Decreased Tobacco-Specific Nitrosamines by Microbial Treatment

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Decreased Tobacco-Specific Nitrosamines by Microbial Treatment with Bacillus amyloliquefaciens DA9 during the Air-Curing Process of Burley Tobacco Xuetuan Wei,‡,∥ Xiaowu Deng,∥ Dongbo Cai,∥ Zhixia Ji,∥ Changjun Wang,§ Jun Yu,§ Jinping Li,§ and Shouwen Chen*,†,∥ †

Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan 430062, People’s Republic of China ‡ College of Food Science and Technology and ∥State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China § Tobacco Research Institute of Hubei Province, Wuhan 430062, People’s Republic of China ABSTRACT: Tobacco specific nitrosamines (TSNA) mainly consisting of N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are a group of toxic components threatening human health. To inhibit TSNA formation in tobacco leaves, a high nitrite reductive strain with low nitrate reduction ability was isolated and applied to tobacco leaves in an attempt to lower the nitrite precursor of TSNA. By morphology, physiology, biochemistry, and 16S rDNA sequence analysis, the strain DA9 was identified as Bacillus amyloliquefaciens. Under the optimized fermentation parameters (glucose 40 g/L, NH4Cl 4 g/L, corn steep liquor 8 g/L, MnSO4 0.01 g/L, KH2PO4 1.0 g/L, MgSO4 0.3 g/L, initial pH 7.0, inoculum age 6 h, inoculum size 3%, temperature 37 °C), the maximum cell dentisity of 1.2 × 109 CFU/mL was obtained at 36 h. The DA9 cell suspensions were applied in the air-curing process of the Burley tobacco (Eyan 6) leaves. The treatment by DA9 cells lowered 32% of the nitrite content and 47% of total TSNA content in the tobacco leaves, and the concentrations of the NNN, NNK, and NAT were decreased by 48%, 12%, and 35%, respectively. Collectively, this study provides a promising strain and a novel strategy for decreasing TSNA during the aircuring process. KEYWORDS: TSNA, Bacillus amyloliquefaciens, nitrite, microbial treatment, air-curing process

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as processing techniques,6 cultivation conditions,7 tobacco lines, and producing areas.8 Various strategies have been developed to lower the TSNA contents, such as genetic breeding,9,10 control of nitrogen fertilization level,7 implementation of curing conditions,6 and addition of nitrite or alkaloid scavenger,11,12 and more novel methods are needed to further decrease the TSNA contents. As the direct precursor of TSNA, the nitrite contents in tobacco leaves directly correlate with TSNA formation,13 and a decreased nitrite content would result in a decreased TSNA formation. It has been well-known that nitrite in tobacco leaves was formed from nitrate reduction action which is mediated by the nitrate reductases from tobacco leaves and, more importantly, their symbiotic microbes.7 Indeed, various microbes have been reported to exist in tobacco leaves, such as Pseudomonas, Enterobacter, and Bacillus species.14 The tobacco cells contain various nutrients,15 which are released to promote the growth of symbiotic microbes, and this will eventually result in the accumulation of nitrite from the nitrate reduction action.16 On the other hand, nitrite can be further transformed into ammonia or N2 by microbial nitrite reduction

INTRODUCTION Tobacco specific nitrosamines (TSNA), mainly consisting of Nnitrosonornicotine (NNN), N-nitrosoanatabine (NAT), Nnitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK), is a group of important components in tobacco and tobacco smoke, of which NNN and NNK are reported to be the main carcinogens.1 The NNN and NNK have been listed as harmful components by International Agency for Research on Cancer (IARC),2 and US Food and Drug Administration (FDA).3 Decreasing TSNA content in tobacco and tobacco products is, therefore, essential to reduce their harmful effects. Though designing new cigarette filters can lower the TSNA content in the main stream of tobacco smoke,4 the side stream of smoke still contains a significant amount of TSNA. Therefore, decreasing the TSNA contents of tobacco products by inhibiting TSNA synthesis in tobacco leaves is a more efficient way to lower the harmful effects of TSNA. It has been reported that alkaloids as the precursors react with nitrite to synthesize TSNA, and the formation of different TSNA components depends on the different types of alkaloids reacting with nitrite. For example, nornicotine, anatabine, and anabasine react with nitrite to form NNN, NAT, and NAB, respectively, and NNK is formed by nitrosation of pseudo-oxo nicotine (PON), an oxidative compound from nicotine.5 In general, the TSNA contents are affected by many factors such © XXXX American Chemical Society

