RU2519767C1 - Method of determining polyphenols in tea - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method involves extracting polyphenols from a crushed tea sample; determining concentration of polyphenols in the extract by a colorimetric method using Folin-Ciocalteu reagent, wherein the extract is obtained using a crushed tea sample weighing 1.0-1.5 g and 50-75 cm³ water at 95-100°C; steeping for 5 minutes at room temperature and filtering; diluting the obtained extract with water 25 times, collecting 0.5-0.6 cm³ of the diluted extract; adding 3.0 cm³ of a 0.5 M Na₂CO₃ solution and 0.3 cm³ of Folin-Ciocalteu reagent and measuring optical density of the solution at wavelength 765 nm after 2-3 min; determining concentration of polyphenols in the diluted extract from a calibration curve of optical density of tannin solution versus weigh concentration of tannin in the solution; the amount of polyphenols in tea transferred into the aqueous extract is expressed through its weight fraction in the analysed tea sample X, % per dry substance, which is calculated using a formula.

EFFECT: faster analysis, reducing the number of simple operations and low consumption of reagents.

2 tbl, 2 ex

Description

The invention relates to the tea industry and is directed to the development of new accelerated methods for assessing the quality of raw materials and finished products.

Closest to the proposed is a method for determining the content of tea polyphenols [1], which provides for the extraction of polyphenols from a ground sample of leaf tea with a 70% methanol solution at a temperature of 70 ° C, centrifugation and decantation of the liquid, repeated extraction of polyphenols from the insoluble residue of tea. The total duration of double extraction and subsequent separation of the extract from the insoluble residue by centrifugation and decantation of the liquid is 80-90 minutes The content of polyphenols in the extract is determined by the colorimetric method using the Folin-Ciocalteu reagent, which contains tungsten phosphoric acids that oxidize the OH groups of phenols and form the tungsten blue as a result of the reaction. The interaction of the extract polyphenols with Folin-Ciocalteu reagent occurs within 60 minutes. Gallic acid is used as a standard. For gallic acid solutions of different concentrations, a calibration curve is constructed for y = f (x), where y is the optical density of standard gallic acid solutions at a wavelength of 765 nm minus the optical density of a blank sample, x is the mass concentration of gallic acid in standard solutions, μg / cm ³ .

Mass fraction of polyphenols in a tea sample ω _τ ,%, calculated by the formula (1)

$${\omega }_{\tau }=\frac{(D_{sample}-D_{\mathrm{int}\text{A.}ercept})\cdot V_{sample}\cdot d\cdot \mathrm{one\; hundred}}{S_{std}\cdot m_{sample}\cdot \mathrm{10,000}\cdot W_{DMsample}},\text{(one)}$$

where D _sample is the optical density of the solution;

D _intercept is the optical density corresponding to the point of intersection of the calibration dependence with the y axis;

V _sample - extract volume (10 cm ³ ), cm ³ ;

d is the dilution coefficient used for colorimetric analysis;

S _std is the slope of the calibration line;

m _{sample sample} mass, g;

W _DMsample - mass fraction of dry matter in the sample,%.

The disadvantage of this method is the duration, the complexity, the large number of stages of the work, the use of a toxic reagent methanol for the extraction of polyphenols, the inconsistency of the result of the analysis with the yield of polyphenols from a tea sample into an aqueous extract under the conditions of traditional tea brewing in a teapot or cup, as in solution methanol passes both water-soluble and water-insoluble tea polyphenols.

The technical result of the invention lies in the fact that the proposed method for the determination of tea polyphenols can reduce the analysis time, reduce the number of basic operations and the consumption of reagents, increase work safety, obtain reliable data on the amount of polyphenols entering the human body when consuming tea extract. Due to the new ratio of reagents used in the interaction of polyphenols with Folin-Ciocalteu reagent, the reaction product - tungsten blue - is formed within 1-2 minutes, instead of 60 minutes according to the prototype, while the optical density of the colored solution does not change for 30-35 minutes , which is also convenient for analysis; in addition, reducing the time of this reaction avoids the possible interaction of tea proteins with Folin-Ciocalteu reagent, which takes more than 30 minutes.

