Tea chemistry – What do and what don’t we know? – A micro review

Journal Pre-proofs Tea chemistry – what do and what don’t we know? – A micro review Ulrich H. Engelhardt PII: DOI: Reference:

S0963-9969(20)30145-9 https://doi.org/10.1016/j.foodres.2020.109120 FRIN 109120

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Food Research International

Received Date: Revised Date: Accepted Date:

15 December 2019 18 February 2020 19 February 2020

Please cite this article as: Engelhardt, U.H., Tea chemistry – what do and what don’t we know? – A micro review, Food Research International (2020), doi: https://doi.org/10.1016/j.foodres.2020.109120

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Tea chemistry – what do and what don’t we know? – A micro review Ulrich H. Engelhardt, Institute of Food chemistry, Schleinitzstr. 20, D-38106 Braunschweig. voice: +49 531 3917203 mail: [email protected]

Abstract: Progress in analytical tools have led to a deeper insight into the chemical constitution and reaction pathways during the tea manufacture. However, the challenges have also changed as “new” teas are traded internationally which makes the authentication much more complicated. This micro-review demonstrates that despite all the achievements in the field of validated methods, authenticity, non-targeted methods we still have some gaps. New reactions products have been detected and those might be useful for authenticity purposes. As regards definitions of certain types of tea it makes sense to combine compositional data generated by validated targeted methods with non-targeted work to get a clearer view. Some more work seems to be necessary to get e.g. a deeper insight in the fate of proanthocyanidins during different types of processing and to develop a concept to quantify the thearubigins. There was progress in our knowledge of the thearubigin fraction in the last decade, however, there are still concepts to develop.

Keywords: Camellia sinensis; definitions; flavonoids; thearubigins; non-targeted analysis; authentication; chromatography; mass spectrometry. 1.

Introduction

Tea is one of the mostly consumed beverages in the world, both in the traditional ways of drinking and also as a constituent of ready-to-drink beverages. The number of papers dealing with potential health benefits of tea or its constituents is still growing. Some types of tea became increasingly sold in the western world, such as white tea, dark tea, yellow tea and matcha. Teas can be divided in aerated/fermented teas (see also section 2a), such as oolong, white and black teas, postfermented teas, such as dark teas and non-fermented/-areated teas, such as green tea. Most of the teas new to western consumers do have a tradition in the countries of origin. Due to globalization those teas come around the globe, together with some information and research results. In earlier days our knowledge regarding the 1

research in China was quite poor as most of the papers were in Chinese. This has dramatically changed and lots of papers from China are in the international literature now. There are a few databases around which include data for tea constituents, such as the USDA (United States Department of Agriculture) flavonoid database (Bhagwat & Haytowitz, 2015), however, it is far from comprehensive. Modern analytical devices and different approaches are widely in use, such as nontargeted metabolomics employing Ultra-High-Performance Liquid Chromatography (UHPLC) hyphenated with Quadrupole Time-of-Flight Tandem Mass Spectrometry (qTOF) and the results of those studies might contribute to definitions to be set-up. The aim of this micro review is to shed a bit of a light on the current status and the work necessary in the future. It is based on a presentation held at the CoCoTea 2019 in Bremen. It does cover only selected items and some aspects are completely ignored, such as the volatile tea constituents and the health benefits of tea, among others. A more comprehensive review covering both chemical constituents and biological activities has been published recently (Zhang, Ho, Zhou, Santos, Armstrong & Granato, 2019), another one focusses on health properties of green tea (Xing, Zhang, Qi, Tsao, & Mine, 2019). Not really covered here are the next set of products produced from those teas – the tea powders, which can be divided in e.g. hot and cold water soluble black tea powders, white and green extracts, possibly with an enrichment of certain compounds by the extraction process (Engelhardt, 2007). Those have to be also covered by the analytical approaches and definitions, but they will not be discussed in this paper.

2.

Definitions

2a. Green and black teas To set up definitions of a certain types of tea data are necessary, to generate data we need methods. Definitions are necessary for authentication. Figure 1 illustrates the interdependencies. In 2011 the ISO has published two international standards, one on green tea definition and requirements (ISO, 2011) and the other on black tea (ISO 2011a). Those standards contain the definition and the basic requirements for 2

