李宗平覃光炯陈茂胜张俊杰彭灏吴哲宽杨丽萍. Effects of curing methods on conversion rate of nicotine and TSNAs contents of tobacco

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1 Chinese Journal of Eco-Agriculture, Oct. 2015, 23(10): DOI: /j.cnki.cjea TSNA * 李宗平覃光炯陈茂胜张俊杰彭灏吴哲宽杨丽萍 (/ ) 为进一步明确调制方式对不同品种 ( 系 ) 烟草烟碱转化和特有亚硝胺 (TSNA) 的影响, 采用裂区试验设计, 以白肋烟 [B37LC( 低烟碱转化 ) 品系 B37HC( 高烟碱转化 ) 品系 ] 马里兰烟[Md609LC( 低烟碱转化 ) 品系 Md609HC( 高烟碱转化 ) 品系 ] 烤烟( 云烟 87 K326 ) 和晒烟 ( 深色公会晒黄烟 浅色公会晒黄烟 ) 等 4 种烟草类型的 8 份材料为主处理, 烤制 晾制和晒制 3 种调制方法为副处理, 进行了生物碱和 4 种 TSNA 含量检测与研究 结果表明 : 品种 ( 系 ) 是影响生物碱含量 烟碱转化率和 4 种 TSNA 含量的主要因素, N 亚硝基降烟碱 (NNN) 是 4 种烟草类型的主要 TNSA, 所占比例在 54.35%~97.36% 不同烟草类型中以白肋烟 马里兰的 HC 品系与 LC 品系间差异最大, 其中 LC 品系的 NNN 含量比 HC 品系下降 90.93% 和 91.54% 3 种调制方法中, 烟碱含量为烤制 > 晒制 > 晾制, 降烟碱及烟碱转化率则为晾制 > 晒制 > 烤制 ; 除 N- 亚硝基假木贼碱 (NAB) 含量烤制显著高于晾制外, NNN 4-(N- 甲基亚硝胺基 )-1-(3- 吡啶基 )-1- 丁酮 (NNK) N- 亚硝基新烟碱 (NAT) 及 TSNA 总量均为晾制 > 晒制 > 烤制, 差异达显著或极显著水平 ; 调制方法对白肋烟 马里兰烟的 HC 品系影响最大, 对 LC 品系影响最小 综合认为 : 在烟叶特别是白肋烟 马里兰烟生产上积极推广烟碱转化率低的品种, 同时努力改善调制条件 改进调制工艺是降低烟叶 TSNA 含量 提高烟叶安全性的关键措施 白肋烟马里兰烟烟碱转化类型调制方式生物碱 TSNA : S56 : A : (2015) Effects of curing methods on conversion rate of nicotine and TSNAs contents of tobacco LI Zongping, QIN Guangjiong, CHEN Maosheng, ZHANG Junjie, PENG Hao, WU Zhekuan, YANG Liping (Hubei Province Tobacco Research Institute, CNTC Burley Tobacco Experimental Station, Wuhan , China) Abstract Tobacco alkaloids and nitrite are the main precursors of tobacco-specific nitrosamines (TSNA). Nitrite is formed by nitrate reduction reaction in tobacco leaves. Nicotine, nornicotine, anatabine and anabasine react with nitrous acid to form 4-(N-methyl nitrosamines)-1-(3-pyridyl)-1-butyl ketone (NNK), N-nitrosonornicotine (NNN), N-nitroso new nicotine (NAT) and N-nitroso false horsetail alkali (NAB), 4 main forms of TSNAs. Nicotine is the main tobacco alkaloid. Although nornicotine content is usually very low, the transformation of nicotine to nornicotine can occur through tobacco gene mutation, which activates and greatly increases the contents of nornicotine, NNN and total TSNA. The conversion of nicotine to nornicotine and nitrite reduction reactions occur during the processes of tobacco curing and storage. This is especially the case for the formation and accumulation of TSNA during the period of curing. In order to further clarify effects of tobacco types and varieties, curing methods on TSNA, TSNA contents and nicotine variations during curing were analyzed. The study used split-plot experimental design involving 4 tobacco types (burley, Maryland tobacco, flue-cured tobacco and sun-cured tobacco) and 8 varieties (lines) [B37LC (low nicotine conversion line) and B37HC (high nicotine conversion line) of burley; Md609LC (low nicotine conversion line) and Md609HC (high nicotine conversion line) of Maryland tobacco; Yunyan87 and K326 of flue-cured tobacco varieties; dark sun cured tobacco and light colored sun cured tobacco of sun-cured tobacco varieties] with 3 curing methods (flue-curing, air-curing and sun-curing) as secondary treatments. Then the 4 TSNA and alkaloid contents in leaves under both the main and secondary treatments were investigated. The results showed that tobacco varieties * (027Y ), li63@163.com : :

