This research was supported by the National Natural Science Foundation of China (32002094, 32102444), the China Agriculture Research System of MOF and MARA (CARS-19), and the Innovation Project for Chinese Academy of Agricultural Sciences (CAAS-ASTIP-TRI).

1. Preparation of the internal standard and tea infusion
<ol>
	<li>Stock solution: Dissolve 10.0 mg of paraxylene-d10 (see Table of Materials) in 10.0 mL of anhydrous ethanol to prepare a 1,000 ppm stock solution of the internal standard.</li>
	<li>Working solution: Dilute 1 mL of the stock solution (step 1.1) to 100 mL with pure water to prepare a 10 ppm working solution of the internal standard. 
	NOTE: The working solution must be prepared on the same day as the analysis.</li>
	<li>Place 3 g of tea leaves (both for green tea and black tea, see Table of Materials) into an Erlenmeyer flask, and add 150 mL boiling water. Cover the flask with a glass stopper.</li>
	<li>After 5 min, quickly filter out the tea infusion through a 300-mesh sieve.</li>
	<li>Wash the spent tea leaves twice with 30 mL of water, and combine the wash solution with the tea infusion.</li>
	<li>Cool the tea infusion to room temperature quickly in an ice water bath.</li>
	<li>Add 1.00 mL of working solution (step 1.2) into the tea infusion, and mix them well.</li>
</ol>
2. Distillation of the tea infusion by SAFE and liquid-liquid extraction of the distillate
<ol>
	<li>Prepare the SAFE assembly following the steps below.
	<ol>
		<li>Install the SAFE assembly (Figure 1), and connect the distillation bottle at the lower left (Figure 1[3]) and the collection bottle at the lower right (Figure 1[4]). Connect the circulating water tube at the rear of the SAFE glass assembly. Install the cold trap (Figure 1[5]), and connect the tube to the vacuum pump (see Table of Materials) at the upper right of the glass assembly. 
		NOTE: Check the connection of the circulating water tube; ensure that the inlet enters the top and the outlet exits from the bottom. Use deionized water for the circulation to prevent the scale from blocking the white tube in the SAFE assembly, which would result in poor circulation of the circulating water and the eventual explosion of the SAFE assembly.&#160;The distillation bottom (Figure 1[3]) can be stirred by a stirring bar to facilitate the evaporation of the sample.</li>
		<li>Set the temperature of the circulating water to 50 &#176;C and that of the water bath for the sample flask to 40 &#176;C. Close the vacuum valve (Figure 1[2]).</li>
	</ol>
	</li>
	<li>Perform the vacuum pump operation.
	<ol>
		<li>Power on the vacuum pump.</li>
		<li>Gradually increase the speed to the maximum speed of 100%. 
		NOTE: If the speed does not reach 100%, check whether the system is airtight and whether there is solvent residue inside the system.</li>
		<li>After reaching a high vacuum (preferably 10-3 Pa) 
		NOTE: The vacuum will improve when the liquid nitrogen is added to the cold trap.</li>
	</ol>
	</li>
	<li>Perform sample distillation.
	<ol>
		<li>Start the water circulation.</li>
		<li>Add liquid nitrogen to the cold trap to cover the outside of the collecting bottle.</li>
		<li>Pour the tea infusion into the sample funnel at the top left (Figure 1[1]), and then cover it with a glass stopper.</li>
		<li>Introduce the sample into the distillation flask dropwise. Control the sample drop speed so that the vacuum is kept in the proper range of around 10&#8722;3 Pa. 
		NOTE: Add liquid nitrogen during the process to ensure that the right collecting bottle is always submerged in liquid nitrogen. Try to avoid condensate formation in the cold trap.</li>
	</ol>
	</li>
	<li>Turn off the vacuum pump after the distillation is completed.
	<ol>
		<li>Press the power switch. When &#34;STOP&#34; flashes, press the Enter key to confirm.</li>
		<li>Unplug the power cord when the speed of the molecular pump decreases to &#34;0&#34;. 
		NOTE: Restart only when the speed decreases to &#34;0&#34;.</li>
	</ol>
	</li>
	<li>Restore the system to atmospheric pressure.
	<ol>
		<li>Remove the grinding plug above the sampling bottle.</li>
		<li>Unscrew the knob of the vacuum valve slowly to restore the system to atmospheric pressure.</li>
	</ol>
	</li>
	<li>Take down the collecting bottle with the sample.
	<ol>
		<li>Remove the liquid nitrogen outside the collection bottle after recovering the system to atmospheric pressure.</li>
		<li>Unscrew the collection bottle slowly. Take down the collecting bottle with the sample carefully.</li>
		<li>Close the circulating water.</li>
	</ol>
	</li>
	<li>Perform liquid-liquid extraction of the SAFE distillate.
	<ol>
		<li>Let the SAFE distillate in the bottle warm to room temperature.</li>
		<li>Extract the SAFE distillate thrice with 50 mL of dichloromethane (see Table of Materials).</li>
		<li>Combine the dichloromethane layers. Dry the extract with anhydrous sodium sulfate (see the Table of Materials). 
		NOTE: The anhydrous sodium sulfate in the solvent is considered dry enough when it is no longer cemented and can flow freely.</li>
		<li>Concentrate the extract to about 2 mL using a gentle nitrogen stream.</li>
		<li>Transfer to a sample vial of 1-2 mL, and further concentrate to 200 &#181;L using a gentle nitrogen stream.</li>
	</ol>
	</li>
</ol>
3. GC-MS analysis and data processing
<ol>
	<li>Analyze the aroma concentrates prepared in protocol section 2 using a GC-MS system (Figure 2) equipped with fused silica capillary columns (see the Table of Materials).</li>
	<li>Use helium as carrier gas with a linear velocity of 40 cm/s.</li>
	<li>Inject 3 &#181;L of the concentrate in splitless injection mode.</li>
	<li>Set the GC oven temperature program: (1) hold at 40 &#176;C for 5 min; (2) increase to 200 &#176;C at 5 &#176;C/min; (3) increase to 280 &#176;C at 10 &#176;C/min; (4) hold at 280 &#176;C for 10 min.</li>
	<li>Operate the mass selective detector in positive EI mode13 with a mass scan range from 30 m/z to 350 m/z at 70 eV.</li>
	<li>Deconvolute the GC-MS data using the Automated Mass Spectral Deconvolution and Identi&#64257;cation System (AMDIS, see the Table of Materials).</li>
	<li>Match and qualify the data after deconvolution using the NIST (National Institute of Standards and Technology) 17 mass spectrometer search program3.</li>
	<li>Calculate the retention index of the compounds14 based on the result of a set of n-alkanes (C5-C25, see the Table of Materials) under the same GC conditions.</li>
	<li>Identify the GC peaks using the NIST mass spectrometry library and the retention index database based on the simultaneous matching of the mass and retention indexes.</li>
	<li>Calculate the concentration of each volatile component in the SAFE sample relative to the internal standard using the TIC (total ion chromatography) peak area.</li>
	<li>Repeat the analysis three times, starting from the tea infusion preparation.</li>
</ol>