Received: August 25, 2014 Revised: December 4, 2014 Accepted: December 4, 2014

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action.17 Therefore, inhibiting nitrite formation from nitrate reduction, as well as enhancing nitrite reduction, can be efficient to decrease the final nitrite content. The aim of this study is to lower the TSNA content of tobacco leaves during the air-curing process using a high nitrite reductive strain with low nitrate reduction capability.

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Leaves sprayed with the same volume of sterile distilled water were used as the control. The fresh leaves (0 day) and the leaves at different air-curing stages (7, 20, and 45 day) were collected, and the concentrations of total cells, nitrate, and TSNA compounds (NNN, NNK, NAB, and NAT) were determined. Determination of Nitrate and Nitrite. Nitrate was determined by the salicylic acid method.21 In brief, 0.1 mL of sample and 0.4 mL of salicylic acid solution (5%) were mixed and added into 9.5 mL of NaOH solution (8%). The reaction mixture was incubated for 20 min and determined for its absorbance at 420 nm. NaNO3 was used as the standard substance. For determination of the nitrite, 4 mL of sulfanilamide solution (1%) was mixed with 2 mL of sample solution in the test tube, supplied with 4 mL of α-naphthylamine solution (0.02%), and maintained at 25 °C for 30 min. The absorbance of the sample was determined at 540 nm with NaNO2 being used as the standard.22 Analysis of the TSNA Concentration. The tobacco leaf samples were dried at 45 °C to reach constant weight, smashed, and sieved. 0.25 g of tobacco powder was placed into an erlenmeyer flask and combined with 0.2 mL of internal standard solution (5000 ng/mL) and of 19.8 mL ammonium acetate solution (100 mM). The mixture was shaken at 150 rpm for 1 h, filtered, and collected for analysis of the contents of TSNA compounds by an LC−MS/MS system (Agilent Technologies Corporate, Santa Clara, CA, USA). The HPLC was carried out on an Agilent 1290 system using an Agilent ZORBAX XDB-C18 column (Agilent Technologies, Santa Clara, CA, USA). The column temperature was set as 65 °C with an injection volume of 5 μL. 0.1% acetic acid water solution and 0.1% acetic acid methanol solution were used as the mobile phases A and B respectively. The gradient elution conditions were programmed as follows: 0.0−0.5 min, 60−70% B; 0.5−1.0 min, 70% B; 1.0−1.5 min, 70−90% B; 1.5−2.3 min, 90−60% B; 2.3−2.5 min, 60% B. MS/MS was performed using the Agilent 6460 triple quadrupole tandem mass spectrometer (Agilent Technologies Corporate, Santa Clara, CA, USA), and the conditions were controlled as follows: ESI ion source, positive ion mode, 4000 V voltage, 35 psig nebulizer pressure, 10 L/min gas flow rate, and 350 °C vaporizer temperature. The deuterated equivalents of NNN-D4, NNKD4, NAB-D4, and NAT-D4 were used as the internal standard to determine the contents of NNN, NNK, NAB, and NAT, and the total TSNA contents were calculated by summing the NNN, NNK, NAT, and NAB. Statistical Analyses. Each experiment was carried out at least in triplicate, and t test was performed to analyze the difference in means at the 95% confidence level using Statistica 6.0 software package.

