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Cardiotonic glycosides are extracted mostly from leaves of Digitalis plants. Commercial production of bioactive secondary metabolites by traditional agriculture is an inefficient process and can be affected by climatic and soil conditions. Strategies, based on in vitro culture methods, have been extensively studied to improve the production of specific plant derived chemicals. The aim of the present research was to obtain biomass of D. purpurea using the temporary immersion system (TIS) and to determine the content of cardiotonic glycosides (digitoxin, digoxin and lanatoside C) as secondary metabolites of commercial value for the pharmaceutical industry. Shoots were cultured in 1,000 ml TIS during 28 days. The effect of four immersion frequencies (once every 2, 4, 6, and 12 h) was studied. Biomass accumulation was influenced by immersion frequency. The maximum biomass accumulation (values in respect of fresh and dry weight) was obtained with immersions every 4 h (six immersions per day). HPLC analysis revealed the presence of digoxin and digitoxin for all immersion frequencies. No lanatoside C was detected in the biomass cultured in TIS. Digoxin concentrations varied depending on the frequencies tested. In contrast, the digitoxin content showed no dependency on the immersion frequency. Net production of digoxin and digitoxin per TIS were found to be higher with immersions every 4 h. The best net production of digitoxin and digoxin per TIS were 167.6 and 119.9 lg, respectively. The development of organ culture based on temporary immersion system can be a reliable method for the steady production of biomass for cardiotonic glycosides production, which is reported for the first time for Digitalis genus in this communication.

Submerged fermentation in a stirred bioreactor of the white rot fungus Cerrena unicolor C-139 was done at a 120-L scale in the presence of wheat bran as a cheap lignocellulosic substrate for fungus growth and laccase production. Enzyme monitoring showed that laccase production started after 2 days of cultivation, attaining a maximum activity of 416.4 UÁmL À1 at day 12 of fermentation. After treatment of culture liquid by successive micro-and ultrafiltration (5 kDa), a liquid concentrate containing 22203176 units of laccase was obtained. Obtaining large amount of laccase is essential for various industrial applications, including detoxification of industrial effluents, textile and petrochemical industries, polymer synthesis, bioremediation of contaminated area, stabilization of beverages, production of cosmetics, manufacture of anti-cancer drugs, and nanobiotechnology. The cultivation method and the fungal strain used here provided a substantial amount of enzyme produced at a price lower than 0.01 s cent/unit enzyme.

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