2 10 : TSNA 1269 (lines) were the main factor influencing alkaloid content, nicotine conversion rate and contents of 4 kinds of TSNA. Nornicotine contents and nicotine conversion rates of HC lines of Maryland tobacco and burley tobacco were highest, and those of HC lines was lowest. NNN was the main TSNA in all the tobacco types, accounting for 54.35% 97.36% of total TSNA. It was followed by NAT (2.33% 38.46%), NNK (0.17% 5.47%) and NAB (0.14% 5.92%). Comparison of different tobacco types in terms of proportions of four forms of TSNA showed that NNN was highest in HC lines of burley tobacco and Mayland tobacco, accounting for 93.93% and 96.99% of total TSNA content. Compared with HC lines, LC lines fell by 90.93% and 91.54% in NNN contents, respectively, in burley and Maryland tobacco. The contents of NNK, NAT and NAB in HC lines were higher than in LC lines, although the difference was smaller than that of NNN. Comparison of 3 curing methods showed that nicotine content of flue-cured tobacco was greater than that of sun-cured which was in turn greater than that of air-cured tobacco. The nornicotine content and conversion rate of nicotine were highest for air-cured tobacco, followed by sun-cured tobacco and then flue-cured tobacco. Except NAB, the contents of NNN, NNK, NAT and total TSNA in flue-cured tobacco were significantly higher than those of air-cured tobacco, which were in turn significantly higher than those of sun-cured tobacco. Also while total TSNA content of air-cured tobacco was 41.85% higher than that of flue-cured tobacco, NNN content of air-cured tobacco was 45.45% higher than that of flue-cured tobacco. Curing method had greatest impact on HC lines and minimal impact on LC lines of burley tobacco and Maryland tobacco. It was beneficial to decrease TSNA content and promote tobacco safety to plant low nicotine conversion varieties of burley tobacco and Maryland tobacco and improve curing conditions. Keywords Burley tobacco; Maryland tobacco; Nicotine conversion type; Curing method; Alkaloid; TSNA (Received Mar. 13, 2015; accepted Jul. 2, 2015) (TSNA) 8 TSNA, N-(NNN) N-(NAT) N- (NAB) 4-(N- )-1-(3- )-1- (NNK) 4, NNN NNK, NAT NAB, TSNA,, TSNA, (NO X ),, NNK NNN NAT NAB [1 6], (Nitotiana tobacum), 93% ;,,, 3.5%,, 1980 [1 4],,, NNN,,, TSNA [5 10] [11] TSNA : >> >,, NNN NATTSNA, TSNA 96%, TSNA, NNN,,, TNSA [1 2,5,12],, 3, [13 15] Bush [16],, TSNA [17], [18],, TN86 TSNA 46.8%, NNN [19], TSNA,, ; TSNA, ; TSNA [20] MHz,, 90 s TSNA TSNA, TSNA