MATERIALS AND METHODS

Samples and Chemicals. Tobacco leaves and involved rhizosphere soil samples were collected from Shiyan, Enshi, and Wuhan of Hubei Province in China for strain isolation. The standard substances of NNN, NNK, NAB, and NAT were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). The deuterated equivalents of NNND4, NNK-D4, NAB-D4, and NAT-D4 were bought from C/D/N Isotopes Inc. (Pointe-Claire, Quebec, Canada). All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Isolation of the Nitrite Reductive Strain. The tobacco and soil samples were added into the liquid screening medium (NaNO2 0.5 g/ L, MgSO4·7H2O 0.2 g/L, K2HPO4 0.5 g/L, KNaC4H4O6·4H2O 20.0 g/L, pH 7.2), cultured at 37 °C and 180 rpm for 48 h. The cultures were then diluted, and spread onto the solid screening medium containing 15 g/L agar, and incubated at 37 °C for another 24 h. The single clone was then inoculated in the nitrate medium (NaNO3 1 g/L, peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.2) and nitrite medium (NaNO2 0.5 g/L, peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.2), respectively. After culture at 37 °C and 180 rpm for 12 h, the concentrations of nitrate and nitrite in the medium were determined. The high nitrite reductive strain with low nitrate reduction ability was selected. Fermentation of Tobacco Leaves. The isolated nitrite reductive strains were cultured in LB liquid medium (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.2) at 37 °C and 180 rpm for 12 h, and subcultured for 24 h. The cells were collected by centrifugation at 7656g for 10 min and diluted with sterile distilled water to obtain the suspension of bacteria (1 × 108 CFU/mL). The suspension of bacteria was sprayed onto the Burley tobacco (Eyan 6) leaves and incubated at 30 °C with 30% moisture content and 80% relative humidity. After incubating for 5 days, the contents of nitrite and various TSNA compounds (NNN, NNK, NAB, and NAT) were determined. Identification of the Strain. The isolated nitrite reductive strain was identified by physiological, biochemical, and 16S rDNA sequence analysis. The traditional physiological and biochemical experiments were performed based on Bergey’s Manual of Systematic Bacteriology.18 The 16S rDNA sequence analysis was carried out according to our previous report.19 The 16S rDNA sequence was amplified using the primers of 27f (AGAGTTTGATCMTGGCTCAG) and 1492r (GGTTACCTTGTTACGACTT), and the PCR was performed as follows: 95 °C for 5 min; 95 °C for 45 s, 54 °C for 45 s, 72 °C for 1.5 min, 28 cycles; 72 °C for 10 min; and 4 °C for 10 min. The DNA products were then purified, recovered, and sequenced by the BGI Tech Solutions Co., Ltd. (Shenzhen, China). The sequence similarity analysis was carried out by the Blastn program (http://blast.ncbi.nlm. nih.gov/Blast.cgi), and the phylogenetic tree was constructed using MEGA 4.0.20 Batch Culture. The selected strain was inoculated into the LB liquid medium, cultured at 37 °C and 180 rpm for 12 h as the seed cultures. With 3% (v/v) inoculum size, the seed cultures were transferred into the initial fermentation medium (glucose 20 g/L, NH4Cl 10 g/L, corn steep liquor 10 g/L), incubated at 37 °C and 180 rpm for 36 h, and the medium components as well as culture conditions were optimized for maximum cell growth. After culture at the optimized fermentation parameters, the cells were collected by centrifugation at 7656g for 10 min, and applied in the air-curing process of tobacco. Microbial Treatment of Tobacco Leaves during the AirCuring Process. The cells collected from batch culture were diluted to 2 × 108 CFU/mL, and 30 mL cell suspensions were sprayed onto 300 g of fresh Burley tobacco (Eyan 6) leaves for air-curing treatment.