This result is achieved in that the proposed method for determining tea polyphenols involves the extraction of polyphenols from a ground tea sample, the determination of the content of polyphenols in the extract by a colorimetric method using Folin-Ciocalteu reagent, while the polyphenols are extracted from a ground tea sample weighing 1.0-1.5 g adding to it 50-75 cm ^{3 of} distilled water with a temperature of 95-100 ° C, insisting for 5 minutes at room temperature and filtering. The extract obtained is diluted with distilled water 25 times, 0.5-0.6 cm ^{3 of the} diluted extract is taken, 3.0 cm ^{3 of a} 0.5 M solution of Na ₂ CO ₃ and 0.3 cm ^{3 of} Folin-Ciocalteu reagent are added to it and after 2-3 minutes, the optical density of the solution is measured at a wavelength of 765 nm. The concentration of polyphenols in the diluted extract is determined by the calibration graph of the optical density of the tannin solution with a molar mass of 400-500 g / mol from the mass concentration of tannin in the solution. The amount of tea polyphenols transferred to the aqueous extract is expressed by their mass fraction in the analyzed tea sample X,% on dry matter, which is calculated by the formula (2)

$$X=\frac{c\cdot 25\cdot \mathrm{fifty}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot {\omega }_{\mathrm{from}\mathrm{at}}}\text{(2)}$$

where c is the mass concentration of polyphenols in the diluted tea extract, mg / cm ³ ;

25 - breeding ratio of the original tea extract;

50 - the total volume of the initial tea extract, cm ³ ;

m is the mass of the sample, mg;

ω _St - mass fraction of dry matter in the sample,%.

The proposed method is as follows. Prepare 50 cm ³ working solution of tannin (1) with a concentration of 0.5 mg / cm ³ . Use tannin with a molar mass of 400-500 g / mol, which corresponds to the characteristics of tea tannin [2]. Diluting the working solution of tannin with distilled water in the proportions indicated in table 1, receive solutions of tannin of a lower concentration.

Table 1 Tannin solutions No. tannin solution The volumes of the working solution of tannin (1) and water used for the preparation of solutions of tannin Mass concentration of tannin in solution, mg / cm ³ one tannin working solution (1) 0,500 2 5.0 cm ^{3 of} solution (1) and 5.0 cm ^{3 of} water 0.250 3 3.0 cm ^{3 of} solution (1) and 7.0 cm ^{3 of} water 0.150 four 1.5 cm ^{3 of} solution (1) and 8.5 cm ^{3 of} water 0,075 5 1.0 cm ^{3 of} solution (1) and 9 cm ^{3 of} water 0,050

0.6 cm ^{3 of} each tannin solution is added to 5 tubes, 3 cm ^{3 of a} 0.5 M Na ₂ CO ₃ solution is added, mixed and 0.3 cm ^{3 of} Folin-Ciocalteu reagent is added. The contents of the tubes are mixed and after 2-3 minutes, the optical density of the solutions against water is measured using a spectrophotometer at a wavelength of 765 nm or a photoelectric colorimeter using a light filter that provides measurement at a wavelength closest to 765 nm. They also perform a blank experiment in which distilled water is used instead of a tannin solution. A calibration graph is constructed of the dependence of the optical density of the tannin solution minus the optical density of the blank experiment on the mass concentration of tannin in the solution. The mass concentration of tannin in the solution, shown in table 1, allows you to get a calibration graph of the linear dependence of the optical density of the tannin solution on the mass concentration of tannin in the solution. If the mass concentration of tannin in the solution is less than 0.05 mg / cm ³ , the optical density of the solution approaches zero, if the mass concentration of tannin in the solution is more than 0.50 mg / cm ³ , the optical density of the solution approaches unity. In both cases, the analysis results become unreliable. The indicated ratio of reagents — tannin solution, Na ₂ CO ₃ solution, Folin-Ciocalteu reagent — was obtained during laboratory tests; it provides almost instant formation of tungsten blue and the formation of a stable color of the solution. Moreover, the optical density of the colored solution does not change for 30-35 min, which is also convenient for analysis. Another reagent ratio slows down the reaction.