3 , TSNA, 1 材料与方法 1.1 B37LC( ) B37HC( ), 87 K326, Md609LC( ) Md609HC ( ), 4 8 ( ), <5%, >50% A ( ), B : A: A1, B37LC; A2, B37HC; A3, 87 ; A4, K326 ; A5, Md609LC; A6, Md609HC; A7, ; A8, B: B1, ; B2, ; B3, 8, 3, 3, 40 16~32, 75%~80%, 6 8 d; 70%~75%, 18~22 d; 40%~50%, 11~13 d; d,, 35~42, 33~37, 48~72 h; 45~55, 37~39, 36~48 h; 55~68, 40~43, 24~36 h; 120~160 h,, 5~25 d [21] [22] 1.2.2, (1.2 m 0.55 m) (187.5 kg hm 2 ) (N.. P 2 O 5.. K 2 O= ) ( ) (22 ) 1.2.3, 2 ( 14~15 ), TSNA TSNA : 0.20 g 100 ml, 5 ml 10% 20 ml ( )/ (V.. V=3.. 1), 1 h 1 h, 0.22 mm, 2 ml, Agilent 7890A FID, Burton [23], : =[ /(+ )] 100% (1) TSNA : [24], 0.20 g 100 ml, 0.2 ml 4 TSNA, 100 mmol L ml, 1 h 0.22 mm, Agilent 6460A N-(NNN) N-(NAT) N- (NAB) 4-(N-)-1- (3- )-1- (NNK) DPS 7.0 (LSD) 2 结果与分析 2.1, ( ) ( ) (P<0.05), F > > ( ) ( ) B37HC Md609HC 87 K326,, Md609LC> B37LC> > K326 > 87 > >Md609HC>B37HC; 87,

4 10 : TSNA 1271 ( ), Md609HC B37HC, mg g mg g 1, 3.00 mg g 1 HC LC, HC LC55.13%, ; HC LC51.40%, > >, > > 3 ( ) ( ), B37HC Md609HC,, B37LC Md609LC > >, 87 3, ( ) ; > >, B37HC Md609HC 3, ( ) 1 Table 1 表 1 不同调制方式对不同品种 ( 品系 ) 烟草生物碱及烟碱转化率的影响 Effects of different curing methods on contents of alkaloids and conversion rate of nicotine of different varieties (lines) of tobacco ( ) Variety (or line) Curing method Nicotine (mg g 1 ) Nornicotine (mg g 1 ) Conversion rate (%) B37LC Flue-curing 60.05±0.92aA 2.29± ±0.06 Air-curing 57.78±0.93bB 2.35± ±0.07 Sun-curing 59.38±0.74aAB 2.32± ±0.03 B37HC Flue-curing 39.98±0.48aA 33.94±0.08cC 45.97±1.89cC Air-curing 20.44±0.16cC 53.19±0.09aA 72.24±0.13aA Sun-curing 25.71±0.39bB 49.52±0.31bB 65.83±0.21bB 87 Flue-curing 41.94± ± ±0.03 Yunyan 87 Air-curing 40.90± ± ±0.09 Sun-curing 41.55± ± ±0.02 K326 Flue-curing 42.76±0.35a 1.11± ±0.05 Air-curing 41.38±0.26b 1.21± ±0.04 Sun-curing 42.71±0.42a 1.18± ±0.05 Md609LC Flue-curing 65.83±0.52aA 2.36± ±0.01 Air-curing 63.66±0.25bB 2.44± ±0.02 Sun-curing 64.13±0.47bAB 2.39± ±0.01 Md609HC Flue-curing 28.83±0.20aA 32.73±0.02cC 42.75±0.12cC Air-curing 19.38±0.33cC 57.70±0.03aA 74.86±0.31aA Sun-curing 23.67±0.04bB 53.06±0.06bB 69.15±0.06bB Flue-curing 48.22±0.65aA 1.33± ±0.04 Dark sun cured tobacco Air-curing 45.68±0.19bB 1.42± ±0.07 Sun-curing 47.51±0.40aA 1.39± ±0.00 Flue-curing 41.46±0.36aA 1.27± ±0.01 Light colored sun cured Air-curing 39.18±0.16bB 1.36± ±0.08 tobacco Sun-curing 41.11±0.66aA 1.33± ±0.04 B37LC 59.07bB 2.32dC 3.78cC Variety effect B37HC 28.71gF 45.55bB 61.34bB 87 Yunyan eDE 1.33eD 3.11dDE K dD 1.17fE 2.69eE Md609LC 64.54aA 2.40cC 3.58cCD Md609HC 28.96gF 47.83aA 62.25aA Dark sun cured tobacco 47.14cC 1.38eD 2.85deE Light colored sun cured tobacco 40.58fE 1.32eD 3.15dDE Flue-curing 48.01aA 9.54cC 13.37cC Curing effect Air-curing 41.05cC 15.13aA 20.90aA Sun-curing 43.22bB 14.07bB 19.27bB ( ) , Different capital and lowercase letters indicate significant difference among treatments or varieties (lines) at 0.01 and 0.05 levels, respectively. The same below.