Unveiling Tea Aroma Complexity with Solvent-Assisted Flavor Evaporation Method

<ol>
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C., Wei, G., Yang, X., Schieberle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+the+key+aroma+compounds+in+aLongjinggreen+tea+infusion+(Camellia+sinensis)+by+the+sensomics+approach+and+their+quantitative+changes+during+processing+of+the+tea+leaves.">Characterisation of the key aroma compounds in aLongjinggreen tea infusion (Camellia sinensis) by the sensomics approach and their quantitative changes during processing of the tea leaves.</a> European Food Research and Technology. 246 (12), 2411-2425 (2020).</li><li>Feng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tea+aroma+formation+from+six+model+manufacturing+processes.">Tea aroma formation from six model manufacturing processes.</a> Food Chemistry. 285, 347-354 (2019).</li><li>Wang, J. -. 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Part I: Comparison of headspace-solid phase microextraction and solvent assisted flavour evaporation.</a> Lwt-Food Science and Technology. 94, 178-189 (2018).</li><li>Li, Z. W., Wang, J. 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The authors have nothing to disclose.

This article describes an efficient method for analyzing volatile compounds in tea infusions using SAFE and GC-MS analysis.
Tea infusions have a complex matrix with a high content of non-volatile components. Several methods have been described in the literature for isolating the volatile components from tea infusions. A common method is simultaneous distillation extraction (SDE)15,16. However, it is not suitable for the analysis of tea...