Leaf tea is ground according to ISO 1572 [3] and stored in airtight containers, providing protection from sunlight. Next, they take a sample of ground tea weighing 1.0-1.5 g, transfer quantitatively to a beaker with a capacity of 150-200 cm ³ , add 50-75 cm ^{3 of} distilled water with a temperature of 95-100 ° C, mix, insist for 5 minutes at room temperature and filtered. The ratio of the mass of the sample of ground tea and water (1.0-1.5 g and 50-75 cm ³ ), as well as the temperature of the water and the duration of extraction, are determined on the basis of technical recommendations for brewing tea during a tasting assessment and when consuming this drink [4]. When reducing the weight of the tea sample to less than 1.0 g, the result will be inaccurate due to a decrease in the volume of tea leaves, its accelerated cooling during extraction and, as a result, a decrease in the yield of polyphenols in the extract. With an increase in the weight of the tea sample of more than 1.5 g, the yield of tea polyphenols in the extract does not change, and the consumption of the analyzed tea sample increases.

The resulting tea extract is diluted with distilled water 25 times. The recommended dilution of the initial tea extract is determined by the need to obtain a concentration of polyphenols, at which after the action of Folin-Ciocalteu reagent on them, the optical density of the solution corresponding to the calibration schedule is achieved. When diluting the initial tea extract more than 25 times, the optical density of the solution approaches zero, and when diluting less than 25 times, it approaches unity. In both cases, the accuracy of the results is reduced.

0.5-0.6 cm ^{3 of} diluted tea extract was transferred to a test tube, 3.0 cm ^{3 of a} 0.5 M Na ₂ CO ₃ solution was added, stirred and 0.3 cm ^{3 of} Folin-Ciocalteu reagent was added. The contents of the test tube are mixed and after 2-3 minutes, the optical density of the solution against water is measured using a spectrophotometer at a wavelength of 765 nm or a photoelectric colorimeter using a light filter that provides measurement at a wavelength closest to 765 nm. Using the calibration graph, the concentration of polyphenols (according to tannin) in the diluted tea extract is found by the optical density of the diluted tea extract minus the optical density of the blank sample. The amount of tea polyphenols transferred to the aqueous extract is expressed by their mass fraction in the analyzed tea sample X,% on dry matter, which is calculated by the formula (2)

$$X=\frac{c\cdot 25\cdot \mathrm{fifty}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot {\omega }_{\mathrm{from}\mathrm{at}}}$$

where c is the mass concentration of polyphenols in the diluted tea extract, mg / cm ³ ;

25 - breeding ratio of the original tea extract;

50 - the total volume of the initial tea extract, cm ³ ;

m is the mass of the sample, mg;

ω _St - mass fraction of dry matter in the sample,%.

The analysis is carried out in at least two replicates. The arithmetic mean of the results of parallel determinations is taken as the final result of the analysis. The calculation result is rounded to the first decimal place.

The proposed method for the determination of tea polyphenols will reduce analysis time and reagent consumption, increase work safety, and obtain reliable data on the amount of tea polyphenols passing into the aqueous extract when brewing tea for tasting analysis or consumption of this drink.

Example 1 (prototype). Prepare the initial standard solution of gallic acid with a mass concentration of 1 mg / cm ³ . Transfer the initial standard solution of gallic acid in the volumes indicated in Table 2 to volumetric flasks with a capacity of 100 cm ³ , bring to the mark with distilled water with constant stirring.

table 2 Standard gallic acid solutions Gallic acid, standard solution The volume of the initial standard solution of gallic acid, cm ³ Mass concentration of gallic acid in a standard solution,
μg / cm ³ BUT 1,0 10 AT 2.0 twenty FROM 3.0 thirty D 4.0 40 E 5,0 fifty

Leaf tea is ground according to ISO 1572 [3] and stored in airtight containers, providing protection from sunlight.