5 TSNA 4 TSNA ( ) ( ) (P<0.05), ( ) ( ) NNN TSNA, NAT, NNK NAB 2 ( ) NNN TSNA B37HC> Md609HC>B37LC>Md609LC> > > 87 > K326, 87 Table 2 表 2 不同调制方式对不同品种 ( 品系 ) 烟草亚硝胺及其组分含量的影响 Effects of different curing methods on contents of nitrosamine and its components of different varieties (lines) of tobacco ng g 1 ( ) Variety (or line) B37LC B37HC 87 Yunyan 87 K326 Md609LC Md609HC Dark sun cured tobacco Light colored sun cured tobacco Variety effect Curing effect Curing method NNN NNK NAT NAB Total Flue-curing ±15.62cB 40.82±0.84bB ±5.72cC 31.11±0.94bB ±18.06cC Air-curing ±10.00aA 43.62±0.39aA ±9.31aA 30.52±1.03bB ±11.09aA Sun-curing ±10.00bA 41.42±0.87bB ±5.07bB 33.35±1.91aA ±6.21bB Flue-curing ±30.00cC 39.30±1.37bB ±3.88cC 45.21±1.04aA ±27.38cC Air-curing ±60.00aA 45.03±0.05aA ±14.59aA 44.11±0.40aA ±68.03aA Sun-curing ±101.49bB 44.54±1.35aA ±10.46bB 32.22±1.98b B ±90.15bB Flue-curing ±5.01cC 32.93±0.98bB ± ±0.97aA ±14.69cC Air-curing ±1.58aA 35.82±0.21aA ± ±0.50aA ±8.13aA Sun-curing ±6.00bB 33.61±0.41bAB ± ±1.30bB ±2.84bB Flue-curing ±10.44cC 29.71±1.14bB ±1.57bB 35.30±0.33bB ±10.46cC Air-curing ±8.66aA 32.67±0.90aA ±7.00aA 38.68±0.23aA ±15.25aA Sun-curing ±10.03bB 32.02±1.89AB ±5.07aA 31.47±0.87cC ±11.98bB Flue-curing ±4.48cB 22.74± ±12.00bB 22.51±0.34aA ±10.11cB Air-curing ±9.79aA 23.75± ±0.01aA 22.34±0.87aAB ±9.97aA Sun-curing ±29.70bA 22.94± ±12.47aA 20.22±1.18bB ±41.66bA Flue-curing ±9.33cC 23.72±0.55bB ±7.06cC 22.51± ±17.06cC Air-curing ±10.05aA 26.30±0.21aA ±5.90aA 21.65± ±16.95aA Sun-curing ± 9.60bB 25.39±0.36abAB ±20.26bB 22.32± ±17.48bB Flue-curing ±10.05bB 34.82± ±5.18bAB 26.96±1.31aA ±17.26bB Air-curing ±3.05aA 36.03± ±10.63aA 24.76±0.09bB ±6.93aA Sun-curing ±16.29bB 36.39± ±9.63bB 23.86±0.88bB ±14.92bB Flue-curing ±9.69bB 34.11±0.07cB ±0.39bB 30.77±0.08aA ±9.31bB Air-curing ±4.93aA 38.29±0.94aA ±10.11aA 25.70±0.49bB ±5.18aA Sun-curing ±9.78bB 36.08±0.54bAB ±4.81bB 30.66±0.73aA ±11.84bB B37LC cC 41.95bA bB 31.66cC cC B37HC aA 42.96aA aA 40.51aA aA 87 Yunyan gG 34.12dC gG 24.33fE gG K gG 31.47eD fF 35.15bB gG Md609LC dD 23.14gF eE 21.69gF dD Md609HC bB 25.14fE dD 22.16gF bB Dark sun cured tobacco eE 35.75cB cC 25.19eE eE Light colored sun fF 36.16cB cC 29.04dD fF cured tobacco Flue-curing cC 32.27cC cC 30.01aA cC Air-curing aA 35.19aA aA 29.25bA aA Sun-curing bB 34.05bB bB 26.90cB bB NNN: N-; NAT: N-; NAB: N- ; NNK: 4-(N-)-1-(3- )-1-NNN: N- nitrosonornicotine; NAT: N-nitroso neonicotinoids; NAB: N-nitroso anabasine; NNK: 4-(n-methyl nitrite amine)-(3-pyridyl)-1-ketone. The same below.

