Effect of ultrasonic-assisted and microwave-assisted technology on bioactive compounds and aroma quality of instant green tea

The production of instant green tea requires hot-water extraction, which is time consuming and contributes to losses in aromatic compounds. In this study, an ultrasonic-assisted technology was used to improve the extraction efficiency of green tea, thereby shortening extraction time from 45 to 15 min. In pure water, the dissolution of caffeine and theanine did not change significantly, but total catechin dissolution increased by 0.23 mg/mL and total tea polyphenol dissolution decreased by 3.2 mg/mL. In 76.2% ethanol, the dissolution of caffeine and theanine did not change significantly, but total catechin dissolution increased by 1.57mg/mL and total tea polyphenol dissolution decreased by 1.5 mg/mL. Additionally, we used microwave-assisted technology to further improve the extraction efficiency of green tea, which shortened the extraction time to 2 min. However, the extraction rate remained unchanged. In pure water, the dissolution of caffeine and theanine did not change significantly, but the dissolution of total catechins increased by 0.41 mg/mL and the dissolution of tea polyphenols decreased by 2.9 mg/mL. Ultrasonic treatment increased the proportion of 3-hydroxybutan-2-one, (5S)-5-(hydroxymethyl)oxolan-2-one and 2-phenylethanol, which were the main aroma compounds in tea. Microwave treatment changed the aroma compounds in tea, causing losses in aroma compounds with low boiling point and maintaining (5S)-5(hydroxymethyl)oxolan-2-one. The taste and aroma of instant green tea improved based on sensory evaluation results.


Ultrasonic-assisted extraction
Tea leaves (5 g) were placed in a 50-mL centrifuge tube. Extraction solvent was added at a tea-to-water ratio of 1:7 for the first extraction and 1:5 for the second extraction (hereinafter, the ratio of tea to water represents the ratio of tea to 76.2% ethanol solution). The ultrasonic extraction conditions consisted of 70℃, 120 w, and 40 kHz. The first extraction time ranged from 5 to 30 min with a 5-min interval. Following the first extraction, the tea leaves were filtered and transferred to a reagent bottle, and mixed with extraction solvent (tea-to-water ratio of 1:5) for a secondary extraction (extraction temperature did not change). The secondary extraction time was set to 0, 1, 3, 5, 7, and 9 min, where 0 was the filtrate obtained by rinsing the tea residue with solvent after the first extraction. We detected the compounds in the filtrate and calculated the extraction rates and set three replicates for each sample.

Microwave-assisted extraction
Tea leaves (5 g) were placed in a 50-mL centrifuge tube, and the extraction solvent, which was mixed with the tea leaves, was added at a tea-to-water ratio of 1:7 for the first extraction and 1:5 for the second extraction. The microwave extraction conditions were 600 w (low power). The first extraction time ranged from 0.5 to 3 min with a 0.5-min interval. Following the first extraction, the tea leaves were filtered and transferred to a reagent bottle, and mixed with extraction solvent (tea-to-water ratio of 1:5) for a secondary extraction. The secondary extraction time ranged from 0 to 1 min with an interval of 0.25 min, where 0 was the filtrate obtained by rinsing the tea residue with solvent after the first extraction. We detected the compounds in the filtrate and calculated the extraction rates and set three replicates for each sample. After determining the optimal extraction time, we optimized the microwave power (750 w, medium power and 900 w, high power) and the tea-to-water ratios (1:7, 1:8, 1:9, 1:10, 1:11, and 1:12).

Extraction rate
Water extraction was performed based on method GB/T 8305-2013. A clean empty aluminum pan with a lid was weighed (m1). Leaching filtrate was added and evaporated to dryness on a boiling water bath pan. The aluminum pan was placed in an oven at 120℃ for 1 h, placed in a dryer for 1 h, and weighed (m2). Subsequently, the aluminum pan was placed in an oven at 120℃ for another 1 h, placed in a dryer for 30 min, and weighed (m3). When m2 − m3 was < 0.001 g, the pan was dried to constant weight. Otherwise, the steps were repeated until the aluminum pan had a constant weight of mn. Leaching rate was calculated using the following equation, where A1 is the absorbance of the test solution after color development, A2 is the absorbance of the base color of the test solution, 1.957 is the factor when absorbance is 0.5 in a 10-mm cuvette (the content of tea polyphenols in 1-mL tea is equivalent to 1.957 mg), and K is the dilution factor.

