Impacts of Environmental Variations on Quality and Chemical Contents of Oriental Tobacco

In 2015 Basma tobacco types in different growing regions of Turkey were screened, morphologically different plants were identified and seeds were collected. The DNA fingerprint analysis of the collected seeds indicated 27 different tobacco lines (Figure 1). Plant material of the study consisted of 21 Basma tobacco lines from the 27 identified lines and four standard tobacco lines (Xanthi 2A, Xanthi 81, Nail and Canik 190–5) (25). Selected plants were isolated using a paper bag to prevent outcrossing. Self-pollinated seeds were harvested after they reached maturity.

Figure 1

Seedlings of the 25 genotypes were grown in a float tray system with a peat medium. Composite fertilizer containing 20% nitrogen (N), 10% phosphor (P) and 20% potassium (K) plus micronutrients (iron 0.4%, manganese 0.4% and zinc 0.4%) was mixed with 500 g/m³ water in float pond water to supply nutrients for the seedlings. The experimental fields, where the study material could be cultivated, were chosen in different altitudes. The research was carried out in Evciler (40°36′43.48″N, 36°36′5.25″E, 581 m altitude) and Karayaka (40°44′16.45″N, 36°33′58.31″E, 302 m altitude), two villages of the Tokat-Erbaa, as well as in Bafra, a town in Samsun Province (41°33′45.29″N, 35°52′18.35″E, 26 m altitude) and in Gümüşhacıköy, a town in Amasya Province (40°53′1.03 ″N, 35°12′47.98E, 848 m altitude). Prior to planting the seedlings, 60 kg/ha N, 40 kg/ha P₂O₅ and 60 kg/haK₂O were applied to the experimental fields (26). The field experiments were carried out in a randomized block design with three replications. The seedlings were planted with 45 cm inter-row and 12 cm intra-row spacing on 5-m long plots.

The seedlings were planted on May 21, 2017 in Evciler, May 19, 2017 in Karayaka, July 4, 2017 in Bafra and June 29, 2017 in Gümüşhacıköy. The harvest of mature leaves was completed in three different intervals, each of which was completed in one day.

The leaves were sun-cured, they were threaded onto needles and string and were hung under direct sunlight after being kept in shade for two days. The cured tobacco was baled and stored in a closed room. Data obtained in the different locations were not homogeneous; therefore, the locations were separately subjected to variance analysis using the SAS 9.0 software (59). Duncan's multiple comparison test was used to compare the parameters obtained in different locations and they were graphed with GraphPad Prism 8 program. Soils in the Evciler experimental field had a clay-loam texture and the other three locations had a sandy loam texture. Electrical conductivity levels indicated a very low soil salinity. All soils were slightly alkaline and the highest organic matter content was recorded in the experimental field of Gümüşhacıköy. Soils in Bafra and Karayaka fields had low and Evciler had very low organic matter content. Soils in the Evciler, Gümüşhacıköy and Bafra locations were moderately calcareous while soil in the Karayaka location was calcareous. Potassium contents of all experimental fields were high. Phosphorus contents of the experimental field in Karakaya were moderate while other three fields were low in phosphorus content (Table 1).

The temperature values during the seven-month period covering seedling, field and curing periods of the tobacco were similar to long-term averages, while the relative humidity values were higher than the long-term average values. The average relative humidity during this period was 69.86% in Erbaa, 64.43% in Gümüşhacıköy and 82.24% in Bafra. Total precipitation during the period of vegetative growth was 222.2 mm in Erbaa, 256.5 mm in Gümüşhacıköy and 222.1 mm in Bafra. Compared to the long-term average, there was less rain by 36.1 mm in Erbaa, by 47.1 mm in Gümüşhacıköy and by 152.2 mm in Bafra (Table 2).

The grade index is the quality score of organoleptic assessments carried out by a tobacco expert on the cured leaves rating properties as stalk position, leaf size, texture, odor, moisture condition, leaf shape, leaf tip angle, leaf venation, color and brightness status, leaf integrity, degradation and disease/pest status of leaves. The expert provides two-digit ratings for each tobacco unit, the first digit representing the stalk position and the second digit representing the quality group according to the aforementioned properties. The grade, which is the basis for the pricing of the product, is calculated by multiplying these degrees with a certain coefficient. The principle of grading is to determine the proportion of A grade tobacco in a certain amount of tobacco (American grading method). The grading is the key action that governs the direction of the product in all stages from processing, marketing and finally to the end product (27).