Tea is a preferred beverage of many people all over the world1,2. The aroma of the tea is a quality criterion as well as a price-determining factor for tea leaves3,4. Thus, the analysis of the aroma composition and content of tea is of great significance for molecular sensory studies and the quality control of tea. As a result, aroma composition analysis has been an important topic in tea research in recent years5,6,7.
The content of aroma components in tea is very low, as they generally only account for 0.01%-0.05% of the dry weight of the tea leaves8. Furthermore, the large amount of non-volatile components in the sample matrix significantly interferes with analysis by gas chromatography9,10. Therefore, a sample preparation procedure is essential to isolate the volatile compounds in tea. The key consideration for the isolation and enrichment method is minimizing the matrix interference and, at the same time, maximizing the preservation of the original odor profile of the sample.
Solvent-assisted flavor evaporation (SAFE), originally developed by Engel, Bahr, and Schieberle, is an improved high-vacuum distillation technique used to isolate volatile compounds from complex food matrixes11,12. A compact glass assembly connected to a high-vacuum pump (under a typical operating pressure of 5 x 10&#8722;3 Pa) can efficiently collect volatile compounds from solvent extracts, oily foods, and aqueous samples.
This article described a method that combines the SAFE technique with solvent extraction to isolate volatile substances from a black tea infusion, followed by analysis using GC-MS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alkane mix (C10-C25)</td>
			<td>ANPEL</td>
			<td>CDAA-M-690035</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkane mix (C5-C10)</td>
			<td>ANPEL</td>
			<td>CDAA-M-690037</td>
			<td></td>
		</tr>
		<tr>
			<td>AMDIS</td>
			<td>National Institute of Standards and Technology</td>
			<td>version 2.72</td>
			<td>Gaithersburg, MD</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>OHAUS</td>
			<td>EX125DH</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sinopharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous sodium sulfate</td>
			<td>aladdin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Black tea</td>
			<td>Qianhe Tea</td>
			<td></td>
			<td>Huangshan, Anhui province, China</td>
		</tr>
		<tr>
			<td>Concentrator</td>
			<td>Biotage</td>
			<td>TurboVap</td>
			<td></td>
		</tr>
		<tr>
			<td>Data processor</td>
			<td>Agilent</td>
			<td>MassHunter</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>TEDIA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GC</td>
			<td>Agilent</td>
			<td>7890B</td>
			<td></td>
		</tr>
		<tr>
			<td>GC column</td>
			<td>Agilent</td>
			<td>DB-5MS</td>
			<td></td>
		</tr>
		<tr>
			<td>Green tea</td>
			<td>Qianhe Tea</td>
			<td></td>
			<td>Huangshan, Anhui province, China</td>
		</tr>
		<tr>
			<td>MS</td>
			<td>Agilent</td>
			<td>5977B</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Xylene-d10</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SAFE</td>
			<td>Glasbl&#228;serei Bahr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure deionized water</td>
			<td>Milipore</td>
			<td>Milli-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Edwards</td>
			<td>T-Station 85H</td>
			<td></td>
		</tr>
	</tbody>
</table>

tea aroma analysis based on solvent-assisted flavor evaporation enrichment

Tea aroma is an important factor in tea quality, but it is challenging to analyze due to the complexity, low concentration, diversity, and lability of the volatile components of tea extract. This study presents a method for obtaining and analyzing the volatile components of tea extract with odor preservation using solvent-assisted flavor evaporation (SAFE) and solvent extraction followed by gas chromatography-mass spectrometry (GC-MS). SAFE is a high-vacuum distillation technique that can isolate volatile compounds from complex food matrices without any non-volatile interference. A complete step-by-step procedure for tea aroma analysis is presented in this article, including the tea infusion preparation, solvent extraction, SAFE distillation, extract concentration, and analysis by GC-MS. This procedure was applied to two tea samples (green tea and black tea), and qualitative as well as quantitative results on the volatile composition of the tea samples were obtained. This method can not only be used for the aroma analysis of various types of tea samples but also for molecular sensory studies on them.