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RESULTS AND DISCUSSION Isolation of the TSNA-Decreasing Strain. Various microbes coexist with the tobaccos,23,24 and thus the leaves and rhizosphere soils of tobaccos were collected for strain isolation. In this work, we isolated 304 clones from different tobacco and rhizosphere soil samples, and those strains were cultured in nitrite and nitrate selection medium. Among those strains, eight strains had high nitrite reducing capabilities. As shown in Figure 1, the degradation efficiencies of three strains (DA9, 1-4, and CH3) were higher than 30%, while the strain DA9 had the lowest nitrate degradation capability (11%). Due to the unique feature of high nitrite reductivity and low nitrate reductivity, the strain DA9 was selected for further verification by fermentation of tobacco leaves. As shown in Table 1, the nitrite concentration after fermentation with DA9 decreased 39% compared with the control, and the total TSNA content decreased 56% accordingly. Further analysis revealed the significant difference of each individual TSNA compound, and the NNN, NNK, NAT, and NAB content decreased by 57%, 69%, 46%, and 52%, respectively. The results indicated that the strain DA9 had the potential to lower the TSNA content in tobacco leaves. B

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Table 2. Physiological and Biochemical Characterization of the Strain DA-9a sole carbon test

Figure 1. Degradation ratios of different strains on nitrite and nitrate. The isolated strains were cultured in the nitrate medium and nitrite medium at 37 °C and 180 rpm for 12 h respectively, and then the concentrations of residual nitrate and nitrite in the medium were determined to calculate corresponding degradation ratios.

acid productive test

property

characterization

property

characterization

sorbose ribose glucose sucrose maltose glycerol lactose starch arabinose trehalose mannose rhamnose xylose raffinose melizitose

+b + + + + + + + − + − + + + −

sucrose lactose glycerol mannitol glucose other tests Gram stain sporulation indole formation lecithase test cellulose hydrolysis starch hydrolysis gelatin hydrolysis growth at 4 °C growth at 45 °C

+ −c + + + + + − + − + + + +

a

The physiological and biochemical analysis of strain DA9 was carried out according to Bergey’s Manual of Systematic Bacteriology.18 bPositive. c Negative.

Identification of the Strain. The strain DA9 was identified by morphological, physiological, biochemical, and 16S rDNA sequence analysis. After culturing in LB agar medium, white colonies with rough surface and irregular edge were observed. Under the microscope, the bacterial cells showed rod shape with strong motility. The physiological and biochemical characteristics of DA9 strain (Table 2) indicate that DA9 belonged to the Bacillus genus.18 The 16S rDNA sequence of 1423 bp was obtained from DA9, aligned with the sequences in GenBank using the Blastn program, and revealed 100% identity to that of the Bacillus amyloliquefaciens LFB112 (CP006952).25 The phylogenetic tree constructed on the basis of the 16S rDNA sequences shows that the DA9 clustered with B. amyloliquefaciens (Figure 2). Collectively, the above results indicate that the strain DA9 belonged to B. amyloliquefaciens. In a separate report, Bacillus and Pseudomonas were reported to be the two dominant genera, of which B. amyloliquefaciens was also found in the flue-cured tobacco leaves.14 The 16S rDNA sequence of DA9 was submitted into the GenBank database (Accession number KM272942), and this strain was preserved in China Center for Type Culture Collection (CCTCC M2014252). Optimization of Fermentation Parameters. To maximize the growth of the isolated B. amyloliquefaciens DA9, we optimized the medium components for this strain including glucose, corn steep liquor, NH4Cl, MnSO4, KH2PO4, and MgSO4. Based on the preliminary screening results (data not shown), three key components, glucose (A), NH4Cl (B), and corn steep liquor (C), were identified as the factors with significant effects on the cell growth, therefore, these three medium compositions were further optimized using threefactor-three-level orthogonal design. As shown in Table 3, range analysis results were observed as R(C) > R(A) > R(B),

Figure 2. Phylogenetic tree of DA9 based on 16S rDNA sequences. Numbers in parentheses indicates the sequence accession numbers of the representative organisms. The scale bar represents 0.01 nucleotide substitution per position.

