The ability of 20 compounds, all but one tobacco constituents, to inhibit the formation of tobacco-specific N-nitrosamines (TSNA) was investigated in buffer and detergent solution and in tobacco midrib and lamina systems. In solution at pH 5.5, ascorbic acid and the phenolic acids caffeic and ferulic acid were the most potent inhibitors of the reaction between nornicotine and nitrite, with nearly complete inhibition at molar ratios test compound/nitrite > 1:1. Also, cysteine > dihydrocaffeic acid > protocatechuic acid approximately catechin acted as strong inhibitors with >90% inhibition at a ratio of 3:1. Lower inhibitions were observed with chlorogenic acid > p-coumaric acid > sclareol > serine. Rutin showed an inhibition of 34% at a ratio of 0.1:1. Sclareol, alanine, proline, and serine did not significantly affect the N-nitrosonornicotine (NNN) formation. alpha-Tocopherol and glutathione enhanced NNN formation at pH 5.5 but were inhibitors at pH 3. Cinnamic acid, vanillic acid, eugenol, and esculin enhanced NNN formation. Increased NNN formation was also observed for dihydrocaffeic acid, chlorogenic acid, protocatechuic acid, and catechin at a less-than-equimolar ratio of test compound to nitrite. The tobacco matrix experiments were performed with air-cured, ground tobacco midrib and lamina. Caffeic acid, ferulic acid, dihydrocaffeic acid and catechin were potent inhibitors of the formation of TSNA in the midrib as well as in the lamina. Also protocatechuic acid, glutathione, ascorbic acid, p-coumaric acid, chlorogenic acid and cysteine were inhibitors, while alpha-tocopherol and rutin inhibited the reaction in the midrib but not in the lamina. Cinnamic acid, vanillic acid, eugenol, alanine, proline and serine showed small effects only. The molar ratio of secondary alkaloid(s)/nitrite in the test systems were 0.1:1 (solution), approximately 0.25:1 (midrib), and approximately 1:1 (lamina) and is most likely the major contributor to the observed order of inhibition potency (solution> midrib > lamina) of the test compounds. The vicinal phenolic hydroxyl groups of polyphenols and the simultaneous presence of a phenol group and an olefinic bond in hydroxycinnamic acids were the most characteristic structural elements of the potent inhibitors.

Several trials the results of which are compiled in this paper, were carried out at the Tobacco Institute of Bergerac (ITB) and in the area nearby from 1996 to 2003. The objective was to study the formation of tobacco-specific nitrosamines (TSNA) in dark air-cured and burley tobaccos during curing and post-curing treatment under the conditions of cultivation, curing and storage commonly applied by the growers in the south-western part of France. For experimental purposes special treatments were performed in certain trials.The results showed that the main genetic trait involved in the formation of TSNA is the propensity of a variety to convert nicotine to nornicotine (NN). In addition, the ability of a variety to lose water rapidly limits the formation of nitrite and hence also the formation of TSNA. Furthermore, agricultural practices that led to an increase of alkaloid concentrations in the tobacco leaves also led to an increase of TSNA concentration. Priming, a mode of harvest which speeds up the cure, as compared to stalk-cutting, as well as low temperatures during curing, limit the formation of TSNA, but do not yield tobaccos of the best quality. Ventilation in the barn plays a major role, and the leaves cured in well ventilated curing structures, such as plastic sheds, generally contained smaller amounts of TSNA than leaves cured in a conventional curing barn. The results also indicated that the TSNA concentrations may increase after the end of cure, if the cured tobaccos were kept hanging in the barn under humid conditions. The concentration of TSNA may also continue to increase, whereas nitrite concentrations tend to decrease, when the leaves are kept in bales.It can be concluded that the French climatic conditions with moderate temperatures and low relative humidity at the time of curing, are favourable for the production of air-cured tobaccos with a good quality and low TSNA concentrations (1.5-3.5 µg/g), provided that the variety has low NN content, the nitrogen fertilization is moderate, the curing is performed in a well ventilated environment, the tobacco is taken down and stripped as soon as it is cured, and the bales are stored as briefly as possible before the leaves are threshed and stabilized.

The present review deals with studies performed during several consecutive years on the effect of air-curing on tobacco-specific nitrosamine (TSNA) formation and quality of tobacco. Temperature, relative humidity, water content and water activity data were collected during curing of dark tobacco in traditional air-curing barns and bulk-curing barns of different sizes, and chemical analysis of the cured tobacco were performed.