both green and black tea. It is stated, that the plant material has to be Camellia sinensis and also that “acceptable processes” have to be employed, in case of green tea the enzyme deactivation, in case of black tea aeration. The latter term is commonly referred to as fermentation, sometimes as oxidation. The term aeration is included in the latest ISO standard on black tea definition. The common term fermentation is not quite correct in a strict sense as it is just a conversion of constituents by endogenous enzymes. The standards are based on compositional data, e.g. for total catechins and total phenolics. To get those data firstly the methodology had to be set up and validated. In 2005 the ISO standards 14502-1 (Folin-Ciocalteu-Assay for total phenolics) and 14502-2 (HPLC determination of catechins) were issued. A more detailed description of these and other analytical methods can be found in Stodt & Engelhardt (2013). Using this methodologies data were generated and a database was compiled containing each 300 green and black origin teas with a good geographic coverage (Obuchowicz, Engelhardt & Donnelly, 2011). The results were evaluated to include as much teas as possible to fulfill the requirements of the standards. The data also supported the discrimination of green and black teas in the ISO standards, where the minimum amount of total polyphenols (green: 11%, black: 9%) and total catechins (green: 7%) is specified. Other parameters in those definitions are water extract, ash (total, water-soluble incl. alkalinity, acid-insoluble ash) and crude fiber. There are some teas fitting in both categories. Darjeeling black teas do often have more than 7% total catechins and 11% total phenolics, and even the ratio total catechins: total phenolics is more than 0.5. The database shows a huge variation in the compositional data of green and black teas, and also within both groups, which is in tune with the available literature. To give some figures the total phenolics content of teas are in most cases roughly between 10 and 20% (Folin-Ciocalteu Assay, calculated as gallic acid), the sum of the major catechins between 7 and 15% in green and 2 and 4% in black tea (except Darjeeling samples and some other low aerated teas). As regards the analytical tools for total phenolics there are a number of other approaches than Folin-Ciocalteu around, such as the Ferrous tartrate method (Turkmen, Sari & Velioglu, 2006) among lots of other methods used. In case of green or black teas the results are similar (Turkmen, Sari & Velioglu, 2006). Folin-Ciocalteu reagent is known to react with ascorbic acid, which might generate a problem with bottled tea drinks. There have been suggestions on the ISO level to use therefore the 3

ferrous tartrate method for bottled tea drinks as there is no reaction with ascorbic acid. Both methods and other bulk methods possibly in use do certainly have the drawback that there is just one calibrant (e.g. gallic acid monohydrate or ethyl gallate) and a number of analytes with probably different response factors. 2b. Other types of tea For other types of tea, no definitions are available at the moment. For white tea the ISO has issued a technical report (ISO, 2013) but not a standard. In case of white tea and oolong tea working groups on an ISO level are established. It can be anticipated that it is possible to get a definition of white tea which most white teas will fulfill, however, a discrimination of green and white teas might be difficult as long as the approach described under 2.a is followed. This is even more complicated with oolong tea. In the literature often the term “semi-fermentation” was used. This could be interpreted as a half-time fermentation/aeration, which is not really correct. Oolong tea is manufactured by a special process, which includes the so-called bruising. A future definition will certainly describe this step in a greater detail. The problem will be that there are oolong teas with a very light “fermentation”, others with a medium degree of “fermentation” around, moreover, some heavily “fermented” oolongs samples are also available. Consequently, some oolong teas will fulfill the requirements for green tea, others for black tea. It will be necessary to establish other criteria, such as an optical recognition (in case of oolong possibly photographs of oxidized leaves) or other criteria in addition to the chemical requirements. Future research might be able to identify certain reaction products between simple phenolics, amino acids or carbohydrates which are possibly related to a certain step in manufacture (see also section 7). 3.

Catechins, theaflavins and other dimers

As already mentioned in section 2a a validated method for the determination of the major catechins is available since 2005 (ISO 14502-2). It includes gallic acid, caffeine, and the flavanols EGC, +C, EC, EGCG and ECG (formulae and abbreviations see figure 2). Methyl epigallocatechin gallate is not included in the standard despite the fact, that some cultivars contain 3”-methyl epigallocatechin gallate. Data can be found in Lv, Yang, Ma, Wang, Shi, Zhang, Peng, Tan, Guo, & Lin (2014). 4