Author Spotlight: Exploring Tea Aroma Using Solvent-Assisted Flavor Evaporation Technique 

Tea Aroma Analysis Based on Solvent-Assisted Flavor Evaporation Enrichment

We explore the molecular basis of taste and aroma, focusing on tea products derived from the Camellia sinensis plant. Our aim is to characterize the flavor molecules and investigate the biochemical processes affecting the sensory attributes of tea. Analyzing tea aroma requires the separation and enrichment of volatiles present in very low concentrations from the complex matrix of tea samples.The sample preparation method often poses the most significant challenge, and it's critical for obtaining valid data, leading to meaningful research conclusions. Through our systematic study on aroma formation, we successfully identified the methyl epi-jasmonate as the key compound responsible for the tea's orchid-like aroma. We also characterize the aromas of four different white tea varieties.The SAFE technique, unlike the commonly-used methods for tea aroma preparation, provides a clear volatile distillate from a tea infusion, free of non-volatile compounds. Aroma compounds can be easily enriched through solvent extraction. Mostly importantly, the odor profile of the obtained aroma concentrate closely resembles that of the origin sample.The proposed method can contribute to various research objectives, such as understanding the evolution of aroma from the tea plant to the tea cup, investigating the chemistry of specific molecules that impact the sensory ecology, characterizing tea aroma to guide plant cultivation, process improvement, quality control, and the creation of new flavors.

The analytical procedure described above is illustrated in this section using the example of the aroma analysis of black tea and green tea samples.
A representative GC-MS chromatogram is shown in Figure 3. Figure 3A shows a set of n-alkanes, and Figure 3B shows the profile of an internal standard. The evaluation results for the extracts from the green tea and black tea samples are shown in <strong class="...

Watch this Scientific Journal Video about Exploring Tea Aroma Using Solvent-Assisted Flavor Evaporation Technique at JoVE.com

Presented here is a method for enriching and analyzing the volatile components of tea extracts using solvent-assisted flavor evaporation and solvent extraction followed by gas chromatography-mass spectrometry, which can be applied to all types of tea samples.

Tea aroma is an important factor in tea quality, but it is challenging to analyze due to the complexity, low concentration, diversity, and lability of the ...

tea-aroma-analysis-based-on-solvent-assisted-flavor-evaporation

Research

JoVE Journal

Chemistry

Distillation of Tea Infusion by Solvent-Assisted Flavor Evaporation (SAFE)

Begin by installing the solvent-assisted flavor evaporation, or SAFE, glass assembly. Then, connect the distillation bottle at the lower left connection site and the collection bottle at the lower right connection site of the assembly. Connect the circulating water tube to the rear portion of the assembly, ensuring the inlet enters the top and the outlet exits from the bottom.After installing the cold trap, connect the upper right opening of the trap to the vacuum pump with a tube. Set the temperature of the circulating water to 50 degrees Celsius and that of the water bath for the sample distillation flask to 40 degrees Celsius. Then, close the vacuum valve.Switch on the vacuum pump to initiate the vacuum application. To prepare for sample distillation, first, start the water circulation. Add liquid nitrogen into the cold trap, as well as surround the collecting bottle with liquid nitrogen.Pour the tea infusion into the sample funnel at the top left and then cover it with a glass stopper. Next, gradually introduce the sample into the distillation flask drop-wise. Control the sample drop speed to maintain the vacuum within the proper range.After the distillation completes, press the control button of the vacuum pump. And when stop flashes, press the Enter key to confirm. When the speed of the vacuum pump decreases to zero, unplug the power cord.Remove the grinding plug above the sampling bottle. Then, unscrew the knob of the vacuum valve slowly to restore the system to atmospheric pressure. Once atmospheric pressure is restored in the system, remove the liquid nitrogen surrounding the collection bottle.Slowly, unscrew the collection bottle and carefully remove the bottle containing the sample. Finally, stop the water circulation.

2.0K Views.  Tea Research Institute Chinese Academy of Agricultural Sciences.  Presented here is a method for enriching and analyzing the volatile components of tea extracts using solvent-assisted flavor evaporation and solvent extraction followed by gas chromatography-mass spectrometry, which can be applied to all types of tea samples.

Unveiling Tea Aroma Complexity with Solvent-Assisted Flavor Evaporation Method

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