BG65649B1 - Method for the assessment of the colour and the colour classification of tobaccos - Google Patents

Abstract

The method is used for objective digital assessment of the colour of Oriental tobaccos and offers the opportunity for their classification by colour of types and Oriental and Virginia type tobacco varieties. Sample of dry powder ground tobacco is analyzed by means of liquid chromatography for the determination of the content of the polyphenols of neochlorogenous acid (Neoch), chlorogenbous acid (Chlor), 4-0-caffeolequinic acid (Caff) and rutin (Rut). Then the numerical value of the colour of the tobacco is determined according to the formula C = 16.15 û 5.81 x [K] Neoch + 5.24 x [K]Caff + 16.00 x [K]Rut û 12.61 x [K]Chlor wherein the concentrations (K) of the above substances are expressed in wt.%. The numerical values produced are related to a reference scale in which the basic coloursare: yellow û mark 10, red - mark 20, orange û mark 15, and intermediate colours: with marks expressed as mean arithmetic values of the basic colours. Then processing of the chromatographic profiles of the polyphenols for the tobacco classification by colour is made, making use of the retention time of each peak (tR) and the percentage participation of area (A) of each peak from the sum of areas of the selected peaks. The results are expressed by similarity indices (Is) between the compared samples. Classification can be made towards the colour of tobacco chosen as a reference unit, the Is of which is taken as 100%, or without a reference unit û by the mutual comparison of the different tobaccos.

Description

(54) METHOD OF ASSESSMENT OF COLOR AND TOBACCO COLORIFICATION

Technical field

The invention relates to a method for evaluating the color of Oriental tobacco and for color classification of types and varieties of Oriental and Virginia tobacco, which is used in the tobacco industry.

BACKGROUND OF THE INVENTION

The prevailing practice of assessing tobacco color so far is based on the organoleptic judgment of a panel of experts who determine the color of tobacco in an approximate manner. This implies the highly subjective nature of the assessments, which is their major drawback,

It is known that physical methods such as colorimetry and reflectance spectrophotometry (1,2) are applied to objectify the evaluation of tobacco color. The use of the Munsell system for the determination of color based on reflectance spectrophotometry is too complex. To facilitate the application of this system, color coefficients have been developed / 1 /. However, their calculation is not less difficult process / 1 /.

The main disadvantages of the physical methods limiting their application are the large scattering of the results due to the variation in color and difficulty in taking a homogeneous sample. Satisfactory results were obtained only after applying a statistical clustering of data allowing the creation of a homogeneous sample / 2 /.

Also known is the dependence of tobacco color on the chemical composition and in particular on the content of polyphenols in tobacco (3,4,5). However, this dependency is only used for an approximate verbal (not numerical) description of color.

SUMMARY OF THE INVENTION

The problem to be solved with the invention relates to the creation of a method for objective numerical evaluation of the color of Oriental tobacco and the possibility of color classification of Oriental and Virginia type tobacco and varieties within each of these types.

The method for evaluating tobacco color and color classification according to the invention is as follows. A dry powdered tobacco sample was analyzed by liquid chromatography to determine the polyphenols content of neochlorogenic acid (Neoch), chlorogenic acid (Chlor), 4-0-caffeic acid (Sai) and rutin (Rut). The numerical value of the color of the tobacco is then determined by the formula:

C = 16.15 - 5.81 x | K] + 5.24 x [K | _£ "+ 16,00 x -12.61 x [K] ^ where the concentration (K) of the said substances are expressed in wt. %. The numerical values obtained relate to a reference scale in which the primary colors are: yellow, grade 10, red, grade 20, orange, grade 15, and intermediate colors, with estimates expressed as the arithmetic mean of the base colors. The chromatographic profiles of the polyphenols are then processed to classify tobacco by color, using the retention time of each peak (Q and the percentage of area (A) of each peak of the sum of the areas of the selected peaks. The results are expressed by means of indexes of similarity (I _s ) between the samples compared, the classification can be done in two ways: by the color of the tobacco selected as the standard whose Is is taken as 100%, without the standard by comparing the different tobacco between them.

The advantages of the process according to the invention are expressed in the following. The proposed chemimetric method combining high performance liquid chromatography (HPLC), multivariate regression analysis (MPA) and pattern recognition (MPO) methods allows for an objective numerical assessment of Oriental tobacco color and digital color classification, expressed in digital color , between

65649 Bl

Oriental and Virginia types, as well as between varieties within each type. The content of typical tobacco substances - Neoch), Chlorogenic (Chlor), 4-0-Caffeic and Acid (Caff) and rutin (Rut) polyphenols is used to objectively determine the color of Oriental tobacco. determined by a reliable method (HPLC). A regression model was developed to calculate the color of Oriental tobacco, including the concentration of said polyphenols. Image recognition methods are applied to classify tobacco by color, using information from all components in the HPLC profile of polyphenols. For this purpose, a medium sample of ground tobacco was taken, whereby the error from the sample homogeneity and hence the scattering of the results is negligible. The same sample can be used to determine other major indicators of tobacco quality (nicotine, carbohydrates, total nitrogen), giving complete information on the tobacco tested. To perform the process according to the invention, standard liquid chromatography apparatus is used, the chromatographic determinations being fast, reproducible and of low cost. This implies the high cost-effectiveness of the proposed method.