Other relevant constituents, such as theogallin (Fig. 1), theobromine, and the flavanols GC, GCG, CG can be separated and quantified, however, those have not been included in the original standard. The major advantage of this international standard method was the RRF (Relative Response Factor) concept. Briefly, really pure catechins, characterized by MS (mass spectrometry), NMR (Nuclear Magnetic Resonance), chromatographic methods and with a known moisture content have been used to establish Relative Response Factors versus caffeine based on measurements of a number of expert labs worldwide. Those factors can be used by other labs to quantify the catechins, caffeine and gallic acid. The gold standard would certainly be that all labs worldwide had pure standards in sufficient amounts so everyone can prepare calibration solutions. However, due to the price of most of the pure catechin standards this is often not the case. So analysts might tend to use very small amounts of the standards to prepare the calibration solutions. The results are in this case depending on the specification of the balance used and a certain variation of the results by this reason can be anticipated. This has been overcome by using the RRF concept. Another crucial thing is the use of stabilization solutions to ensure the stability of the analytes during the time on the autosampler. Catechins and theaflavins are known to be relatively susceptible to degradation in dilute solution. ISO 14502-2 makes use of a so called stabilization solution consisting of EDTA and ascorbic acid in aqueous/organic media. The method does just rely on extraction and no further clean-up is included. This works with major constituents quite well, however, if minor constituents are relevant is has to be checked very carefully whether or not a clean-up gives better results. The original method was an HPLC method. Figure 3 shows a UHPLC chromatogram of an instant green tea. The separation of the catechins is possible within roughly 6 mins or even faster compared to 25 mins with conventional HPLC. Moreover, with both HPLC and UHPLC, using a 2nd detection wavelength most of the flavonol glycosides can be determined. Using a slightly modified gradient also the 4 major theaflavins can be separated, however, those methods are currently not validated. Future development of targeted methods should consider to quantify not just the catechins but also flavonol glycosides and possibly other analytes in one run, so time and money is saved. In fresh tea leaves and in green tea some of the relevant dimers are not present as those are reaction products of the enzymatic conversion. In green tea relevant 5

amounts of proanthocyanidins (Fig. 2) have been detected, in average between 1 and 2% as a sum of 20 compounds (Engelhardt, Lakenbrink & Lapczynski, 2000). Not too many papers on proanthocyanidins in tea can be found in the literature. Moreover, it is clear that the average amount of proanthocyandins is higher in green compared to black tea (Engelhardt, Lakenbrink & Pokorny, 2004). It is also known that the bisflavanols or theasinensins are formed during the enzymatic oxidation. However, the fate of the proanthocyanidins during the oxidation remains unclear (see chapter oxidation products). This is one of the gaps in our knowledge. On one hand around 20 different proanthocyanidins even in black teas can be quantified on the other hand the compounds are not often mentioned in the non-targeted work and the information on oxidation products of the compounds is really scarce. Other relevant dimers are the well-known theaflavins (see figure 4). Theaflavins have been named by Roberts in the late 1950ties (and the structures have been elucidated by Collier et al. some years later (Collier, Bryce, Mallows, Thomas, Frost, Korver & Wilkins, 1973). In black tea manufacture the theaflavins are formed from a pair of catechin precursors, one gallocatechin and one catechin, e.g. TF is formed from EGC and EC (Collier, Bryce, Mallows, Thomas, Frost, Korver & Wilkins, 1973). There are 4 major theaflavins present in oxidized teas, constituting in average usually around 1%. Other TF compounds are isotheaflavin, neotheaflavin, epitheaflavic acid and theaflagallins, for which not many quantitative data are in the open literature (Engelhardt, 2013). Higher concentrations of the major TFs can be found in CTC (Crush, Tear, Curl) teas from Assam or Kenya, lower concentrations are present in Darjeeling samples. More recently theaflavins were detected in white tea samples, depending on the type of white tea the sum of the 4 major TFs is 0.03% (Silver needle, n=10), 0.10% (white Peony, n=17), 0.18% (ShouMai, n=26) and 0.18% (compressed brick tea) according to Tan, Engelhardt, Lin, Kaiser & Maiwald (2017). Currently no validated method for the TF has been published, however, an international ring test with a simple methodology is on the way. It employs the same concepts as mentioned for the catechins (no clean-up but just extraction, RRF concept, stabilization solution). There are data in the literature, however, some have been generated using the flavognost assay which just gives a sum of the TFs (Steinhaus & Engelhardt, 1988; Robertson & Hall, 1989). Others make use of calibration standards which might not be really pure. Again, pure theaflavins are meanwhile available but are not too 6

cheap. The synthesis and function of the theaflavins have been reviewed recently (Takemoto & Takemoto, 2018). This paper also shows, that more recently some biosynthetic methods have been published to get higher yields of theaflavins. This might also help to overcome the calibration problem.

4.

