Three fungal strains isolated from tobacco were cultured on tobacco water extracts. In these cultures, the mycelium weight was shown to be correlated with the concentration of a steroid, ergosta-5,7,22-trien-3ss-ol [ergosterol]. This steroid is not a tobacco constituent, but tobacco samples where mold or yeast infections had occurred exhibited significant amounts of it. A method is proposed to quantify ergosterol in tobacco samples by reverse-phase high-performance liquid chromatography (HPLC) with UV detection at 282 nm. 7-Dehydrocholesterol can be used as internal standard. When found in a tobacco sample, the ergosterol concentration exhibits a good correlation with that of another related steroid, ergosta-4,6,8(14),22-tetraen-3-one [ETO], for which an HPLC quantification method in tobacco is proposed. Because it is highly fluorescent, ETO is also amenable to a sensitive and quick determination by thin-layer chromatography (TLC). Once produced on tobacco, ergosterol concentration remains stable through storage under normal conditions, and even primary processing does not alter it appreciably. Possible applications of ergosterol analysis to the detection of fungal infections or the monitoring of fungal growth on tobacco are outlined. In addition, TLC estimation of the ETO concentration in a sample may constitute a convenient and fast screening method for fungal infections.
The cigarette ingredients cocoa powder, glycerol, and saccharose were investigated regarding their potential effect on the resulting mainstream smoke, i.e., smoke chemistry (Hoffmann analytes), mammalian cell cytotoxicity (Neutral Red Uptake assay), and bacterial mutagenicity (Ames assay). Each ingredient was added at three concentrations to the tobacco of a 6 mg and 10 mg ‘tar’ yield experimental American blend filter cigarette (obtained under ISO/FTC smoking regime). The lowest application concentration was equivalent to the normal approximate use level of the ingredients; the highest application level was up to 5-fold higher. The resulting data were compared with the respective control cigarettes without addition of the ingredients. The addition of cocoa powder did not lead to any consistent effects on the measured mainstream smoke analytes. Neither the in vitro cytotoxicity nor the in vitro mutagenicity was affected by cocoa addition. The addition of glycerol resulted in a decrease in the delivery of several smoke constituents (generally around 20%), e.g. aldehydes, phenolics, and N-nitrosamines. Water in the particulate phase (TPM) was distinctly increased (up to +150%). The cytotoxicity of the TPM was decreased (approx. !15%). Mutagenicity was not affected. Saccharose addition consistently increased formaldehyde delivery in smoke by up to 40% and decreased tobacco-specific N-nitrosamines by up to approximately 20%. The increase in formaldehyde is discussed in the context of the human smoker. The cytotoxicity was not affected by the addition of saccharose, while the mutagenicity of the TPM was decreased in tester strain TA98 with metabolic activation (!15%). The results are in agreement with currently available literature. Some investigations summarized in this publication are novel and have not yet been reported in the literature. Based on the total evidence, it can be concluded that the three ingredients added at their current use levels do not increase the inherent toxicity of the cigarette smoke.