Determination of tea polyphenols in milk tea based on spectrophotometer

In this study, the characteristics of colorimetric change in turbid solution can be determined by spectrophotometer. The content of tea polyphenols in milk tea without protein precipitation is determined by using ferrous tartrate solution as the chromogenic agent at a certain temperature. The influence of dilution ratio of milk tea, the amount of chromogenic agent, the chromogenic time and the chromogenic temperature on the determination result is explored, and the analytical method for determining the content of tea polyphenols in milk tea and other tea drinks by spectrophotometer is established. The results showed that the best test results were obtained when the dilution ratio of milk tea was 2 times, the addition amount of ferrous tartrate solution was 10 mL, the color development temperature was 25 ± 1 ℃, and the color development time was 20 min. The method was applied to the determination of tea polyphenols in milk tea and other tea drinks. The linear range of tea polyphenols was 0.1-0.9mg, and the linear correlation coefficients of the obtained standard curves were above 0.9815. The recovery rate of the sample was 98.2% – 107.9%, and the relative standard deviation (RSD) was 1.7% – 6.1% (n=6). The method established in this study has high precision and accuracy, and is suitable for the determination of tea polyphenols in milk tea and other tea drinks.

Tea drink is one of the popular drinks, and tea polyphenols is one of the important ingredients of milk tea, which has the functions of antioxidation, anti-cancer, anti-inflammatory, anti-radiation, and regulation of intestinal micro-ecology [1,2,3,4]. China’s current national standard GB/T 21733-2008 stipulates that the content of tea polyphenols in flavored tea drinks (milk and milk flavor) should not be less than 200mg/kg, and describes its detection method in detail, that is, tea drinks need to be precipitated by ethanol and filtered to obtain clear samples, and then determined by ferrous tartrate colorimetry [5].

The existing research on the determination method of tea polyphenol content in tea drinks mainly focuses on the improvement of the protein precipitation link of the national standard method. For example, Du Jianzhong [6] uses anhydrous ethanol and potassium chloride solid to enhance the salting-out effect, destroy the beverage emulsification system, and precipitate proteins and other substances; Liang Ruiting et al. [7] used acetic acid as precipitator, Du Shuxia et al. [8] used acid acetone as demulsifier and precipitator to obtain clear solution. However, with the improvement of tea beverage processing technology, the stability of tea beverage systems such as milk tea has been enhanced by adding high-quality emulsion stabilizer and using homogenization technology [9,10], which also increases the difficulty of precipitation protein to obtain clear solution, and thus has a negative impact on the test results.
The principle of determining the content of tea polyphenols by using the UV-visible spectrophotometer and ferrous tartrate colorimetry is that the ferrous tartrate solution reacts with the tea polyphenols in the precipitated clear solution to form a stable purple-blue complex with a characteristic absorption wavelength of 540 nm, and the absorbance value at the characteristic absorption wavelength is in direct proportion to the content of tea polyphenols in the solution, The content of tea polyphenols in the system can be calculated by measuring the absorbance value of the sample at 540 nm with the UV-visible spectrophotometer [11]. The spectrophotometer uses the chromaticity values L, a and b to indicate the color change in the visible light area, where L represents black and white (brightness), a represents red and green, and b represents yellow and blue [12,13,14]. The spectrophotometer can not only accurately measure the chromaticity change in the solution system, but also the chromaticity change in the turbid solution system, and has been well applied in the chromaticity detection of apple turbid solution [15]. As a kind of tea beverage, milk tea has the dual nutrition of milk and tea, and has unique flavor and taste. It is an important representative of tea beverage. In this study, the content of tea polyphenols in the milk tea system was determined by spectrophotometer. The characteristic of this method is that the milk tea sample does not need sedimentation, which can reduce the complexity of sample pretreatment, increase the detection accuracy and reduce the detection cost.

1. Materials and methods

1.1 Materials and reagents

Ferrous sulfate, potassium sodium tartrate, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate (analytical reagent, Tianjin Damao Chemical Reagent Factory); Ethyl gallate (analytical reagent, Shanghai McLean Biochemical Technology Co., Ltd.); Milk tea drinks sold in the market (a supermarket in Dongguan).