A test tube with a 70% aqueous methanol solution is placed in a water bath at a temperature of 70 ° C and incubated for 30 minutes. 0.200 ± 0.001 g of ground leaf tea was transferred to an extraction tube, after which it was placed in a water bath at a temperature of 70 ° C. 5.0 cm ^{3 of a} heated aqueous methanol solution are added to the extraction tube, closed with a stopper and mixed on a shaker. Continue heating the extraction tube in a water bath for 10 minutes. Additional mixing on a shaker is carried out after 5 and 10 minutes. Remove the tube from the water bath and cool to room temperature. The tube is removed, the tube is placed in a centrifuge and centrifuged for 10 minutes at a speed of 3500 rpm. Decant the liquid into a graduated tube. Repeat the extraction. The extracts are combined, the volume is adjusted to 10.0 cm ^{3 with a} colloidal aqueous methanol solution and mixed. The tea extract thus prepared is diluted 100 times with distilled water. Place 1.0 cm ^{3 of} standard solutions of gallic acid A, B, C, D and E in duplicate in separate graduated tubes. Pour 1.0 cm ^{3 of} distilled water in two replicates into separate graduated tubes for blank samples. Contribute 1.0 cm ^{3 of} diluted tea extract in duplicate to separate graduated tubes. 5.0 cm ^{3 of an} aqueous solution of Folin-Ciocalteu reagent is added to each tube and mixed. After 3-8 minutes, 4.0 cm ^{3 of a} 7.5% aqueous solution of Na ₂ CO _{3 is} added to each tube, and the tubes are closed corks and mix. The solutions are aged for 60 minutes at room temperature, and the optical density of the solutions relative to water is measured with a spectrophotometer at a wavelength of 765 nm.

A linear calibration graph is constructed of the dependence y = f (x), where y is the optical density of standard solutions of gallic acid minus the optical density of a blank sample, x is the mass concentration of gallic acid in standard solutions, μg / cm ³ .

Mass fraction of polyphenols in a tea sample ω _τ ,%, calculated by the formula (1)

$${\omega }_{\tau }=\frac{(D_{sample}-D_{\mathrm{int}\text{A.}ercept})\cdot V_{sample}\cdot d\cdot \mathrm{one\; hundred}}{S_{std}\cdot m_{sample}\cdot \mathrm{10,000}\cdot W_{DMsample}},$$

where D _sample is the optical density of the solution;

D _intercept is the optical density corresponding to the point of intersection of the calibration dependence with the y axis;

V _sample - extract volume (10 cm ³ ), cm ³ ;

d is the dilution coefficient used for colorimetric analysis;

S _std is the slope of the calibration line;

m _sample - _sample weight, g;

W _DMsample - mass fraction of dry matter in the sample,%.

The analysis is carried out in triplicate. The obtained results of the content of polyphenols in the analyzed tea samples are: ω _τ1 1 = 15.41%; ω _τ2 = 14.90%; ω _τ3 = 14.82%; ω _τ cf. = 15.04%. Deviations of the results from the average value: δ = 0.37%; δ ₂ = 0.14%; δ ₃ = 0.22%. The time that went into the experiment is 220 minutes. The number of basic operations is 7.

Example 2 (proposed). Prepare 50 cm ³ working solution of tannin (1) with a concentration of 0.5 mg / cm ³ . Use tannin with a molar mass of 485 g / mol. The working solution of tannin in test tubes is diluted with distilled water in the following proportions:

1) a working solution of tannin (0,500 mg / cm ³ );

2) 5.0 cm ^{3 of} solution (1) and 5.0 cm ^{3 of} water (0.250 mg / cm ³ );

3) 3.0 cm ^{3 of} solution (1) and 7.0 cm ^{3 of} water (0.150 mg / cm ³ );

4) 1.5 cm ^{3 of} solution (1) and 8.5 cm ^{3 of} water (0.075 mg / cm ³ );

5) 1.0 cm ^{3 of} solution (1) and 9 cm ^{3 of} water (0.050 mg / cm).

0.6 cm ^{3 of} each tannin solution is added to 5 tubes, 3 cm ^{3 of a} 0.5 M Na ₂ CO ₃ solution is added, mixed and 0.3 cm ^{3 of} Folin-Ciocalteu reagent is added. The contents of the tubes are mixed and after 3 minutes the absorbance of the solutions against water is measured on a spectrophotometer at a wavelength of 765 nm. They also perform a blank experiment in which distilled water is used instead of a tannin solution. A calibration graph is constructed of the dependence of the optical density of the tannin solution minus the optical density of the blank experiment on the mass concentration of tannin in the solution.

Leaf tea is ground according to ISO 1572 [3] and stored in airtight containers, providing protection from sunlight. A sample of ground leaf tea weighing 1.0 g is taken, transferred quantitatively to a beaker with a capacity of 150 cm ³ , 50 cm ^{3 of} distilled water with a temperature of 95 ° C are added, mixed, insisted for 5 minutes at room temperature and filtered.