Examples of carrying out the invention

The method for evaluating the color of Oriental tobacco and for color classification of tobacco types and varieties is as follows.

Medium sample: An average sample of dry ground tobacco powder in an amount of 50 to 100 g is prepared according to the requirements of the sampling standard for analysis.

Analysis of the polyphenols by high performance liquid chromatography: (HPLC) (6.7): 0.1 g of tobacco powder was extracted with a mixture of methanol and water at a ratio of CH ₃ OH: H ₂ O = 60:40 (v / v). The resulting extract was separated into fractions by the solid phase extraction (TFE) method with cartridge C18 in mobile phase CH ₃ OH: H ₂ O = 60:40 (v / v). The first 10 ml fraction was filtered through a 0.45 µm membrane filter.

An aliquot portion of the solution determines the polyphenols content. Separation by components is done by reverse phase HPLC. A Perkin Elmer liquid chromatograph (Perkin Elmer Ltd., Beaconsfield, Buckinghamshire, England) consisting of a LC 290 biner pump, an LC 290 UV / VIS detector and an LCI-100 integrator or other adequate configuration is used. Separation and detection were performed under the following conditions: Kromasil LC18 analytical column, 150 mm, 5 µm, 4.6 mm id (Supelco Park, Bellefonte, PA, USA) or adequate; wavelength 340 nm; mobile phase velocity 1.0 ml min ¹ ; sample volume 20 microl; composition of the mobile phase A - CH ₃ OH: H ₂ O: UNCCD = 5: 93: 2; B - CH _} OH: Hp: CH ₃ COOH = 86: 12: 2. Multiple phase gradient applied: 100% A, 0 min; 20 min to 85% A; 5 min to 80% A; 17 min to 45% A; 5 min to 100% A and 5 min to 0% A for cleaning.

The identification and quantification of the peaks of chlorogenic acid and routine are performed with standard solutions of pure substances (Merck), at a concentration corresponding to that in the sample. The peak of the isomers of chlorogenic acid (neochlorogenic acid and 4-0-caffeic acid) is used to identify and quantify the peaks of the chlorogenic acid.

Each sample is analyzed in at least two replicates. The relative standard deviation of the results is 2 to 5%. The analytical yield of polyphenols is from 95 to 100%.

Defined parameters:

- Retention time and peak area of the individual components present in the chromatographic profile of the polyphenols.

- Content of the following polyphenolic components by weight. %:

Chlorogenic acid (Chlor.) Neochlorogenic acid (Neoch.) 4-0-Caffeic acid (Caff.) Routine (Rut.)

Calculation of the color (C) of Oriental tobacco using the regression model using the formula:

C - 16.15 - 5.81 χ [Κ] _ΝΜΛ + 5.24 x (K] ^

16.00 χ [Κ] ", - 12.61 hVD ^,)

The regression equation has a high determination coefficient (R ** ² = 0.88).

65649 Bl

The numerical values obtained refer to a reference scale in which the color is represented as follows:

Basic colors

Grade 10 - yellow

Rating 20 - Red

Grade 15 - Orange

Intermediate colors

The estimate of intermediate colors is expressed as the arithmetic mean of the primary colors, for example orange to orange-red [15+ (15 + 20) / 2] / 2 = 16.2; yellow-orange to orange [15+ (10 + 15) / 2] / 2 = 13.7; orange-red to red [(15 + 20) / 2 + 20] / 2 = 18.7.

Processing of chromatographic profiles by MPO for classification of tobacco by color.

The image recognition method (MPE) is implemented through the Patreco software package.

The MPO matrix is compiled using the retention time of each peak (Q and the percentage of area A for each peak of the sum of area A of the selected peaks in the processing of all chromatograms. In the absence of peak area (A), The following criteria / 8,9 / shall apply to the selection of peaks for MPOs:

- high degree of peak separation and good repeatability of t _R and A;

- statistically significant value of area A (twice the noise of the detector signal);

- elimination of peaks with significant participation in all samples, the amount of which does not vary greatly.

MPO results are expressed by similarity indices (Is,%) between the compared samples.

