38 :38 43 Research report VARIABLES AFFECTING FLUORIDE IN TURKISH BLACK TEA Yunus Hudaykuliyev, a Mustafa Tastekin, b Ender S Poyrazoglu, a Ensar Baspinar, c Y Sedat Velioglu a Ankara, Turkey SUMMARY: The effects of tea grade, processing method, growing elevation, and plucking season on the fluoride content of Turkish black teas were investigated and analyzed statistically. The mean fluoride content overall was 156.3±34.76 mg/kg dry tea (range 87.6 to 289.2 mg/kg). The leaf rolling method had no effect on the fluoride content, but low-land teas contained more fluoride than high-land teas. Teas plucked in May had the highest fluoride content. In general, grade 7 tea (dust-tea) contained more fluoride than the other grades. Keywords: Fluoride in tea; Tea grade; Tea growing elevation; Tea plucking season; Tea rolling method; Turkish black tea. INTRODUCTION The tea plant, Camellia sinensis L, selectively absorbs fluorine from the soil and progressively accumulates it in the leaves with age. 1 The fluoride (F) content of tea plants is reported to range between 3.2 and 260 mg/kg mostly between 100 and 200 mg/kg. In some publications, the F content is reported to be as high as 400 mg/kg. 2 Recently, it was found that the F content in tea infusions accounts for 24 to 83% of the total F in the original leaf samples. 3 Another recent study revealed that tea is an important source of dietary fluoride, providing 31 to 38% of total dietary intake in four-year old children. 4 Turkish black tea infusions indicated the presence of 53-98 mg water-soluble F/kg tea after extraction for 20 min at 80 ºC with soft water. 5 With respect to methods of tea processing, the modern conventional method is a mechanical development of the original hand rolling procedure, the primary objective of which is to wring out juices from the tea leaves. In the orthodox method, the withered leaves are subjected to twisting, unrolling, re-rolling, and breaking up. The expressed juice, which is then easily oxidized, spreads out on the leaf surfaces making them wet and sticky. This method is still one of the most commonly used methods. In the CTC (cut, tear, curl) method the main idea is intensive maceration of the withered leaves to ensure rapid and complete oxidation. An obvious way to break up the leaves is by a mincing machine, and Rotorvane rollers depend on this technique. Some methods apply a combination of two methods, such as rotorvane and CTC. 6,7 The Cay-Kur method is a combination of the orthodox and Rotorvane methods. In the present study, the effects on the F content of 84 samples of Turkish black tea resulting from two different tea processing techniques, two different growing elevations, and seven different grades of Turkish black tea harvested during three different plucking periods were investigated. a For Correspondence: Y Sedat Velioglu, a Ankara University, Faculty of Engineering, Department of Food Engineering, Ankara, Turkey. E-mail: velioglu@eng.ankara.edu.tr b Ankara University, Faculty of Science, Department of Chemistry, Ankara. c Ankara University, Faculty of Agriculture, Department of Biometry and Genetics, Ankara.