1.2 Instruments and equipment

ME104E analytical balance (Mettler Toledo Group, Switzerland); CS-500 handheld spectrophotometer, (Hangzhou CHNSpec Technology Co., Ltd.); Constant temperature heating magnetic stirrer (Heigoph, Germany).

cs 500 portable spectrocolorimeter - Determination of tea polyphenols in milk tea based on spectrophotometer

1.3 Experimental method

1.3.1 Reagent preparation

Ferrous tartrate solution [16]: Weigh 0.1g of ferrous sulfate and 0.5g of potassium sodium tartrate into a beaker, dissolve them with a small amount of distilled water, transfer them to a 100mL volumetric flask, and fix the volume to the scale. Use as you go.
1mg/mL tea polyphenol standard solution: use ethyl gallate as the standard of tea polyphenol [17], accurately weigh 250mg of ethyl gallate in a beaker, dissolve it with a small amount of distilled water, transfer it to a 250mL volumetric flask, and fix the volume to the scale. Use as you go.
PH=7.5 phosphate buffer solution [18]: weigh 23.87g of Na2HPO4 solution into a beaker, dissolve it with a small amount of distilled water, transfer it to a 1L volumetric flask, and fix the volume to the scale; Weigh 9.08g of KH2PO4 solution into a beaker, dissolve it with a small amount of distilled water, transfer it to a 1L volumetric flask, and bring it to the scale; Take 850mL of the above Na2HPO4 solution and 150mL of KH2PO4 solution and mix them evenly.

1.3.2 Method optimization

  • 1) Optimization of dilution ratio of milk tea. Take a certain amount of milk tea and dilute it with distilled water to obtain diluents A, B, C, D and E, with dilution ratios of 1, 2, 4, 6 and 8 respectively. Take 2mL of the above A, B, C, D and E diluents into a 50mL volumetric flask, add 0.4mL of standard solution, 8mL of distilled water, and 10mL of ferrous tartrate solution to each experimental group, fully shake them up, dilute them with pH7.5 phosphate buffer solution to the scale, and develop color at 25 ± 1 ℃ for 20min. In the blank control group, replace the standard solution with phosphate buffer solution.
  • 2) The addition amount of color developer is optimized. Take 2mL of milk tea diluent B into a 50mL volumetric flask, add 0.4mL of standard solution and 8mL of distilled water, respectively add 5mL, 10mL, 15mL, 20mL and 25mL of ferrous tartrate solution, shake well, dilute to volume with pH7.5 phosphate buffer solution, and develop color at 25 ± 1 ℃ for 20min. In the blank control group, replace the standard solution with phosphate buffer solution.
  • 3) Color rendering time optimization. Take 2mL of milk tea diluent B into a 50mL volumetric flask, add 0.4mL of standard solution, 8mL of distilled water, and 10mL of ferrous tartrate solution, shake well, and dilute to the scale with pH7.5 phosphate buffer solution, and color at 25 ± 1 ℃ for 10min, 20min, 30min, 40min, and 60min respectively. In the blank control group, replace the standard solution with phosphate buffer solution.
  • 4) Color rendering temperature optimization. Take 2mL of milk tea diluent B into a 50mL volumetric flask, add 0.4mL of standard solution, 8mL of distilled water, and 10mL of ferrous tartrate solution, shake well, dilute to volume with pH7.5 phosphate buffer solution, and then develop color for 20min at 5 ± 1 ℃, 25 ± 1 ℃, and 37 ± 1 ℃, respectively. In the blank control group, replace the standard solution with phosphate buffer solution.
  • Use the spectrophotometer to measure the chromaticity values of the above experimental group and the blank group, and record them as L, a, b and L0, a0, b0 respectively, and calculate the △ E value according to the publicity (1). 20230110232111 53007 - Determination of tea polyphenols in milk tea based on spectrophotometer

1.3.3 Determination of standard curve

1.3.2 The standard curve needs to be determined under the different experimental conditions, as follows: take 2mL of milk tea diluent into a 50mL volumetric flask, add 8mL of distilled water, a certain amount of ferrous tartrate solution, 0.1-0.9mL of standard solution, dilute to the scale with pH7.5 phosphate buffer solution, and then develop color at a certain temperature for a certain time. In the blank control group, replace the standard solution with phosphate buffer solution. Calculate the color difference value △ E of the experimental group relative to the blank group, and draw the standard curve with △ E as the ordinate and the standard concentration as the abscissa.

1.3.4 Determination of spiked recovery

Determination of spiked recovery: substitute each △ E in 1.3.2 into the standard curve to obtain the experimental value X of spiked content, and calculate the spiked recovery according to the following formula.

Recovery rate of spiking=5X/Y6 x100%

In the formula: X, the experimental value of ethyl gallate standard content, in mg; Y. Theoretical value of ethyl gallate standard content, unit: mg.