The resulting tea extract is diluted with distilled water 25 times. 0.4 cm ^{3 of the} diluted extract was transferred into a test tube, 2.0 cm ^{3 of a} 0.5 M Na ₂ CO ₃ solution was added, stirred and 0.2 cm ^{3 of} Folin-Ciocalteu reagent was added. The contents of the test tube are mixed and after 3 minutes, the optical density of the solution against water is measured on a photoelectrocolorimeter at a wavelength of 765 nm. Using the calibration graph, the concentration of polyphenols (according to tannin) in the diluted tea extract is found by the optical density of the diluted tea extract minus the optical density of the blank sample. The amount of tea polyphenols transferred to the aqueous extract is expressed by their mass fraction in the analyzed tea sample X,% on dry matter, which is calculated by the formula (2)

$$X=\frac{c\cdot 25\cdot \mathrm{fifty}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot {\omega }_{\mathrm{from}\mathrm{at}}}$$

where c is the mass concentration of polyphenols in the diluted tea extract, mg / cm ³ ;

25 - breeding ratio of the original tea extract;

50 - the total volume of the initial tea extract, cm ³ ;

m is the mass of the sample, mg;

ω _St - mass fraction of dry matter in the sample,%.

The analysis is carried out in triplicate. The results obtained for the amount of tea polyphenols transferred to the aqueous extract are: ω _τ1 = 8.59%; ω _τ2 = 8.44%; ω _τ3 = 7.97%; ω _τ2avr . = 8.33%. Deviations of the results from the average value: δ ₁ = 0.26%; δ ₂ = 0.11%; δ ₃ = 0.36%. The time spent on the experiment is 110 minutes. The number of basic operations - 6.

The decrease in the number of polyphenols in example 2 (by the proposed method) compared with example 1 (by the prototype) is due to different extraction conditions.

The proposed method allows to reduce the analysis time of tea polyphenols by 110 minutes, reduce the number of basic operations by 14.3%, reduce the consumption of Folin-Ciocalteu reagent by 16 times, obtain reliable data on the amount of tea polyphenols turning into an aqueous extract when brewing tea for tasting analysis or consumption of this drink.

Literature

1. GOST R ISO 14502-1-2010. Tea. Method for determination of total polyphenol content.

2. Tsotsiashvili I.I., Bokuchava M.A. Chemistry and technology of tea. - M .: Agropromizdat, 1989 .-- 391 p.

3. GOST 28550-90 (ISO 1572-80). Tea. Method for the preparation of ground samples and determination of solids.

4. Slavyansky A.A., Vovk G.A., Zhigalov M.S., Moyseyak M.B. Laboratory workshop on the technological control of tea raw materials and finished products of tea production. - M.: Publishing complex MGUPP, 2007. - 56 p.

Claims (1)

A method for determining tea polyphenols, which involves extracting polyphenols from a ground tea sample, determining the content of polyphenols in the extract by the colorimetric method using Folin-Ciocalteu reagent, characterized in that the polyphenols are extracted from the ground tea sample weighing 1.0-1.5 g by adding 50 to it -75 cm ^{3 of} water with a temperature of 95-100 ° C, insisting for 5 minutes at room temperature and filtering, the resulting extract is diluted with water 25 times, 0.5-0.6 cm ^{3 of the} diluted extract is taken, ³ are added to it, 0 cm ³ 0.5 M solution of Na ₂ CO ₃ and 0.3 cm ^{3 of} Folin-Ciocalteu reagent and after 2-3 minutes the optical density of the solution is measured at a wavelength of 765 nm, the concentration of polyphenols in the diluted extract is determined from the calibration graph of the optical density of the tannin solution on the mass concentration of tannin in solution, the amount of tea polyphenols, transferred to an aqueous extract, expressed as their mass fraction in the analyzed sample of tea X,% dry matter, which is calculated by the formula

where c is the mass concentration of polyphenols in the diluted tea extract, mg / cm ³ ;
25 - breeding ratio of the original tea extract;
50 - the total volume of the initial tea extract, cm ³ ;
m is the mass of the sample, mg;
ω _St - mass fraction of dry matter in the sample,%.