indicating that the influential effects of the three components were ranged as corn steep liquor (C) > glucose (A) > NH4Cl (B). The k value analysis showed that the optimal factor levels were glucose (A3), NH4Cl (B1), corn steep liquor (C2). Based on the above results, the optimized medium components were determined as glucose 40 g/L, NH4Cl 4 g/L, corn steep liquor 8 g/L, MnSO4 0.01 g/L, KH2PO4 1.0 g/L, MgSO4 0.3 g/L. The culture conditions were also investigated follwing the medium optimization. It was found that the combined conditions of initial pH 7.0, inoculum age 6 h, inoculum size 3%, and temperature 37 °C were optimal for cell growth (data not shown). Under those optimized parameters, the DA9 cells were cultured, and the maximum cell dentisity reached 1.2 × 109 CFU/mL at 36 h. Those cells were then collected for applying to the tobacco leaves during the air-curing process.

Table 1. Effects of Fermentation with DA9 on the Contents of Tobacco Nitrite and TSNAa sample

nitrite content (μg/g)

TSNA content (μg/g)

NNN content (μg/g)

NNK content (μg/g)

NAT content (μg/g)

NAB content (μg/g)

control treatment

14.05 ± 0.01 8.59 ± 1.09

94.00 ± 2.10 41.64 ± 0.75

81.65 ± 1.02 35.30 ± 0.74

1.31 ± 0.13 0.41 ± 0.01

10.43 ± 1.24 5.64 ± 2.00

0.62 ± 0.03 0.30 ± 0.02

a The DA9 cell suspensions (1 × 108 CFU/mL) were sprayed onto the Burley tobacco leaves and fermented at 30 °C with 30% moisture content and 80% relative humidity for 5 days to determine the concentrations of nitrite and the various tobacco specific nitrosamine (TSNA) compounds Nnitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanatabine (NAT), and N-nitrosoanabasine (NAB).

C

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Table 3. Results of Orthogonal Testa

their symbiotic microbes can mediate the nitrate reduction action during the air-curing process,7 which may be the main reason for the increased nitrite formation. By treating the leaves with DA9 cells, however, the maximum nitrite content just reached 12.06 μg/g, and such an increment was significantly lower than the control (Figure 3A). For the control, the native strain number changed from 9.33 × 105 CFU/g to 1.05 × 105 CFU/g after air-curing of tobacco leaves for 45 d, while the DA9 treated tobacco leaves had bacterial counts ranging from 8.71 × 108 CFU/g to 1.32 × 108 CFU/g, much higher than that of the control. As the dominant microflora, the DA9 cells inhibited the growth of other native bacteria and, thus, lowered those bacteria’s capability of nitrate reduction. This feature, together with the strong nitrite reduction ability, makes the DA9 strain an ideal strain for decreasing nitrite during the tobacco air-curing process. It has been reported that nitrite was the direct precursor for TSNA synthesis.5 Therefore, decreased nitrite content inhibited TSNA formation. As shown in Figure 3B, the total TSNA concentration in the control increased before 20 d, which is in agreement with a previous report.26 However, the maximum TSNA concentration reached 56.94 μg/g at 20 d, while the content decreased to 32.19 μg/g at 45 d, which is a little higher than previous reports.6,27 The Burley tobaccos have higher TSNA levels than other tobacco lines, such as flue-cured and oriental tobaccos.6 Moreover, the average TSNA content of aircured Burley tobacco from Hubei Province is much higher than that of Sichuan, Yunnan Province, and USA.8 The TSNA contents are significantly affected by processing techniques,6 cultivation conditions,7 tobacco lines, and producing areas,8 which may result in the high TSNA levels of air-cured tobacco in Hubei Province. Figure 3B also shows that, after treatment with DA9, the total TSNA content of tobacco leaves was much lower than that of the control at each stage. The final TSNA content at 45 d reached 17.03 μg/g (Figure 3B), which was 47% lower than the control. The above results indicate that treatment by B. amyloliquefaciens DA9 was an efficient way to lower the nitrite and TSNA contents of air-cured Burley tobacco. Effects of Microbial Treatment on the Contents of NNN, NNK, NAT, and NAB. The individual compounds of TSNA, i.e., NNN, NNK, NAT, and NAB, were further analyzed. As the direct substrate, nornicotine shows positive correlation with NNN formation.9 Among the alkaloid precursors of TSNA, nornicotine was most abundant, and thus the NNN preponderated among the four kinds of TSNA. Similar to previous results,6 the NNN contents detected were much higher than those of other TSNA components (Figure 4), and thus the change trend of NNN agreed with that of total TSNA contents (Figure 3B). After microbial treatment with DA9, the NNN content was 15.53 μg/g at 45 d, a 48% decrease compared to the control (Figure 4A). Meanwhile, similar inhibitory effects were observed for NNK and NAT, which decreased by 12% and 35% after 45 d (Figure 4B and Figure 4C), while no significant difference was found in NAB contents (Figure 4D). NAT and NAB show little biological activity, while NNN and NNK are the main carcinogens.1 In summary, our results show that microbial treatment with DA9 can efficiently lower the contents of the main carcinogens of TSNA in Burley tobacco. Tobacco leaves treated with DA9 have the potential to be applied in cigarette as the filler, which may help to decrease the probability of smoking-related cancer. The health implications of smoking tobacco leaves treated with B.