For nicotine analysis (% DM), a 200-mg ground moisture-free tobacco sample was weighed into a 50-mL Falcon tube and 1% aqueous acetic acid and acetonitrile (85:15, v/v) was added. The mixture was kept in an ultrasonic water bath for 30 min. Samples removed from the water bath were centrifuged at 4000 rpm for 10 min. The remaining solution (supernatant) atop the precipitated sample was taken up by a syringe. The supernatant was passed through the filter (Nylon, 0.45 μm) and transferred into the vial marked with the sample number. Extractions were analysed using an Agilent technology 1260 series high-performance liquid chromatography (HPLC) system (Agilent Technologies, Boeblingen, Germany) equipped with an ACE C18 column (250 × 4.6 mm i.d. dimensions and 5 μm particle size) with a flow rate of 1 mL/min, a column temperature of 35 °C and a DAD (diode array detector) detector. The mobile phase consisted of 1% acetic acid in water (solvent A) and acetonitrile (solvent B). A wavelength of 324 nm was set for UV detection of alkaloids and nicotine was quantified using an authentic standard (28, 29).

For glucose and fructose analysis (% DM), 1 g of ground moisture-free tobacco sample was weighed into a Falcon tube and 1% acetic acid and methanol (75:25, v/v) was added to the weighed sample. After adding the solvents, the mixture was stirred, then placed in an ultrasonic water bath and kept there for 30 min. The samples were removed from the water bath and centrifuged at 4000 rpm for 8 min.

The remaining solution (supernatant) atop the precipitated sample was subsequently removed with a syringe. The supernatant was passed through a filter (Nylon, 0.45 μm) and transferred into the vial marked with the sample number. The content of glucose and fructose was analysed by HPLC using a RID (refractive index detector) with a Zorbax Carbohydrate column (250 × 4.6 mm i.d. dimensions and 5 μm particle size) at 1.5 mL flow and 40 °C column temperature (29, 30). The mobile phase consisted of 1% acetic acid in water (solvent A) and acetonitrile (solvent B) with 25% A and 75% B.

For chlorogenic acid and rutin analyses, 200 mg ground moisture-free tobacco sample was weighed into a Falcon tube and 5% acetic acid and methanol (85:15, v/v) was added into the tubes. After adding the solvents, the mixture was stirred, placed in an ultrasonic water bath and kept there for 30 min. Samples were removed from the water bath and centrifuged at 4000 rpm for 10 min. The remaining solution (supernatant) atop the precipitated sample was removed with a syringe. The supernatant was passed through the filter (Nylon, 0.45 μm) and transferred into a vial marked with the sample number. The extract was analysed by HPLC with a poroshell 120 EC C18 column (2.7 μm, 150 mm × 3.0 mm i.d.) and a guard precolumn using a diode array detector (DAD) at a 0.3 mL/min flow rate and a 35 °C column temperature. The mobile phase consisted of 1% acetic acid in water (solvent A) and acetonitrile (solvent B) using an isocritical elution with 85% A and 15% B (20, 29).

The peaks obtained from the sample chromatograms were identified by comparison with the peaks obtained from the standards and the area of each peak was calculated according to their standard calibrations (r²; 0.999 and 1.0). The concentrations of nicotine, glucose and fructose were given as percent (% DM) and those of chlorogenic acid and rutin as ppm. Extraction recovery rates, that indicate the reliability of the analysis, were 101% for nicotine, 106% for glucose, 102% for fructose, 83% for chlorogenic acid and 96% for rutin.

Pricing of tobacco marketed for its degustative properties is practically based on organoleptic estimates, which is a subjective assessment (31). The products with a 60% or higher grade A evaluation obtained the highest market price ($ 4.86/kg) during the study season in 2017. Therefore, genotypes providing a 60% grade A rating have been categorized as high-quality. The A-grade rates of ERB-9, ERB-11, ERB-18, ERB-25 and ERB-27 lines were lower than 60%. Despite the slight differences in the grade values of the remaining 20 genotypes, they were all very alike in quality and commercially on the same scale (Table 3, Figure 2).

Figure 2

The yield and grade values had an inverse relationship, indicating that factors such as soil and climate contributing to the yield increase, had a negative impact on quality (3, 32). The highest grade values and the lowest yield values were obtained in the Erbaa-Evciler location which corroborates the aforementioned inverse relationship between yield and quality grade. Environmental effects induce the plants to maintain their performance by developing biochemical and morphological mechanisms, which are responsible for the quality characteristics that distinguish Oriental tobacco from others (4, 8).

Rainwater contributes to the growth of more elastic and aromatic leaves in Gümüşhacıköy. This location benefits from more regular and continuous rainfall compared to the other locations and it is located in the highest altitude among all the locations (33).