Variables affecting fluoride in Turkish black tea 39 MATERIALS AND METHODS Black tea samples included in this study were obtained from four different tea processing factories of the Turkish General Directorate of Tea Establishments (CAY-KUR). Two of the factories process teas obtained from highlands (140 m to 480 m above sea level), and the other two process teas obtained from lowlands (6 m to 14 m above sea level). Two of the factories that process teas from both highlands and lowlands use the orthodox method, and the other two use the Cay- Kur method. All these factories are located northeast of the Black Sea region. The tea samples were obtained from the May, July, and September plucking seasons. After heating to 95-100 ºC, seven different black tea samples were obtained from each sample by using the Middleton sieving system during sorting and grading. This system can be fitted with 5 to 10 trays, and the aperture size of the sieves varies from 8 mesh (2.36 mm) to 40 mesh (0.425 mm). Here five sieve sizes were used with aperture sizes of 8 (largest), 10 (2.0 mm), 12 (1.7 mm), 20 (0.85 mm), and 30 (0.6 mm) mesh. Black teas retained by the 10, 12, and 20 mesh sieves were separated as tea grades 1, 2, and 3, respectively. Since these teas do not contain any broken parts, they have the best quality characteristics. Teas passing through the 30 mesh sieve are designated as grade 7 or dust-tea. The largest particles of black tea remaining over the 8 mesh sieve were passed through the breaker crushers and screened again in the same system. Black teas then remaining over the 10, 12, 20, and 30 mesh sieves were classified as grades 4, 5, 6, and 7, respectively. Coding of samples: Black teas were coded according to their grade as G1,G2, G3, G4, G5, G6, and G7. The Cay-Kur and orthodox rolling methods were coded as R1 and R2, respectively. Teas obtained from higher and lower elevations were coded respectively as E1 and E2. Teas plucked in May, July, and September were coded as P1, P2, and P3, respectively. These descriptions and coding of the tea samples are summarized in Table 1. Table 1. Description and coding of tea samples Factor Tea samples Code Rolling method (R) Cay-Kur R1 Orthodox R2 Elevation (E) Highland E1 Lowland E2 Plucking season (P) May P1 July September P2 P3 Fluoride analyses: Tea samples were ground using a laboratory mill (Falling Number type KT 30, Stockholm, Sweden) and screened from a No. 40 sieve. For F analysis, 0.5-g powdered samples were weighed in 100-mL plastic containers,
40 Hudaykuliyev, Tastekin, Poyrazoglu, Baspinar, Velioglu 20 ml of 0.05 N HNO3 was added, and the mixture shaken for 20 min on a rotating shaker. Next, 20 ml of 0.1 N KOH was added, and agitation was continued for an additional 20 min. Afterward, 5.0 ml of 0.4 M sodium citrate solution containing 1 ppm F adjusted to ph 5.5 was added, and the mixture was vortex mixed for 1 min. Five ml of 0.2 N HNO3 was added and vortex mixing was repeated for 1 min. The mixture was then filtered through Whatman No. 40 filter paper, and 10 ml of the filtrate was mixed with 10 ml of TISAB solution. Fluoride determinations were conducted potentiometrically with a Jenway 3040 ion analyzer electrometer, an Orion Ion plus No: 9609 BN selective fluoride ion electrode, and an Orion Ion plus No: 940907 (100 mg F/kg) fluoride standard solution. 