factor

number 1 2 3 4 5 6 7 8 9 k1 k2 k3 R

A: glucose (g/L)

B: NH4Cl (g/L)

C: corn steep liquor (g/L)

20 20 20 30 30 30 40 40 40 5.53 5.58 8.10 2.56

4 8 12 4 8 12 4 8 12 7.76 5.59 5.87 2.16

4 8 12 8 12 4 12 4 8 4.56 8.33 6.32 3.77

OD600 4.26 8.07 4.27 8.22 3.92 4.62 10.78 4.80 8.71

a

The experiments were performed using three-factor-three-level orthogonal design. The R value indicates the range, and the k value represents the average biomass of each factor level.

Effects of Microbial Treatment with DA9 on the Contents of Nitrite and Total TSNA in the Tobacco Leaves. The B. amyloliquefaciens DA9 cells were sprayed onto the tobacco leaves; the same volume of distilled water was also sprayed onto the leaves as control. After the air-curing process, the nitrite content of the control increased from 10.20 μg/g to 17.77 μg/g (Figure 3A). The nitrate reductases of tobaccos and

Figure 3. Effects of microbial treatment on the nitrite (A) and tobacco specific nitrosamine (TSNA) (B) concentrations of the tobacco leaves during the air-curing process. 30 mL cell suspensions (2 × 108 CFU/ mL) were sprayed onto 300 g of fresh Burley tobacco leaves for aircuring treatment. At 0, 7, 20, and 45 days, the control without treatment and the tobacco leaves treated with DA9 cells were sampled to determine the concentrations of nitrite and total TSNAs. D