Oxidation products – thearubigins (TR)

The thearubigins got their name by E.A.H. Roberts in the 1950ties (Roberts & Wood, 1951). For black tea TRs are always referred to as the most abundant group of compounds. I agree to that, however, real robust figures are not available from my point of view. Using liquid-liquid extraction a fractionation scheme was developed (Roberts, Cartwright & Oldschool, 1957), separating the TRs into fractions (SI, SII and SIa) based on their solubility in ethyl acetate and n-butanol. This “nomenclature” is still used by some groups nowadays. In earlier times it was believed that TRs are of high molecular mass (polymers), which is not quite true. In the literature a number of papers dealing with the chemistry of thearubigins have been published. A remarkable paper was the “thoughts on thearubigins” by Haslam published in 2003. Attempts to separate individual compounds from the thearubigin fraction have failed. Thearubigins elute even on relatively high resolution HPLC or UHPLC as a Gaussian hump in the chromatogram – not separated at all. It is even a task to get a pure TR fraction free from resolved peaks on the hump. The isolation of a pure TR fraction by countercurrent chromatographic (CCC) techniques was firstly published in the early 1990ties (Wedzicha, Lo & Donovan, 1990). This methodology was further developed by addition of a XAD-7 clean-up prior to CCC (Degenhardt, Engelhardt, Wendt & Winterhalter, 2000). Another method to get TR factions is the caffeine precipitation (Kuhnert, Drynan, Obuchowicz, Clifford, & Witt, 2010). More recently a combination of caffeine precipitation and size-exclusion chromatography was employed for that purpose (Wang, Zhang, Lv & Sang, 2018). It was possible to get hold of pure TR fractions by the methods mentioned, however, it was far away from a structural elucidation or a quantification. The first real progress was in 2010 when highresolution mass spectrometry entered this game introduced by the Clifford and the Kuhnert group.

7

According to this work we have four building blocks for the TRs: the theaflavins, the bisflavanols or theasinensins, the theanaphthoquinones and the theacitrins (all in Figure 4). These compounds are formed during the enzymatic oxidation, which is also the first steps of the TR formation (Kuhnert, 2010). The mechanism is firstly a reaction of a o-quinone generated from a flavanol with another flavanol in the original state. The intermediate can react to a theaflavin by CO extrusion and formation of a benzotropolone system, it can form bisflavanols (theasinensins) via a rearomatisation or theacitrins. Next step in the so-called oxidative cascade is a successive oxidation of the four types of building blocks mentioned (Kuhnert, Drynan, Obuchowicz, Clifford, & Witt, 2010). To understand the huge variety of compounds formed it has to be kept in mind that there is not only one theasinensin present but at least 8 different compound. The same is true in principal for the other building blocks. More recently additional products of the oxidative cascade the so-called theatridimensins have been identified (Verloop, Vincken & Gruppen, 2016). TR fractions were isolated from black tea by caffeine precipitation and different mass spectrometric measurements on FTICR (Fourier transform ion cyclotron resonance mass spectrometry) and other mass spectrometric techniques, such as ion-trap were employed. The evaluation was done via van Krevelen and Kendrick plots. It was concluded from the experiments that TR are formed via oxidation formally yielding an insertion of oxygen. Therefore, thousands of different oxidation products are built which are very similar to each other and unresolvable by chromatographic techniques. Later the hypothesis was confirmed via the LC-MS/MS analysis of a TR fraction with a molecular mass below 1000, where polyhydroxylated theanaphthoquinones and theasinensin C derivatives were identified, among other compounds generated by the aid of H2O2 during processing. (Yassin, Koek, Jayaraman & Kuhnert, 2014). It was stated that the peroxidase can perform the hydroxylation step in the oxidative cascade (Verloop, Vincken & Gruppen, 2016a). Furthermore, techniques like ion mobility MS have been employed to get a deeper insight (Yassin Grun, Koek, Assaf & Kuhnert, 2014). In model fermentations the formation of the building blocks could be confirmed along with degradation kinetics of catechins and theaflavins as well as the formation kinetics of TRs (Stodt, Blauth, Niemann, Stark, Pawar, Jayaraman, Koek, & Engelhardt, 2014). 8