The highest-quality tobaccos were grown in class II and III agricultural land, while the quality was lower in I class land (34, 35). Karayaka was the location where the best quality grade values were obtained due to the effects of the aforementioned factors. The Bafra location has class I agricultural land, therefore this location was included in the study. Water requirement of Oriental tobaccos for a maximum yield is around 400 to 600 mm depending on the length and the climate of the growing season. About 50% of the total water required in the field should be applied during the period of 30–40 days after planting to obtain healthy and resistant plants. The water demand rises to the highest level at 50–70 days after transplanting, and afterwards gradually decreases (36). Excessive irrigation increases the total leaf area, causes thinning of leaves, reduces leaf density and results in quality losses (33).

Color in tobacco is used as a guide in all stages, and defined as the maturity index in cases such as determining the harvest time of stalk positions. The most apparent characteristic of the change in parallel to the advancement in maturity is transformation of leaf colors to yellow, orange and brown following the breakdown of chlorophyll. The vitality, brightness and lightness or darkness of leaf color are important criteria that determine the quality of a particular tobacco type (37). The plants in turgor, due to the continuous water supply, grow more rapidly, and thus the leaves cannot exhibit the type characteristics. In this case, the product will be fine-textured, very low or flavor-free, easily burning, of low-nicotine content and light-colored, which is markedly different from the color of plants grown under regular circumstances (33). The leaf color of Samsun type tobaccos changes from yellow to dark red as cultivation locations are progressing from the lowland to the slopy lands. The curing period of tobacco plants grown in lowland is usually shorter and the color of leaves quickly turns into yellow due to the faster curing (38). Therefore, mostly B grade and partially Kapa group products have been produced in Bafra location.

Each type of tobacco within a blend has defining effects on the physical and chemical quality composition, as well as reducing/increasing effects on the amount of nicotine. This fact also has a significant impact on the market demand for the Oriental tobacco types. The desired nicotine content in Basma type tobaccos in the market for the Turkish private sector is in the range of 2.00 to 2.75% DM (26). The nicotine content of tobacco grown in the Karayaka location meets this requirement (1.09–3.15% DM; average 2.06% DM), while the nicotine content of 11 lines and 3 standards (Xanthi 2A, Nail, Xanthi 81) were 2.00% DM or higher (Table 3, Figure 3). On the other hand, nicotine content of tobacco grown in Evciler, Gümüşhacıköy and Bafra locations remained below the expectations of the sector. Nicotine content of tobacco can be increased to meet the market demand of 2.00–2.75% DM by agrigultural measures such as e.g., nitrogenous fertilization (26), by wider planting distances (39) and by topping (40). Since the assimilation of nitrogen takes place in the root system and the photosynthesis takes place in the leaves, organic compounds carrying carbon are synthesized only in the leaves, and nitrogenous compounds are synthesized in part in the root system. Therefore, various tobacco types rich in certain desired contents can be produced by modifying the external conditions (41). Topping on time and to a sufficient degree can cause the moving to a lower plant part of the photosynthesis products in the leaves, thus improving root growth and consequently facilitating assimilation of the nitrogenous compounds (41, 42).

Figure 3

Nicotine content, which is synthesized at the root tips, is closely related to root growth (41). Despite the expectation of high nicotine content in tobacco from the Evciler location (42) due to drier conditions compared to other locations, root and therefore plant growth were weak which hampered the increase in nicotine content.

Oriental tobaccos have a different flavor, lower nicotine levels compared to commercial flue-cured Virginia and air-cured Burley tobaccos and compared to Burley and bright tobaccos they also have lower tobacco specific nitrosamine (TSNA) levels. Therefore they are generally used in American blend type cigarettes by blending them with Virginia and Burley tobaccos (43). Precipitation and high humidity, especially during the harvest period, can delay maturation and leach down the resin and nicotine substances on the surface of the leaves. (44). Bafra is the location with the highest humidity.

If tobacco is cultivated in lighter soils, where the roots are well aerated and can take up the available water easily (under both limited nitrogen fertilization or under fertilized conditions), then tobacco with low levels of nitrogenous compounds is produced (41). The lower nicotine content of tobaccos grown in lowlands similar to the Bafra location has been attributed to a weaker root system as well as the lower fertility and less available nitrogen in lighter soils (8). Camas (45) studying regional tobaccos and their hybrids in Bafra stated that the lowest nicotine value of genotypes in the F1 generation was 0.47% DM (mean 1.04% DM) in Basma 192–23 varieties. The decrease in nicotine content was reported for the hybrids related to the Basma variety, and the results were considered promising to obtain tobacco with a low nicotine content. Camas et al. (46) and Esendal et al. (47,48,49) reported that nicotine content in tobacco grown in the Bafra region ranged from 0.3 to 2.1% DM. The nicotine contents obtained in this study were similar to those reported in these studies carried out in Bafra.

