Changes in TSNA contents during tobacco storage and the effect of temperature and nitrate level on TSNA formation

Transcription

1 Changes in TSNA contents during tobacco storage and the effect of temperature and nitrate level on TSNA formation HONGZHI SHI 1, RUIYUN WANG 1, LOWELL P. BUSH 2, JUN ZHOU 3, FRANKLIN F. FANNIN 2, RUOSHI BAI 3, HUIJUAN YANG 1 1 Henan Agricultural University, National Tobacco Cultivation & Physiology & Biochemistry Research Center, Zhengzhou, China; 2 University of Kentucky, Lexington, KY 40546, USA; 3 Beijing Cigarette Factory of Shanghai Tobacco Group, Beijing China 2013 CORESTA Agro-phyto Joint Meeting, Oct 12-17,2013,Italy

2 Tobacco specific nitrosamines are important toxic components in tobacco and maintained their status as important main stream smoke toxicants due to its specificity in tobacco and tobacco products Reduction of TSNA formation and accumulation in tobacco leaves and their products have been important topics for many researchers around the world Tobacco-specific nitrosamines (TSNAs) are formed from the nitrosation of tobacco alkaloids. They could be formed during both air-curing and leaf storage.

3 In China air-cured leaves generally are stored on farm for 2-3months before selling to tobacco companies. Then, they are stemmed and re-dried, followed by 18 months storage in the company warehouses before being processed for cigarette production. The period of storage after air-curing is an important time of TSNA formation; previous research revealed that the TSNA level may increase several fold compared with that in the freshly air-cured leaves. What is the mechanism of TSNA formation during storage.

4 Our hypothesis : Both factors (high nitrate content in leaves,high temperature) are essential for TSNA formation during tobacco storage. Burley tobacco had much higher levels of nitrate than that in flue-cured tobacco, nitrate is a chemically unstable compound and may produce gaseous nitrogen oxides under high temperatures which could react with alkaloids to form TSNAs by nitrosation. This may be the cause of TSNA increase during storage under relatively high temperatures.

5 In our research : A series of experiments were designed to investigate the changes in TSNA contents during a one year storage and the effect of temperature and nitrate on TSNA formation to elucidate the mechanism of TSNA formation during storage and to provide theoretical basis for the adoption of environment and chemical control measures to minimize TSNA formation and accumulation after air-curing.

6 Chemical Measurements TSNA measurement: Tobacco samples were sent to University of Kentucky of USA and run for TSNA in the Tobacco Testing Laboratory of the Department of Agronomy and Soil Science. GC- TEA method was used to conduct the analysis Nitrate and nitrite: Also tested in the Tobacco Testing Laboratory in University of Kentucky Alkaloid measurement: MTBE was used as extraction agent and quinoline as internal standard. gas chromatography equipped with a DB-5 capillary column was used to determine the alkaloid contents

7 Results Changes in TSNA contents in storing tobacco leaves of different types Air-cured burley tobacco and sun-cured tobacco produced in 2011 season were put into storage in the cold season after the first sampling, and then samples were taken every 4 months. Individual TSNA in all the collected samples were analyzed after one year storage.

8 Changes in NNK contents of burley and sun-cured tobacco during storage in ambient environment With increased storage time, NNK contents in both burley and sun-cured tobacco increased throughout the year, and that the largest increase occurred during the second 4 month period which coincided with the high temperature season

9 Changes in NNN contents of burley and sun-cured tobacco during storage in ambient environment For NNN content, there was no significant change occurring during the first 4 month time period; while the second period which was from middle of April to middle of August when the air temperature was high, NNN contents increased very significantly.

10 Changes in NAT contents of burley and sun-cured tobacco during storage in ambient environment NAT contents of burley and sun-cured tobacco were greatly increased during one year storage and magnitude was the largest in the high temperature season

11 Changes in NAB contents of burley and sun-cured tobacco during storage in ambient environment The NAB contents of burley and sun-cured tobacco increased slowly in the first 4 month period, followed by a rapid increase in the second 4 month, similar to others.

12 Changes in total TSNA contents of burley and suncured tobacco during storage in ambient environment During the one year period of storage the total TSNA contents increased continuously, but with the second 4 month period having a greater rate of increase for both burley and suncured tobacco. The total TSNA content in burley tobacco was consistently higher than that in sun-cured tobacco.

13 The increase of TSNA contents during the one-year ambient storage of burley and sun-cured tobacco coincided with the changes in temperature. -- First 4-month period, average temperature Second 4-month period,average temperature 26 --Third 4-month period, average temperature 15.4 Apparently, the increase of TSNA contents during the one year storage time was highly related to the storage temperature, and the higher temperatures were favorable to the accumulation of TSNA.

14 Results Response of TSNA formation to high temperature treatment for different tobacco types Samples of burley, flue-cured and suncured tobacco were put in temperature controlled chambers for 36 days at 45 and 10.

15 Effect of temperature on NNK formation in flue-cured, burley and sun-cured tobacco during 36 days of storage Flue-cured tobacco had very low levels of NNK content, and remained constant in both temperature treatments,. In contrast, burley and sun-cured tobacco had much higher levels of NNK contents than flue-cured tobacco and showed dramatic increase when the samples were stored at high temperature (45 ) compared with that stored at 10.