Determination of Caffeine
Caffeine was determined according to method GB/T 5009.139-2014. To 1 mL filtrate, we added 9 mL of trichloroacetic acid, prepared according to method GB/T 5009.139-2014. The solution was filtered. We injected a standard series of working solutions of caffeine into the liquid chromatograph and measured the corresponding peak areas. We generated a standard curve by plotting the concentration of the working solutions against the peak areas.
Caffeine concentration in the samples was calculated according to the following formula, where c is the mass concentration of caffeine in the sample solution (μg/mL), V is the total volume of the tested sample (mL), and v is the volume of the sample (1 mL).

Determination of Catechin
Catechin was determined based on method GB/T 8313-2018. An aliquot of filtrate (2 mL) obtained using a 0.45-μm membrane was added to a 10-mL volumetric flask after filtration followed by 8 mL of stabilizer. The solution was passed through a 0.45-μm membrane after shaking into chromatographic column for testing. Stabilizers and catechin working solution were prepared based on method GB/T 8313-2018. Catechin concentration was determined using the following formula, where A is the peak area of the tested component in the sample, A0 is the peak area of the corresponding tested component in the blank, fstd is the correction factor of the measured component (concentration/peak area, concentration unit is μg/mL), v is the volume of sample extract (mL), V is the total volume of the sample to be tested (mL), and d is the dilution factor (5). The total amount of catechins was the sum of five catechins. where c is the concentration of the measured sample (mg/mL), V is the volume of the sample after final volume determination (mL), m is the mass of the sample (g), and w is the dry matter content of sample (%).

Aroma Analysis
Extract (30 g) obtained by different treatment methods was transferred to a 60-mL bottle and placed in a 50℃ water bath for 10 min. Adsorption and desorption time were 30 and 5 min, respectively.
Gas chromatographic conditions: chromatography was performed using a DB-WAX elastic Shi Ying capillary column (30 m×0.25 mm×0.25m) with a carrier gas flow rate of 1.0 mL/min. The program was set to 50℃ for 5 min, increased to 160℃ at 5℃ per min, held at 160℃ for 5 min, increased to 250℃ at 10℃ per min, and held at 250℃ for 5 min. The inlet temperature was 250℃.
Mass spectrometry conditions: the ionization energy was 70 eV with an interface temperature of 280℃, an ion source temperature of 300℃, a temperature of the four-stage pole at 150℃, and a scanning quality range of 30-500 u.

Sensory Evaluation
Samples were concentrated and dried under similar conditions. Sensory analysis was performed using a doubleblind test design, and the scoring standard referred to the sensory evaluation method for green tea recorded in GB/T 23776-2018. A total of 10 participants (five were knowledgeable on tea) from the research and development and sales personnel of Hangzhou Branch of Zhejiang Minghuang Natural Food Development Co., Ltd. participated in the analysis.

Statistical analysis
All measured or calculated parallel data were analyzed using SPSS19.0 for single-factor significance difference.