The limit value for the similarity indices (Is ₀ ) is estimated on the basis of five times the repetition of one sample. At Is? The ₀ samples are considered indistinguishable because the differences are statistically insignificant.

When processing HPLC profiles of polyphenols, all 12 peaks present in most samples were used. The limit value (Is ₀ ) for Is from polyphenols is 98%.

The invention is illustrated by the following specific examples which do not limit it.

Oriental Tobacco Color Assessment Example 1.

Plovdiv variety 50 [* U _ch = 0.09%; [K] _caff = 0.22%; [K] _Route = 0.60%; [K] _Chlor = 1.23%

C = 10.8 (Corresponding to color scale) Example 2.

Krumovgrad variety 988

IKU »-» · <»%; [«U = about 1b%; 14 »= ° .78%; - ο. "%

C = 16.8 (corresponds to the orange-red color on the scale)

Oriental Tobacco Classification:

The MPO comparison matrix contains 12 chromatographic data:

retention time - t _R grade L 880 - 10.40; 14.53; 17.38; 20.16; 24.27; 28.86; 29.95; 34.53; 36.22; 37.37; 38.40; 39.55.

grade L 413 - 10.40; 14.61; 17.33; 20.05; 24.27; 29.13; 30.35; 34.64; 36.13; 37.24; 39.54.

% area A participation:

grade L 880 - 7.50; 0.55; 37.22; 15.55; 2.41; 0.80; 0.90; 1,23; 27.69; 1,23; 0.60; 6,23.

grade L 413 - 9,67; 0.40; 39.44; 17.91; 2,7; 10.80; 1.14; 1.92; 20.63; 1.57; 0.00; 4.96.

The similarity index (Is,%) of 92.4% indicates the color difference of the two Oriental tobacco varieties.

Claims (9)

Claims 1. Method for color assessment and color classification of tobacco, characterized in that a sample of dry powdered tobacco is subjected to liquid chromatography analysis to obtain a chromatographic profile for the determination of the content of the neochlorogen polyphenols4 65649 B1 is a new acid, chlorogenic acid, 4-0 which determines the numerical value of the color pheoylchloric acid and rutin, the time of the cured tobacco according to the formula: harvesting and peak area of polyphenols, after C = 16.15 - 5.81 x [K] _{Neodi +} 5.24 x [K] _Caff + 16.00 χ [K] _Rut -12.61 x [K] _SIog ), where the concentrations (K) of the mentioned substances are expressed in weight. %, and the numerical values obtained refer to a reference scale in which the primary colors are: yellow, grade 10, red, grade 20, orange, grade 15, and intermediate colors, with estimates expressed as the arithmetic mean of the base colors, and the classification of tobacco by color is done by treatment of the chromatographic profiles of said polyphenols, using the retention time of each peak (Q and the percentage of area (A) of each peak of the sum of the areas of selected peaks, and the results expressed chre h the indexes of similarity (I _s ) between the samples compared, the classification being made by the color of tobacco selected for the standard whose Is is taken as 100%, or without the standard by comparing the different tobacco between them. Literature 1. Guzelev, L., T. Drachev: Method for objective determination of tobacco color and system for its labeling; Bulgarian. Tobacco 2 (1967) 83-42. 2. Garcia,,., R. Barreiro and M, RuizAltisent: Development of a virtual expert for color classification of tobacco leaves. Validation against human experts; and the Sensoral 98-Intemational Workshop on Sensing Quality of Agricultural Products, Montpellier France. Actes de Collogne, TechnologyAgronomie section, 1, 2000, 3086. 3. Drachev, T .; Relationship between chemical composition and color of tobacco; Bulgarian. Tobacco 1 (1967) 33-37. 4. Drachev, T.: Application of the method for $ 1 objective determination of the color of tobacco during fermentation; Bulgarian. Tobacco 4 (1967) 26-32. 5. Grabuloski, T. and Z. Taseska; Study of the correlation between tobacco color and polyphenol content of prilep tobacco; Tutun 3- ₂₀ 4 (1977) 227-234. 6. Court, W. A., J. G. Hendel, and R. Poce: Fractionation of flue-cured tobacco samples using Sep-Pak cartridges; Tob. Sci. 35 (1991) 59-62. 7. Snook, Μ. E. and Ο. T. Chortyk: An improved extraction-HPLC method for tobacco polyphenols. Tob. Sci. 16 (1982), 25-29. 8. Dimov, Yu., E. Stojanov, G. Velikov: Differentiation of commodity production by one of the methods of image identification; Chimia and ₃₀ Industria 5 (1984) 210. 9. Dimov, N., A. Tsoutsoulova and E. Stojanov: Pattern recognition methods for the discrimination of essential oils (rose oils) by their gas chromatograms; Perfumer & Flavorist 12 (1987) 45-48.