentration (3...20 mg/kg fresh weight), and not an accidental contamination.In both the systems studied the application of HPLC in reverse phase mode made possible the use of the original microbial activity test as detector with minimal manipulation of the isolated fractions.Therefore investigations could be directed to the correct fractions in these complex mixtures