Sugars, one of the most important components of the chemical structure of leaf tobacco, are primary metabolites (24) that contribute to the growth and development of tobacco plants. The reducing sugar composition is directly related to the taste and flavor of tobacco (30, 50, 51). The sugar content in different tobacco types is quite variable and depends primarily on the curing process (14) where longer curing periods cause a decrease in the sugar content (41, 42, 44).

Tobacco types are generally classified based on their curing methods. Reducing sugar in Oriental (sun-cured) and Virginia (flue-cured) tobaccos are relatively high (10–25% DM) compared to the reducing sugar content (< 2% DM) in Burley and Maryland (air-cured) tobaccos (53). Fructose content in this study was between 2.60 and 8.66% DM (Table 4). The fructose content of American blends, of which Virginia, Burley and Oriental tobacco are the main raw materials, is between 3.98 and 5.76% DM (52). Reducing sugar composition, together with phenolic compounds, resins and essential oils, determines the taste and flavor of tobacco (30, 50, 51). The reducing sugar content of an American blend is between 4.92 and 8.28% DM (52).

Late planting in the Gümüşhacıköy location caused a delay in the harvest and curing period and as a consequence the curing of the 2^nd hand harvest was postponed to September. Decreasing temperatures in September made for a longer curing period. The temperatures in Erbaa after the 2^nd hand harvest went up to 25.9 °C, but the temperatures in August/September, when the 2^nd hand is cured in Gümüşhacıköy, were only at 22.3 and 20.8 °C. Similar conditions were found in the Bafra location where the 2^nd hand harvest was harvested on September 4. There the temperature in the curing period was 21.2 °C (Table 2). The excessive relative humidity in addition to the lower ambient temperatures during the curing period caused a decrease of the sugar content. This was due to the prolonged curing period (41, 42). Bafra is the location with the highest relative humidity among the locations (Table 2). The lowest reducing sugar content of Bafra, compared to the other locations, can be attributed to those low temperatures and high relative humidity throughout the curing period. The reducing sugar content in high demand by leaf tobacco companies for the Basma type tobacco is between 8 and 13% DM (26), and indeed the average reducing sugar content in the study ranged from 6.60 to 10.57% DM (mean 8.58% DM) (Table 4, Figure 4).

Figure 4

Secondary metabolites, in addition to nicotine and reducing sugars that directly affect the color, taste and flavor of tobacco and thus determine the characteristics of the smoking product, should be taken into consideration during the planning and production processes. The most important of these secondary metabolites are polyphenols and among them the most abundant in tobacco are chlorogenic acid and rutin (17). The chlorogenic acid values in this study were between 156.70 and 426.73 ppm (Table 5).

The chlorogenic acid contents reported in various studies for Oriental tobacco were between 0.16 and 0.25% DM (54), 1.56 mg g⁻¹ (15), 30.2 mg g⁻¹ (55), 7.9 mg g⁻¹ (56) and between 4.5 and 12.3 mg g⁻¹ (57). Rutin contents in Oriental tobacco were reported as 1.28–1.52% DM (44), 0.49–0.98% DM (58), 0.55–0.66% DM (54), 4.240 mg g⁻¹ (15), 2.8 mg g⁻¹ (55), 6.2 mg g⁻¹ (56), and 5.6–9.3 mg g⁻¹ (57). The mean rutin content of lines in all locations was between 399.94 and 582.60 ppm, while the variation of each line at each location was between 121.05 and 1021.53 ppm (Table 5).

The content of chlorogenic acid + rutin, which constituted approximately 85% of the total polyphenols (44), was similar or lower compared to those reported in previous studies (15, 44, 54,55,56,57,58). The mean total contents of chlorogenic acid + rutin were 1018.95 ppm in Evciler, 1246.38 ppm in Karayaka, 478.42 ppm in Gümüşhacıköy and 328.96 ppm in Bafra (Table 5, Figure 5).

Figure 5

Chlorogenic acid + rutin content in Oriental tobacco was reported as 0.84–2.41% DM (58), 0.71–0.91% DM (54), 33.00 mg g⁻¹ (55), 14.10 mg g⁻¹ (56) and 10.10–21.60 mg g⁻¹ (57). Xie et al. (15) reported chlorogenic acid + rutin content as 2.3% DM for flue-cured, 0.054% DM for Burley and 1.08% DM for Oriental tobaccos. Phenolic compounds directly contribute to the formation of leaf color, and oxidation of phenolic compounds causes mutations towards dark brown tones. The amount of chlorogenic acid increases rapidly, depending on the severity of the enzymatic activity on the first and second day during curing and subsequently decreases related to the browning reaction (44). The duration and regime of climatic events have a significant impact on the chemical structures of tobacco lines grown in the experimental locations. The prolongation of curing processes in Gümüşhacıköy and Bafra locations had a negative effect on the accumulation of phenolic compounds.