Effect of Ultrasonic Time on Extraction Rate
In the industry, the optimal extraction time for traditional one-time extraction is 30 min. Our findings showed that the extraction rate during a 30-min period was lower with water than with 76.2% ethanol. The extraction rate with water followed the equation f(t) = -0.0019t 2 +0.0847t+0.0295, R 2 =0.9903 (Fig 1). At t1=15 min, the extraction rate was 16.8% (maximum value without auxiliary extraction), and at t1 = 22.3(min), the extraction rate was 19.4% (maximum value with ultrasonic treatment). Similarly, the extraction rate with 76.2% ethanol had the following equation, f(t)= -0.0025t 2 +0.1179t+0.0536, R 2 =0.9919. At t1=15 min, the extraction rate was 25.6% (maximum value without auxiliary extraction), and at t1=23.6(min), the extraction rate was 28.8% (maximum value with ultrasonic treatment). The extraction rates of the water extraction and 76.2% ethanol extraction groups were significantly different at the two time points. However, considering that the secondary extraction could be carried out and the Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 3 March 2020 doi:10.20944/preprints202003.0041.v1 extraction time could be reduced to improve product quality, t1=15(min) was selected, followed by a secondary extraction to assess whether the total extraction rate reached a maximum value.  To conserve water, it is necessary to increase the tea-to-water ratio in the extraction conditions. However, due to the imbibition effect of tea leaves, decreasing the water volume during each extraction would contribute to a reduction in yield. Therefore, to assess the feasibility of increasing the ratio of tea to water, we combined the two extractions. The water consumption for one extraction was increased, and the total water consumption was lower than the total water consumption for the original two extractions. Under the assumption that the optimal extraction time remained at 15 min, the effect of the tea-to-water ratio on extraction rate was investigated. As shown in Figure   3, by decreasing the tea-to-water ratio, the extraction rate gradually increased. At 1:12, the extraction rates with water and ethanol increased to 22.4% and 30.4%, respectively. The total volume of water and ethanol reached the total volume of the two extractions without assistance, but the extraction rates were lower than 29.2% and 39.8% of the two extractions. Therefore, the ratios of tea to water were 1:7 and 1:5 for the two extractions. Figure 3. Effect of tea-to-water ratio of two solvents on extraction rate Considering the average temperature in the workshop of factory throughout the year, the normal leaching temperature was set at 30℃. For energy consumption, the maximum temperature did not exceed 70℃ for leaching without auxiliary conditions. The change of leaching rate at this temperature range was investigated. As shown in Figure 4, the extraction rate of the water extraction and 76.2% ethanol extraction groups increased with increasing temperature. The trend of the two groups was linear, with R 2 =0.9934 in the water extraction group and R 2 =0.9977 in the 76.2% ethanol extraction group. The effect of temperature on extraction rate had no maximum value in the selected temperature range; therefore, 70℃ was set as the optimal extraction temperature.   We investigated the effect of microwave treatment at 750 W between 0.5 and 3 min ( Figure 6). In the water group, extraction rate increased before 1.5 min, decreased from 1.5 min to 2 min, and remained relatively constant after 2 min. The extraction rate at 1.5 min was 19.5%, which was 16.8% higher than the optimal extraction rate at 15 min of ultrasonic treatment, and the energy consumption was 67.5 kJ. In the ethanol group, extraction rate gradually increased before 2 min. After 2 min, the solution began to boil. The extraction rate could not be accurately measured. The extraction rate at 2 min was 23.2%, which did not reach the maximum extraction rate at one time during ultrasonic treatment, and the energy consumption was 90 kJ. The maximum extraction rate of the microwave first extraction was reached at 1.5 min at medium power. The secondary extraction time ranged from 0 to 1 min. At time 0, the tea residue was rinsed with 1:5 solvent to obtain the filtrate. The filtrates of the two extractions were pooled, and the extraction rate was determined. Figure 7 shows that at 2 min, the extraction rate reached a maximum value of 29.1%. With increasing secondary extraction time, the total extraction rate stabilized. When total extraction time was 1.5 min, the extraction rate was 26.3%. The energy consumption was 90 kJ and 67.5 kJ in the first and second extractions, respectively, and the energy consumed per unit extraction rate was 3.1 kJ and 2.5 kJ, respectively. Therefore, the 1.5-min total extraction time resulted in the lowest energy consumption, which may improve production efficiency, but the extraction is not sufficient, while the extraction rate at 2 min was higher, which is consistent with that of ultrasonic extraction for 15 min. accurately measured. The extraction rate at 1 min was 21.6%, which did not reach the maximum extraction rate of ultrasonic treatment, and the energy consumption was 54 kJ. The 1.5-min and 2-min total extraction times had advantages and disadvantages. Therefore, the tea-to-water ratio was evaluated at both times. As shown in Figure 9, the change in extraction rate in the water extraction 1.5-min group and the water extraction 2-min group increased gradually with decreasing tea-to-water ratio, i.e., increasing water consumption. The change in the water extraction 1.5-min group was less significant. When the tea-to-water ratio was 1:12, the extraction rate was 22.3% in the 1.5-min group and 27.6% in the 2-min group. The extraction rate of single extraction was lower than that of extraction twice. Therefore, the optimal ratio of tea to water remained at 1:7 for the first extraction and 1:5 for the second extraction. extraction group was 7.9±0.2 mg/mL, which was not significantly different from the tea polyphenol content in the 76.2% ethanol extraction group (8.5±0.1 mg/mL). However, the tea polyphenol content in the water extraction group was reduced to 4.7±0.2 mg/mL following ultrasonic treatment. In the 76.2% ethanol group, the tea polyphenol content was reduced to 7.0±0.2 mg/mL after ultrasonic treatment. The water-extracted tea polyphenols were reduced to 5.0±0.1 mg/mL by microwave treatment. Ultrasonic and microwave treatment conditions in the following section were the same as those in this section. Different letters in Fig. 10 and 11 represent differences among groups (P < 0.01).