8 The TISAB (total ionic strength adjustment buffer) solution was prepared by mixing 58 g of NaCI with 57 ml of glacial acetic acid, and the ph was adjusted to 5.3 using 0.1 M NaOH solution, followed by dilution to 1000 ml with distilled water. Automatic mv readings were made using the F selective electrode with continuous mixing. F concentrations in the dry tea samples were calculated using the equation: ppm F (µg/g) = (C - 0.10) x 50 where C = ppm F from the curve; 0.10 = ppm background F in final solution; 50 = ml of final solution; and w = g sample. Values reported are averages of three replicates. Statistical Methods: The effects of rolling methods x plucking season x elevation and tea grades on the fluoride contents of the tea samples were examined by a four- factor factorial analysis of variance (ANOVA) using the combination of rolling method x plucking season x elevation x tea grade interaction (4th degree of interaction). Comparison of each of the differences between tea grades was made with an LSD (least squares differences) multiple comparison test. 9 RESULTS AND DISCUSSION The interaction between tea grade, rolling method, and elevation was statistically significant (p<0.01) for the fluoride contents of black teas as a result of ANOVA. LSD (least significant differences) test results of the fluoride contents of tea grades according to the subgroups of rolling method, plucking season, and elevation are given in Table 2 and in summary form in Table 3. An LSD value of 17.413 mg F/kg tea was found from an ANOVA table using an LSD equation not shown here. When the absolute differences between groups were greater than 17.413 mg F/kg tea, the results were considered significant. Compared group numbers were 21 on tea grades (G7 to G6, G7 to G5 etc.), 3 on elevations (P1E1 to P1E2, P2E1 to P2E2 etc.), 21 on plucking season (G1P1 to G1P2, G2P1 to G2P2 etc.) and 7 on rolling methods (G1R1 tog1r2, G2R1 to G2R2 etc.). w
Variables affecting fluoride in Turkish black tea 41 Table 2. Descriptive statistics of fluoride content of teas (mg/kg) for rolling method x plucking season x elevation x tea grade combinations and results of the LSD (least squares differences) comparisons for the same rolling x plucking x elevation combination (n=3). a Tea Grade b & (Rolling method b ) Plucking season b P1 P2 P3 Elevation b Elevation Elevation E1 E2 E1 E2 E1 E2 G1 G2 G3 G4 G5 G6 G7 ±S ±S ±S ±S ±S ±S 153.8±6.66 A 146.1±7.29 A 131.5±3.16 A 123.5±4.87 AB 149.6±4.13 A 231.5±3.57 A 158.7±5.63 A 166.7±8.15 B 177.5±0.99 B 108.5±7.21 AD 155.5±2.25 A 157.3±4.04 B 153.1±3.29 A 197.4±8.46 C 136.0±3.27 A 124.4±2.49 AB 150.0±3.51 A 136.3±4.44 C 143.5±5.64 AC 201.2±1.97 C 136.5±8.55 A 130.0±4.23 B 120.7±0.96 B 205.0±5.42 D 120.9±3.69 B 197.9±0.79 C 121.6±2.41 A 114.0±0.39 AB 130.0±3.35 B 136.8±3.49 C 136.7±5.81 BC 199.3±3.69 C 122.7±6.12 A 94.1±3.94 D 136.7±4.32 B 149.0±7.70 BC 177.4±10.00 D 268.8±10.89 D 137.0±2.26 A 178.0±2.34 E 199.1±8.70 C 180.0±3.05 E G1 G2 G3 G4 G5 G6 G7 157.7±5.68 A 265.1±3.68 A 138.7±3.89 A 141.6±6.36 A 183.5±0.95 A 184.4±3.85 AD 151.1±4.52 AB 233.0±3.04 B 135.2±4.78 A 149.1±5.77 A 123.5±4.67 B 166.3±3.83 AB 148.5±8.66 AB 202.7±2.37 CE 143.1±7.97 A 138.9±4.80 A 127.5±5.27 B 167.7±7.91 AB 138.0±1.22 BC 194.7±8.15 C 132.0±2.08 A 131.5±4.80 AB 124.4±7.30 B 164.8±3.03 B 128.4±2.73 C 162.9±5.42 D 134.8±5.72 A 146.3±4.27 A 136.7±5.41 B 144.9±5.15 C 122.0±3.00 C 165.5±0.91 D 133.7±5.77 A 118.8±3.82 B 133.9±4.94 B 147.0±5.64 C 193.2±4.72 D 212.0±2.37 E 172.5±9.12 B 211.1±7.74 C 133.9±2.92 B 194.4±7.72 D a Different letters in the same R i x P j x E k (i=1,2; j=1,2,3; k=1,2) columns indicate the difference between two means is statistically significant (p<0.01) for each of the treatment combinations. b See Table 1 for abbreviations.