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Figure 4. Effects of microbial treatment on the concentrations of N-nitrosonornicotine (NNN) (A), 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) (B), N-nitrosoanatabine (NAT) (C), and N-nitrosoanabasine (NAB) (D) of the tobacco leaves during the air-curing process. The DA9 cell suspensions (2 × 108 CFU/mL) were sprayed onto the fresh Burley tobacco leaves and air-cured for 0, 7, 20, and 45 days, and then the concentrations of NNN, NNK, NAB, and NAT were determined. (4) Wang, C.; Dai, Y.; Feng, G.; He, R.; Yang, W.; Li, D.; Zhou, X.; Zhu, L.; Tan, L. Addition of porphyrins to cigarette filters to reduce the levels of benzo[a]pyrene (b[a]p) and tobacco-specific n-nitrosamines (TSNAs) in mainstream cigarette smoke. J. Agric. Food Chem. 2011, 59, 7172−7177. (5) Hecht, S. S. Biochemistry, biology, and carcinogenicity of tobacco-specific n-nitrosamines. Chem. Res. Toxicol. 1998, 11, 559− 603. (6) Shi, H.; Wang, R.; Bush, L. P.; Zhou, J.; Yang, H.; Fannin, N.; Bai, R. Changes in tsna contents during tobacco storage and the effect of temperature and nitrate level on TSNA formation. J. Agric. Food Chem. 2013, 61, 11588−11594. (7) Lewis, R. S.; Parker, R. G.; Danehower, D. A.; Andres, K.; Jack, A. M.; Whitley, D. S.; Bush, L. P. Impact of alleles at the yellow burley (yb) loci and nitrogen fertilization rate on nitrogen utilization efficiency and tobacco-specific nitrosamine (TSNA) formation in aircured tobacco. J. Agric. Food Chem. 2012, 60, 6454−6461. (8) Shi, H. Z.; Wang, R. Y.; Bush, L. P.; Yang, H. J.; Fannin, F. F. The relationships between TSNAs and their precursors in burley tobacco from different regions and varieties. J. Food Agric. Environ. 2012, 10, 1048−1052. (9) Gavilano, L. B.; Coleman, N. P.; Burnley, L.-E.; Bowman, M. L.; Kalengamaliro, N. E.; Hayes, A.; Bush, L.; Siminszky, B. Genetic engineering of nicotiana tabacum for reduced nornicotine content. J. Agric. Food Chem. 2006, 54, 9071−9078. (10) Lewis, R. S.; Jack, A. M.; Morris, J. W.; Robert, V. J. M.; Gavilano, L. B.; Siminszky, B.; Bush, L. P.; Hayes, A. J.; Dewey, R. E. RNA interference (RNAi)-induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves. Plant Biotechnol. J. 2008, 6, 346−354. (11) Rundlöf, T.; Olsson, E.; Wiernik, A.; Back, S.; Aune, M.; Johansson, L.; Wahlberg, I. Potential nitrite scavengers as inhibitors of the formation of n-nitrosamines in solution and tobacco matrix systems. J. Agric. Food Chem. 2000, 48, 4381−4388. (12) Cui, M.; Burton, H. R.; Bush, L. P.; Sutton, T. G.; CraftsBrandner, S. J. Effect of maleic hydrazide application on accumulation of tobacco-specific nitrosamines in air-cured burley tobacco. J. Agric. Food Chem. 1994, 42, 2912−2916.

amyloliquefaciens DA9 are very important before commercial application, and the toxicity and sensory characteristics will be evaluated in further study. Meanwhile, the detailed mechanism for decreased TSNA in tobacco leaves treated with DA9 will be further investigated in a future study.

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AUTHOR INFORMATION

Corresponding Author

*Tel/fax: +86 027-87280670. E-mail: [email protected]. Postal address: No. 368 Youyi Road, Wuchang District, Wuhan 430070, Hubei, People’ s Republic of China. Funding

This work was funded by the National Program on Key Basic Research Project (No. 2015CB150505), the National Science & Technology Program for the Rural Development during the Twelfth Five-year Plan Period (No. 2013AA102801-52), the Opening Project of State Key Laboratory of Agricultural Microbiology (No. AMLKF201403), the Fundamental Research Funds for the Central Universities (No. 529020900206142), and the Key Project of Hubei Province Tobacco Company (027Y2011-055). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Gupta, P. C.; Murti, P. R.; Bhonsle, R. B. Epidemiology of cancer by tobacco products and the significance of tsna. Crit. Rev. Toxicol. 1996, 26, 183−198. (2) IARC. Smokeless tobacco and some tobacco-specific N-nitrosamines; IARC monographs on the evaluation of carcinogenic risks to humans, Vol. 89; 2007. http://monographs.iarc.fr/ENG/Monographs/vol89/ mono89.pdf. (3) USDHHS. Harmful and potentially harmful constituents in tobacco products and tobacco smoke: Established list; US Food and Drug Administration: 2012. http://www.Fda.Gov/tobaccoproducts/ guidancecomplianceregulatoryinformation/ucm297786.Htm. E

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Journal of Agricultural and Food Chemistry

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dx.doi.org/10.1021/jf504084z | J. Agric. Food Chem. XXXX, XXX, XXX−XXX