6 10 : TSNA 1273 K326, 6 ; NNK NAT, ( ), NNK B37HC>B37LC> > > 87 > K326 > Md609HC>Md609LC, NAT B37HC>B37LC> > >Md609HC>Md609LC> K326 > 87 ; NAB Md609LC Md609HC, ( ), B37HC>K326>B37LC> > > 87 >Md609HC>Md609LC, NNN TSNA,, ; NAT > > > ; NNK NAB> > >, HC NNN ng g ng g 1, LC ng g ng g 1, LC HC 90.93% 91.54%, ; NNK NAT NAB HCLC, NNN 3 ( ), NAB, NNN NNK NAT TSNA, TSNA 41.85%, NNN 45.45%, NAT 17.78%, NNK 9.05%, NAB 2.53% 3 ( )Md609LC NNK 87 NAT Md609HC NAB, B37HC Md609HC, B37LC Md609LC,, 4 TSNA 3, NNN 4 TNSA, 54.35%~97.36%; NAT, 2.33%~38.46%; NNK NAB, 0.17%~5.47% 0.14%~5.92% ( ), NNN HC, 93.93% 96.99%, LC, 60.36% 77.13%, 56.23%~62.96%; NAT B37LC > > Md609LC> B37HC Md609HC; NNK NAB3%~5%,, LC, HC1% 4 TSNA 87 NNN 8.35%, NNN > >, NAT NNK NAB> > 表 3 不同调制方式对不同品种 ( 品系 ) 烟草亚硝胺组分占总量比例的影响 Table 3 Effects of different curing methods on proportions of four components to total nitrosamine content of different varieties (lines) of tobacco % ( ) Variety (or line) B37LC B37HC 87 Yunyan 87 K326 Md609LC Md609HC Dark sun cured tobacco Curing method NNN NNK NAT NAB Flue-curing Air-curing Sun-curing Average Flue-curing Air-curing Sun-curing Average Flue-curing Air-curing Sun-curing Average Flue-curing Air-curing Sun-curing Average Flue-curing Air-curing Sun-curing Average Flue-curing Air-curing Sun-curing Average Flue-curing Air-curing Sun-curing Average Flue-curing Light colored sun cured Air-curing tobacco Sun-curing Average Flue-curing Average Air-curing Sun-curing

7 讨论与结论,, [2 3,7] Jeffrey Tso 1955,,,,,,,,,,, [1 2,5,7 11],, ( ) TSNA, [9 11] HC LC HC LC, HC 50%~75%, LC 5%, HC LC, 20 TSNA,, TSNA [12] HC NNN LC 11.03~11.82, NC95 NNN 0.95 [25] NNN [6], TSNA,, > >, > >, HC HC 4 TSNA,, TSNA NNN, 41.85% 45.45%, ( ) HC HC, ;, ;,,, [6,21 22],, TSNA [1,3 4,26 27] 3,, TSNA [28 30] 30, 40 60% [1] 4 7 TSNA, NNN 14 d [2], (32 ) (83%) TSNA, 2 3 [31] [32] (NR),,, TSNA NNN 4 TNSA, > > ; NAT, > > ; NNK NAB [11] NNN NATTSNA, NNN NAT NNK, NAT [3] TSNA NNK, NNN,,, ( ) TSNA, TSNA, TSNA, TSNA,,, TSNA

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