A small-scale method for quantitation of carotenoids in bacteria and yeasts.

Microbial carotenoids are difficult to extract because of their embedding into a compact matrix and prominent sensitivity to degradation. Especially for carotenoid analysis of bacteria and yeasts, there is lack of information about capability, precision and recovery of the method used. Accordingly, we investigated feasibility, throughput and validity of a new small-scale method using Micrococcus luteus and Rhodotorula glutinis for testing purposes. For disintegration and extraction, we combined primarily mild techniques: enzymatically we used combinations of lysozyme and lipase for bacteria as well as lyticase and lipase for yeasts. Additional mechanical treatment included sonication and freeze-thawing cycles. Chemical treatment with dimethylsulfoxide was applied for yeasts only. For extraction we used a methanol-chloroform mixture stabilized efficiently with butylated hydroxytoluene and alpha-tocopherol. Separation of compounds was achieved with HPLC, applying a binary methanol/tert-butyl methyl ether gradient on a polymer reversed C30 phase. Substances of interest were detected and identified applying a photodiode-array (PDA) and carotenoids quantitated as all-trans-beta-carotene equivalents. For evaluation of recovery and reproducibility of the extraction method, we used beta-8'-apo-carotenal as internal standard. The method provides a sensitive tool for the determination of carotenoids from bacteria and yeasts and also for small changes in carotenoid spectrum of a single species. Corequisite large experiments are facilitated by the high throughput of the method.