1.3.5 Determination of tea polyphenol content in milk tea

Dilute the milk tea to be tested twice with distilled water, take 2mL of the milk tea diluent into a 50mL volumetric flask, add 8mL of distilled water and 10mL of ferrous tartrate solution, shake it well, dilute it to the scale with pH7.5 phosphate buffer, and then color it for 20min at 25 ± 1 ℃. No ferrous tartrate solution was added in the blank control group. Calculate the color difference △ E of the experimental group relative to the blank group, and substitute it into the standard curve to calculate the content of tea polyphenols in the milk tea to be tested.

1.3.6 Data analysis

All experiments were repeated three times or more, and the data were expressed as mean ± standard deviation. Origin 9.1 software was used for mapping, and SPSS 16.0 data processing software was used for one-way ANOVA, P<0.05 showed statistically significant difference.

2. Results and analysis

2.1 Optimization of milk tea dilution ratio

When the dilution ratio of milk tea is small, the concentration of tea polyphenols in the system is high, and the stability of the color correlation value of the system is poor [19]. At this time, the concentration of interference impurities in the milk tea system is also high [20], which affects the determination results. As shown in Table 1, when the dilution ratio is 2, there is no significant difference (P>0.05) between the spiked recovery rate of 99.7 ± 2.6% and the spiked recovery rate when the dilution ratio is 4, both of which are close to 100%. Moreover, the RSD value at this dilution ratio is small, and the test reproducibility is good. Therefore, in the subsequent determination process, the diluted release ratio of milk tea is 2 times.
Table.1 Effect of milk tea dilution ratio on the recovery rate of tea polyphenols

Dilution ratio of milk tea Scaled quantity/ml5mg6 Standard addition recovery rate 1% RSD/%5n=66
1 0. 4 109.4±3.4ab 3. 2
2 0. 4 99. 7 ± 2. 6bc 2. 6
4 0. 4 101. 1 ± 9. 0bc 8. 9
6 0. 4 115. 7 ± 14. 3a 12. 3
8 0. 4 90. 4 ± 8. 0c 9. 3

Note: There is a significant difference between the corner marks on different letters (P<0.05).

2.2 Optimization of colorant addition

Table.2 Effect of color reagent addition on the recovery rate of tea polyphenols

Dosage of developer/mL Scaled quantity/ml5mg6 Standard addition recovery rate 1% RSD/%5n=66
5 0. 4 79. 3 ± 6. 2b 7.9
10 0. 4 99. 7 ± 2. 6a 2.6
15 0. 4 101.4±3.1a 3.1
20 0. 4 93. 9 ± 5. 4a 5.7
25 0. 4 98. 4 ±6. 5a 6.6

Note: There is a significant difference between the corner marks on different letters (P<0.05).
As shown in Table 2, when the amount of chromogenic agent was increased from 5mL to 10mL, the recovery rate of spiking increased by 20.4% (P<0.05), and the RSD value decreased by 5.3%; When the amount of chromogenic agent was continuously increased to 25mL, there was no significant difference in the recovery rate of spiking between the groups (P>0.05) and all were close to 100%, but the RSD value would increase with the increase of the content of chromogenic agent. When the color reagent is insufficient, the color reaction is not complete, resulting in a low measured value of the recovery of spiking. When the amount of chromogenic agent is more than 10mL, the measured value tends to be stable. In order to avoid the impact of excessive ferrous tartrate on the determination result, the amount of chromogenic agent added in the subsequent determination process is set to 10mL.

2.3 Optimization of color development time

As shown in Table 3, when the color development time is 10min, 20min and 30min, there is no significant difference (P>0.05) and the recovery rate of spiking between groups is close to 100%. When the color development time is more than 30min, the recovery rate of spiking gradually decreases with the increase of color development time. Ferrous tartrate interacts with catechol hydroxyl or pyrogallol hydroxyl in the structure of tea polyphenols [21]. The formation and stability of the interaction products are closely related to time. The time is short, and the formation of interaction products is incomplete; If the time is too long, the structure of the interaction products may change and affect the determination results. When the color development time is 20min, the recovery rate of spiking is close to 100% and the RSD value is relatively small, so the color development time is 20min in the subsequent determination process.
Table.3 Effect of color development time on the recovery rate of tea polyphenols

Color rendering time/min Scaled quantity/ml5mg6 Standard addition recovery rate 1% RSD/%5n=66
10 0. 4 97. 1 ± 10. 0ab 10. 3
20 0. 4 99. 7 ± 2. 6a 3. 0
30 0. 4 105.3±6.0a 5. 7
40 0. 4 90. 9 ± 2. 9b 3. 3
60 0. 4 74. 6 ± 5. 0c 6. 7

Note: There is a significant difference between the corner marks on different letters (P<0.05).