16 Effect of temperature on NNN formation in flue-cured, burley and sun-cured tobacco during 36 days of storage NNN content in flue-cured tobacco was low and did not change with storage at either temperature treatment. NNN of burley and sun-cured tobacco both increased with storage at high temperatures but the increase was greater in burley tobacco.

17 Effect of temperature on NAT formation in flue-cured, burley and sun-cured tobacco during 36 days of storage NAT contents in burley and sun-cured tobacco not only had much higher level, but also had a strong response to high temperature treatment.

18 Effect of temperature on NAB formation in flue-cured, burley and sun-cured tobacco during 36 days of storage Burley and sun-cured tobacco had substantially higher levels of NAB than flue-cured tobacco, and the high temperature treatment greatly increased NAB formation in burley and sun-cured tobaccos.

19 Effect of temperature on total TSNA formation in flue-cured, burley and sun-cured tobacco during 36 days of storage As was the case for each individual TSNA, total TSNA in flue-cured tobacco was the lowest among the tobacco types and there was no significant increase when the tobacco was stored at high temperature over tobacco stored at low temperature. Contrarily, burley tobacco and sun-cured tobacco had a significant increase in TSNAs to high temperature treatment.

20 Alkaloid, nitrate and nitrite contents among tobacco types and the response to temperature Flue-cured burley Sun-cured nicotine /mg g nornicotine alkaloids anabasine /mg NOx anatabine /mg Percent nicotine conversion(%) NO 3 -N /µg g NO 2 -N /µg g Burley tobacco had significantly higher nicotine content than flue-cured tobacco The changes in individual alkaloids were not significant between low and high temperature conditions. Burley tobacco had extremely high nitrate content relative to the other tobacco types, and the nitrite contents in burley and sun-cured tobacco were increased substantially in 45 treated tobacco over the 10 treated tobacco.

21 Results Effects of temperature on TSNA formation of air-cured burley tobacco To accurately determine the effect of temperature on TSNA formation in burley tobacco, five shredded tobacco samples were stored in controlled temperature chambers at 10, 27, 30, 45 and 60.

22 NNK content of air-cured burley tobacco stored at different temperatures High temperatures significantly promoted NNK formation. Especially when temperatures exceeded 30, TSNA accumulation increased dramatically.

23 NNN content of air-cured burley tobacco stored at different temperatures

24 NAT content of air-cured burley tobacco stored at different temperatures

25 NAB content of air-cured burley tobacco stored at different temperatures

26 Total TSNA content of air-cured burley tobacco stored at different temperatures High temperatures significantly promoted TSNA formation. Especially when temperatures exceeded 30C, TSNA accumulation increased dramatically. There was a significant, but small, TSNA increase from 10C to 30C. For tobacco stored at 60C for 24 days, the increase was by almost 10 times

27 From the results: Storage temperature as high as 27 was enough to induce increased TSNA formation, while the most significant effect was shown when the temperature was above 30, and the rate of increase became greater as the temperature increased, indicating that the high temperature was very favorable to TSNA formation and that the high temperature was one of the main contributors for the TSNA formation during the storage of air-cured burley tobacco.

28 Results Effect of addition of nitrate and nitrite to flue-cured tobacco on TSNA formation at high temperature Low level of nitrate content is characteristic of cured leaves of fluecured tobacco and they had minimal response of increased TSNA to high temperature treatments. To determine the effect of nitrate content on TSNA formation, we added nitrate to flue-cured tobacco to the levels equivalent to the nitrate content of air-cured burley tobacco to determine if increased nitrate content can trigger the response of TSNA formation to high temperature. Nitrates from 3 different nitrate salts (NH 4 NO 3, KNO 3, NaNO 3 ) were added separately to cut flue-cured tobacco before high temperature treatment.

29 Effect of adding nitrate and nitrite to flue-cured tobacco on TSNA formation under high temperature treatment NNN /μg g -1 NAT /μg g -1 NAB /μg g -1 NNK /μg g -1 TSNAs /μg g -1 Water only NH 4 NO KNO NaNO NaNO High nitrate content in flue-cured tobacco equivalent to burley tobacco resulted in a strong increase of TSNA with high temperature treatment. However, the degree of increase of TSNAs varied with different nitrate source.

30 Conclusion & Discussion Contents of TSNAs in burley and sun-cured tobacco increased substantially during one-year under natural storage. The most rapid increase occurred during the high temperature season. Temperature had significant promoting effect on TSNA formation during storage. Different types of tobacco showed different results in terms of the response of TSNA formation to high temperature. Addition of nitrate onto flue-cured followed by high temperature treatment increased the TSNA concentration.

31 Conclusion & Discussion TSNA formation during leaf storage was the consequence of the interaction between high temperature and abundant nitrate content in cured tobacco. Nitrate is a chemically unstable compound, and high temperature could cause it to give rise to gaseous nitrite or other volatile nitrogen oxides, which may easily react with alkaloids to form TSNAs. Therefore, the control of storage environment, reduction of leaf nitrate content and the scavenging of gaseous nitrosating agents would be effective pathways to reduce or inhibit TSNA formation during the storage of cured tobacco.

32 Thank you! 谢谢各位!