The use of a tobacco agar medium (TAM) was investigated to visually differentiate Cryptococcus species from Rhodotorula and Candida species that can be isolated from tobacco. This study was first conducted with pure isolates of each of the major yeast species that have been isolated from tobacco. All Cryptococcus strains that were tested produced colonies with different degrees of pigmentation ranging from light to dark brown or black. All Candida and Pichia colonies were white to off-white. Candidaparapsilosis colonies were easily differentiated since they had rough contours and surfaces. All Rhodotorula colonies were pink or orange. In order to validate the use of this medium, tobacco was spiked with a mixed culture of Cryptococcus, Candida, Rhodotorula and Pichia. TAM allowed visual detection and enumeration of the four yeast genera based on colony colour and/or morphology.
Canadian tobacco was flue-cured using two different heating systems: direct-fired in which the exhaust gases were in contact with the tobacco and indirect in which only hot air, via a heat exchanger, contacted the tobacco. The concentrations of tobacco-specific nitrosamines (TSNAs) in tobacco cured by indirect heating did not increase during curing and were in the range 0.25-0.35 ppm. There were no changes in TSNA concentrations (range 0.13-0.3 ppm) in tobacco cured by direct firing during the first six days (0-144 h) of curing. However between 168 and 264 h, significant increases in TSNAs occurred (up to 1.91 ppm). TSNA concentrations in leaves at the bottom of the plant were significantly higher than in those found at higher plant position. There were no significant differences in TSNA concentrations in tobacco cured on different farms. The TSNA concentrations in tobacco cured by indirect heat were 87% ± 5% lower than in tobacco cured by direct heat. Subsequent processing of tobacco did not change the relative concentrations of TSNAs.
Bacillus is a predominant genus of bacteria isolated from tobacco. The Gram stain is the most commonly used and most important of all diagnostic staining techniques in microbiology. In order to help confirm the Gram positivity of Bacillus isolates from tobacco, three methods using the chemical differences of the cell wall and membrane of Gram-positive and Gram-negative bacteria were investigated: the KOH (potassium hydroxide), the LANA (L-alanine-4-nitroanilide), and the vancomycin susceptibility tests. When colonies of Gram-negative bacteria are treated with 3% KOH solution, a slimy suspension is produced, probably due to destruction of the cell wall and liberation of deoxyribonucleic acid (DNA). Gram-positive cell walls resist KOH treatment. The LANA test reveals the presence of a cell wall aminopeptidase that hydrolyzes the L-alanine-4-nitroanilide in Gram-negative bacteria. This enzyme is absent in Gram-positive bacteria. Vancomycin is a glycopeptide antibiotic inhibiting the cell wall peptido-glycan synthesis of Gram-positive microorganisms. Absence of lysis with KOH, absence of hydrolysis of LANA, and susceptibility to vancomycin were used with the Gram reaction to confirm the Gram positivity of various Bacillus species isolated from tobacco. B. laevolacticus excepted, all Bacillus species tested showed negative reactions to KOH and LANA tests, and all species were susceptible to vancomycin (5 and 30 µg).
Flue-curing is a post harvest conditioning process which strongly affects the tobacco leaf chemistry, and consequently the chemical properties of tobacco smoke. Several studies identified the major changes in tobacco chemistry occurring during flue-curing. It is not known how flue-curing contributes to changes in bioactivity of cigarette smoke condensate (CSC). In this study, tobacco leaves collected throughout the twelve days of flue-curing were used to prepare cigarettes that were smoked to generate CSC samples. The assessment of mutagenicity was performed using the Bacterial Reverse Mutation / Ames test with Salmonella typhimurium TA98 in the presence of S9 metabolic activation. CSC from cured leaves were significantly more mutagenic than CSC from uncured leaves. The number of revertants was positively influenced by the duration of the curing. The effect of the duration of curing on the number of revertants was more pronounced with increasing CSC concentration.
Tobacco as many other plants has its own microbiota. There are very few studies determining the evolution of this microbiota during tobacco storage, which may affect the quality of tobacco. Polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) were used to determine changes in the microbiota of tobacco during the aging of eleven different tobacco grades stored at three different locations for twelve months. The microbial fraction of these tobacco grades was extracted, and the bacterial 16S and the fungal 18S ribosomal RNA gene (rDNA) sequences were PCR amplified before being segregated by DGGE. The bacterial complexity of the tobacco grades was represented by DGGE migrating banding profiles that varied between 20 and 30 bands. Some variations in the banding profiles were observed between the tobacco grades, but overall no substantial changes occurred in the bacterial population of the different grades during their storage at different locations. Most of the fungal DGGE profiles were identical and had only one dominating band related to the genus Aspergillus. Bacterial and fungal isolates were also derived from the microbial fractions of the tobacco, and part of their respective 16S and 18S rDNA sequences were determined. Bacterial isolates belonged to Bacillales and gamma Protobacteria. Fungal isolates belonged to the genus Aspergillus. Our results showed that the bacterial and fungal biota of tobacco are relatively stable throughout 12 months storage time.