2.4 Color development temperature optimization

As shown in Table 4, within the range of color development temperature selected in the experiment, the color development temperature has little effect on the recovery of spiking. When the color development temperature is 25 ± 1 ℃, the recovery of spiking is 99.7 ± 2.6%, which has no significant difference from the recovery of spiking at 37 ± 1 ℃, and the RSD value is low, with the best reproducibility. Therefore, 25 ± 1 ℃ is selected as the color development temperature for subsequent determination.
Table.4 Effect of color temperature on the recovery rate of tea polyphenols

Color rendering temperature/ Scaled quantity/ml5mg6 Standard addition recovery rate 1% RSD/%5n=66
5 ± 1 0. 4 87. 8 ± 12. 9b 14. 8
25 ± 1 0. 4 99. 7 ± 2. 6a 2. 6
37 ± 1 0. 4 104. 5 ± 6. 9a 6. 7

Note: There is a significant difference between the corner marks on different letters (P<0.05).

2.5 Recovery test

Under the best experimental conditions, that is, when the dilution ratio of milk tea is 2 times, the amount of chromogenic agent is 10 mL, the chromogenic time is 20 min, and the chromogenic temperature is 25 ± 1 ℃, add 0.30, 0.35, 0.40, 0.45, and 0.50 mL of standard solution respectively, and repeat the experiment for 6 times, the average recovery rate of standard addition is 98.2% – 107.9%, and the relative standard deviation (RSD) is 1.7% – 6.1% (n=6), as shown in Table 5.
Table.5 Recovery rate of tea polyphenols

Scaled quantity/ml5mg6 Standard addition recovery rate 1% RSD/%5n=66
0. 30 106. 8 ±2. 4 2. 3
0. 35 100. 9 ±1. 8 1. 7
0. 40 99. 7 ± 2. 6 2. 6
0. 45 98. 2 ± 1. 7 1. 7
0. 50 107. 9 ±6. 6 6. 1

2.6 Determination of tea polyphenol content in milk tea

Take commercially available 1 # bottled milk tea, 2 # bottled milk tea, 3 # milk tea powder, 4 # milk tea powder, 5 # ready-made milk tea, 6 # ready-made milk tea as samples, and draw the standard curve according to the operation steps of 1.3.3 (take 1 # bottled milk tea as an example, see Figure 1). The standard curves obtained are y=6.4x+0.65, R2=0.9917 (1 #); y=5.1253x+0.3694,R2=0.9822(2#); y=2.5717x+0.7058,R2=0.9941(3#); y=3.6301x+0.2049,R2=0.9815(4#); y=5.6936x+0.4814,R2=0.9843(5#); y=4.5533x+0.2335,R2=0.9848(6#)。 It shows that the standard tea polyphenol has a good linear relationship within the range of 0.1-0.9mg.
Determine the content of tea polyphenols in the sample according to the operation steps in 1.3.5 (see Table 6). Polyphenol content of various commercially available milk tea: milk tea powder>bottled milk tea>ready-made milk tea.
20230110234947 68720 - Determination of tea polyphenols in milk tea based on spectrophotometer
Fig.1 Standard curve of ethyl gallate
Table.6 Determination of tea polyphenol content in milk tea

Milk tea sample number Tea polyphenol content/(mg/mL)
1# 0. 48 ±0. 03
2# 0. 54 ±0. 01
3# 1.34 ±0.06
4# 0. 81 ±0. 02
5# 0. 14 ±0. 02
6# 0. 37 ±0. 03

3. Conclusion

In this study, the content of tea polyphenols in milk tea was determined by spectrophotometer. When the dilution ratio of milk tea was 2 times, the amount of chromogenic agent added was 10 mL, the chromogenic time was 20 min, and the chromogenic temperature was 25 ± 1 ℃, the results were good. The average recovery rate of this method is 98.2% – 107.9%, and the relative standard deviation is 1.7% – 6.1%, with high accuracy and precision. This method has high efficiency, low cost, and overcomes the shortcomings of the national standard method that milk tea is difficult to precipitate, and avoids the complicated operation before the experiment. It is suitable for determining the content of tea polyphenols in milk tea and other tea drinks.

Author: Yao Jiaqi, Yang Zebing, Liu Shuxian, Li Yuting, Chen Junru, Ye Ran, Luo Xuanying, Yang Guohua

Source: China Manufacturer of Spectrophotometer – www.spectrumgfa.com

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