42 Hudaykuliyev, Tastekin, Poyrazoglu, Baspinar, Velioglu Table 3. Fluoride content of tea subgroups (mg/kg) a Sub-groups Mean S Min Max Range Effects of the tea grade on F content (n=36) G1 167.3 6.99 116.4 269.4 153.0 G2 156.9 5.12 99.6 239.1 139.5 G3 152.1 4.31 117.6 209.2 91.6 G4 151.8 5.21 114.0 215.8 101.8 G5 139.6 3.77 113.2 199.1 85.9 G6 138.3 4.40 87.6 203.9 116.3 G7 188.1 5.97 129.1 289.2 160.1 Effects of elevation on F content (n=126) E1 143.8 1.87 110.8 213.2 102.4 E2 168.8 3.64 87.6 289.2 201.6 Effects of plucking season on F content (n=84) P1 174.9 4.30 114.4 289.2 174.8 P2 137.9 2.64 87.6 224.8 137.2 P3 156.1 3.14 114.0 238.0 124.0 Effects of rolling method on F content (n=126) R1 154.6 3.21 87.6 289.2 201.6 R2 158.0 2.99 112.0 269.4 157.4 F content of all samples (n=84) a See Table 1 for abbreviations. 156.3 34.76 87.6 289.2 201.6 From Tables 2 and 3, the following conclusions can be drawn: Comparing only tea grades (omitting other variables), the fluoride contents of G7 teas in 6 groups of 21 were higher than in any of the other grades of teas. The fluoride differences between 12 groups of 21 were found non-significant. G7 teas had the highest fluoride content among rolling methods and elevation combinations and are considered the poorest quality tea (dust tea) because they contain more older leaves and pieces of stems. Older tea leaves are known to contain more fluoride, and therefore the G7 teas would be expected to contain more fluoride. 10 In regard to tea-growing elevation and plucking season, in 2 groups of 3, the fluoride contents of teas plucked in the lowlands were significantly higher, whereas the difference in the other group was non-significant. This result indicates that the elevation affected the fluoride content of teas with highland teas having a lower fluoride content than lowland teas. This difference might be due to fluoride contamination of air through evaporation of airborne sea water at lower elevations, or the soil being washed with rain water containing higher amounts of fluoride. Comparison of rolling method results indicated that the difference between 6 of 7 groups was not significant. Calculations without taking into account the eleva-
Variables affecting fluoride in Turkish black tea 43 tion and tea grade showed that the fluoride contents of tea samples processed by rolling method R1 or R2 were not significantly different, thus indicating that the rolling method had no effect on fluoride content. Considering plucking season, the F content for P1 was found higher in 11 of 21 groups, whereas for P3 it was found higher in 3 of 21 groups. The difference was not significant in 7 of 21 groups. It can be concluded that the plucking season had an effect on the fluoride content, which was highest in teas plucked in May. The fluoride content of the 84 tea samples (84 x 3 = 252 analyses) varied between 87.6 and 289.2 mg/kg, with a mean of 156.30±34.7 ACKNOWLEDGMENT The authors would like to thank the Turkish General Directorate of Tea Establishments (CAY-KUR) for providing tea samples. REFERENCES 1 Wong MH, Fung KF, Carr HP. Aluminium and fluoride contents of tea, with emphasis on brick tea and their health implications. Toxicol Lett 2003;137:111-20. 2 Chan JT, Koh SH. Fluoride content in caffeinated, decaffeinated and herbal teas. Caries Res 1996;30:88-92. 3 Fung KF, Zhang ZQ, Wong JWC, Wong MH. Fluoride contents in tea and soil from tea plantations and the release of fluoride into tea liquor during infusion. Environ Pollut 1999;104:197-205. [abstract in Fluoride 1999;32:109-10.]. 4 Zohouri FV, Rugg-Gunn AJ. Sources of dietary flavonoid intake in 4-year-old children residing in low, medium and high fluoride areas in Iran. Int J Food Sci Nutr 2000;51:317-26. 5 Kalayci ş, Somer G. Factors affecting the extraction of fluoride from tea: application to three tea samples. Fluoride 2003;36:267-70. Clarification in: Fluoride 2004;37:238. 6 Tomlins KI, Mashingaidze A. Influence of withering, including leaf handling, on the manufacturing and quality of black teas-a review. Food Chem 1997;60:573-80. 7 Werkhoven J. Tea Processing. FAO Agricultural Services Bulletin. No. 26. Rome; 1978. 8 AOAC. Fluoride in plants potentiometric method (975.04). Official Methods of Analysis. Arlington, VA: AOAC; 1990. 9 Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research. 3rd ed. NewYork: WH Freeman;1995. 10 Fung KF, Wong MH. Effects of soil ph on the uptake of Al, F and other elements by tea plants. J Sci Food Agric 2002;82:146-52. Published by the International Society for Fluoride Research http://homepages.ihug.co.nz/~spittle/fluoride-journal.htm Editorial Office: 727 Brighton Road, Ocean View, Dunedin 9051, New Zealand