Effect of Ultrasound and Microwave on Extraction of Catechins
As shown in Figure 12, 76.2% ethanol promoted a higher degree of catechin dissolution than pure water. The distribution of catechins in the samples was similar. EGC and ECG contents changed following ultrasonic and microwave treatments, and EGC content was slightly higher in the ultrasonic-water extraction group and microwavewater extraction groups than in the pure water extraction samples. The ultrasonic-76.2% ethanol extraction group had higher EGC content than the 76.2% ethanol extraction group. Opposite results were obtained for ECG content.
Ultrasonic and microwave treatments had a promoting effect on the dissolution of EGCG and EGC in water-extracted samples, with little effect on other catechins. Therefore, the total catechin content was higher in the ultrasonic-water extraction group and microwave-water extraction group than in the water extraction group, and the promoting effect of microwave treatment was more obvious. The total amount of catechins in the ultrasonic-76.2% ethanol extraction group reached 5.06±0.06 mg/mL, which was much higher than 3.49±0.04 mg/mL in the 76.2% ethanol extraction group, mainly because the dissolution of EGCG and EGC increased following ultrasonic treatment.

Sensory analysis
The high pigment content in the sample extracted with 76.2% ethanol was easily identified, affecting the sensory analysis results; therefore, the sample extracted with 76.2% ethanol was not included in the sensory analysis. The scores obtained in Table 2 represent average scores. The taste scores were higher than other sensory scores in the water extraction group. The taste scores generally revealed a level of astringency. Sensory analysis scores were higher in the ultrasonic-water extraction group than in the other two groups. The flavor score was lower in the microwave-water extraction group than in the other two groups, but the scores of other indicators were higher IN WHICH GROUP? than in the water extraction group. 2. The taste of the tea after the entrance is not thick and mellow, and the taste is weak, scoring 5-9 points.
3. The taste is not strong, and the liquid is viscous, with a score of 1-4. 2. The fragrance is insufficient, and the aroma is not straight, with different fragrance, etc., 5-9 points.
3. The aroma is not harmonious, and the miscellaneous smell is heavy, 1-4 points.

Ultrasonic-assisted extraction
At 120 w and 40 kHz ultrasonic treatment, pure water and 76.2% ethanol reduced extraction time. The most ideal extraction time was 15 min with a water-to-tea ratio of 1:7 for the first extraction, and 0 min for the second extraction with a water-to-tea ratio of 1:5 at 70℃. At 15-min extraction time, the total extraction rate was the highest.
The maximum extraction rates were 29.2% in the pure water and 39.8% in the 76.2% ethanol, respectively, which were the same with those of treatments without auxiliary. In the presence of pure water, ultrasonic treatment had no significant effects on the dissolution of caffeine and theanine, but it had a promoting effect on the dissolution of total catechins (increasing by 0.23 mg/mL) and an inhibitory effect on the total amount of tea polyphenols (reducing by 3.2 mg/mL). In the presence of 76.2% ethanol, ultrasonic treatment had no significant effects on the dissolution of caffeine and theanine, but it had a considerable promoting effect on the dissolution of total catechins (increasing by 1.57 mg/mL) and a significant inhibitory effect on the total amount of tea polyphenols (decreasing by 1.5 mg/mL).
Ultrasonic treatment promoted the release of the main aroma compounds in tea. The main aroma compounds in the water extraction group were 3-hydroxy-2-butanone, (S)-5-hydroxymethyldihydrofuran-2-one, and 2phenylethanol, which impart, respectively, pleasant milk fragrance, fruit fragrance, and flower fragrance characteristics. The main aroma compounds in the 76.2% ethanol extraction group were neophytadiene, (S)-5hydroxymethyldihydrofuran-2-one, and acetic acid, which impart a fresh sour aroma.

Microwave-assisted extraction
The optimal microwave treatment was achieved at 750 w. The extraction rate of pure water in one extraction reached 19.5% in 1.5 min, which was higher than the optimal extraction rate of 16.8% in 15 min of ultrasonic treatment. The second extraction time was 0.5 min, and the other conditions were the same as in the first extraction.
The tea-to-water ratios of the two extractions were 1:7 and 1:5, and the total extraction rate was 29.1%. The maximum extraction rate of 76.2% ethanol under low microwave treatment was 19.3%, which was lower than 25.6% with ultrasonic treatment for 15 min. Under medium and high power, the boiling phenomenon occurred after 2 min and 1 min, respectively, while the maximum extraction rates before boiling were 23.2% and 21.6%, respectively, which were lower than 25.6% after ultrasonic treatment for 15 min.
In the presence of pure water, microwave treatment had no significant effects on the dissolution of caffeine and theanine, but had a significant promoting effect on the dissolution of total catechins (increasing by 0.41 mg/mL) and a significant inhibitory effect on the total amount of tea polyphenols (reducing by 2.9 mg/mL).

Sensory evaluation
Ultrasonic and microwave treatments improved color and taste of tea. Ultrasonic treatment promoted the release of aroma compounds and enhanced the aroma flavor of the sample, while microwave treatment negatively impacted the flavor, especially aroma compounds of low boiling point. A comprehensive evaluation showed that the total sensory score was the highest and lowest in ultrasonic and microwave treatments, respectively.

Cost of Ultrasonic-and Microwave-Assisted Extraction
The maximum extraction rate with ultrasonic and microwave treatments was basically the same as that without these auxiliary treatments. Under optimum conditions, energy consumption was 108 kJ for ultrasonic treatment and microwave extraction. However, the microwave extraction rate of the 76.2% ethanol extraction group was lower than the ultrasonic extraction rate, and was prone to boiling; therefore, it was difficult to apply microwave-assisted extraction with ethanol solution in production. Ultrasound and microwave treatments require additional energy consumption [21][22]; therefore, it is important to assess whether the value of the increased production capacity due to the improved production efficiency through auxiliary means is higher than the increased energy consumption cost.
Alternative, the increased energy consumption could be accounted for by reasonably pricing the tea products.

Ultrasonic-Assisted Extraction Effect
Compared with the conventional industrial operation conditions, under optimized conditions, the ultrasonic extraction efficiency of tea leaves was greatly improved, from the original total extraction time of 45 min for two times to 15 min. However, total extraction rate did not change significantly: 28.7% for non-assisted water extraction, 29.2% for ultrasonic-water extraction, 40.0% for non-assisted-76.2% ethanol extraction, and 39.8% for ultrasonic-76.2% ethanol extraction. Due to the lack of ultrasonic power selection, there might be a shorter extraction time and higher ultrasonic extraction efficiency, which require further evaluation.
According to the evidence, the effect of ultrasonic treatment on the extraction of green tea components varies [23][24][25]. In this study, the effect of ultrasonic treatment on specific components was mainly concentrated on tea polyphenols. In the water extraction group and the 76.2% ethanol extraction group, the content of tea polyphenols was significantly reduced due to the effect of ultrasonic waves, which significantly reduced astringency and improved tea quality.
The aroma of tea treated by ultrasonic waves increased in proportion to the main components. The flavor of the water-extracted sample was closer to that of the original tea, and the flavor of the alcohol-extracted sample was more suitable for preparing fruit-flavored tea.

Microwave-Assisted Extraction Effect
Compared with the conventional industrial operation conditions, under optimized conditions, the microwave extraction efficiency greatly improved, from the original total extraction time of 45 min for two times to 2 min.
Extraction rate did not change significantly, with 28.7% without auxiliary water extraction and 29.1% with microwave-water extraction. Microwave technology can be easily used in small-scale application. For mass production, large-scale microwave equipment are required.
The effect of microwave on the extraction components of green tea is variable [26][27][28]. In this study, the effect of microwave on specific components was mainly concentrated on tea polyphenols. The content of tea polyphenols in the water extraction group was significantly reduced due to ultrasonic treatment, which significantly reduced astringency. Microwave-assisted extraction might be suitable for making single-cup or large-pot tea.
The aroma of tea extracted by microwave treatment had obvious changes in the main components, and the natural and fresh aroma compounds with low boiling point were lost. The tea retained aroma compounds with high boiling point and had a heavy aftertaste.

Sensory Evaluation
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 3 March 2020 doi:10.20944/preprints202003.0041.v1 Studies have reported that the color and aroma of tea improves following ultrasonic and microwave treatments [29][30][31][32][33][34]. However, in this study, the aroma of tea after ultrasonic and microwave treatments increased and decreased, respectively. The quality of tea obtained by water extraction following traditional methods was acceptable only in terms of taste. The scores of other sensory indicators were significantly lower in the traditional water extraction group than in the ultrasonic extraction group, while the sensory scores of aroma were lower in the microwave extraction group than in the traditional water extraction group. Other sensory indicators were higher in the microwave extraction group than in the traditional water extraction group. The ultrasonic-water extraction group had bright tea with soft taste, weak astringency, and great aroma. Compared with the traditional water extraction group, the microwave-water extraction group had superior flavor, but not aroma. The overall tea flavor of the instant tea sample was improved by ultrasonic-water extraction compared to traditional water extraction. Compared with traditional water extraction, microwave-water extraction results in a better tea flavor.