Currently no real concept to quantify the TR is available. The very early approaches by Roberts (briefly, a combined liquid/liquid extraction and spectrophotometric measurements) are interfered by resolved non-TR compounds, such as flavonol glycosides. It was proposed to quantify the TR indirectly following this scheme (Lakenbrink, Lapczynski, Maiwald & Engelhardt, 2000): - Total flavonoids = total phenolics – (gallic acid + theogallin + chlorogenic acids) - Thearubigins = Total flavonoids - (catechins + theaflavins + flavonols + flavones) Total phenolics were determined by the Folin-Ciocalteu-Assay, all the other analytes by individual HPLC systems. Despite the fact that it is a hell of an effort to generate all the data it is not even quite correct as the data for proanthocyanidins and bisflavanols are missing as do figures for hydrolyzable tannins. However, it was an approximation. Currently one concept could be an estimation of the hump by using an isolated fraction (e.g. a CCC fraction) as a calibrant and just measure the area of the hump at 280 nm, however, this method has not been developed and is has certainly the drawback that it might give figures, but no information on the constituting TR compounds. Recently identified compounds include products from the reaction of phenolic acids and flavanols, e.g. the reaction product from chlorogenic acid (CGA) and flavanols, EGCG-CGA and EGC-CGA (see figure 5) in model oxidations with horseradish peroxidase. The compound had a benzotropolone moiety as with theaflavins and the concentration in black tea were 13.3 and 41.6 mg x kg-1 (Zhang, Yang, Idehen, Shi, Lv, & Sang 2018). In some teas the concentration of coumaroyl quinic acid (CoQA) is higher compared to caffeoylquinic acid (CQA) (Horanni, 2010; Engelhardt, 2010). Therefore, it could be possible that reaction products of coumaric acid are present, depending on the mechanism of formation. Other reaction products from EGC and phenolic acids have been identified in Zijuan green tea (Ke, Dai, Zheng, Wu, Hua, Hu, Chu & Bao 2019). Two pairs of phenylpropanoidated ECGs have been elucidated by spectroscopic techniques, 2 compounds are present in both fresh leaves and made green tea, two compounds only present in the made tea. The structures of 2 of the compounds are characterized by a six-membered lactone attached to the A-ring at C7/C8. It is currently not clear whether or not the compounds can be found in other teas than Zijuan tea.

9

The compounds mentioned have not been analyzed for further reactions. Maybe oxidation products will be identified in the TR fraction contributing to the hump. One remark on the content of hydroxycinnamic acid derivatives content in teas: Not too many quantitative data are available in the open literature. In UHPLC-MS studies the CQA and CoQA isomers are detected, however, without any figures given. The content of chlorogenic acids in tea is usually between 0.5 and 1% (six isomers: 3-, 4and 5 CQA and 3-, 4- and 5 CoQA) with the CoQa being more abundant (Horanni, 2010; Engelhardt, 2010 & 2013). Another compound in tea is theogallin, which is with a content of usually 0.6% (0.1 to 1.5%) sometimes more abundant than the CGA in sum (Engelhardt, Lakenbrink & Lapczynski, 2000). Theogallin is often detected in systems employing a MS-detection, however, quantitative data are scarce. Systematic studies on reaction products of theogallin with e.g. flavonoids and the fate during oxidation with state-of-the art methods are currently not available and need to be initiated. 5. Flavonol glycosides (FOG) and Flavone C-glycosides (FCG) The occurrence of myricetin, quercetin and kaempferol O-glycosides in tea is known since long. Data have been published (Engelhardt, Finger, Herzig & Kuhr, 1992), showing that in some oxidized black teas the amount of FOG is higher or at least in the same order of magnitude than the flavanol content. In those early days overall 14 FOG (3 myricetin, 6 quercetin and 5 kaempferol glycosides) could be detected. The methods included a clean-up by polyamide column chromatography prior to the HPLC separation (Engelhardt, Finger, Herzig & Kuhr, 1992; Jiang, Engelhardt, Thraene, Stark & Maiwald, 2015). As shown in that paper the separation of rutin (quercetin-3-O-rutinoside) and K-grgal (kaempferol-3-O-glucorhamnogalactoside) was not possible and a second HPC system for that was necessary. Later the method was further developed and 18 FOG could be quantified (Engelhardt, Lakenbrink & Lapczinski, 2000; Jiang, Engelhardt, Thraene, Stark & Maiwald, 2015). Typical contents of the sum of FOG detected is between 0.5 and 2% (as aglycone), sometimes the content is a bit lower (Engelhardt, 2010; Fang, Song, Xu & Ye, 2019; Hilal & Engelhardt, 2007). Again, this depends on the geographic origin, the plant material and the processing. Flavonol glycosides seem to be relatively stable during manufacture compared to catechins. Recently a study has been published on the fate of 21 FOG during the 10

manufacture of different types of tea starting from the same leaf material (Fang, Song, Xu & Ye, 2019). Fresh tea leaves were processed in the typical way for green, yellow, white, oolong and black tea. As the starting material was the same the influence of manufacture could be assessed. Under the conditions in the study black tea process led to a reduction in FOG (sum) of roughly 38%, oolong process yielded a reduction by 25% while this in white green and yellow tea was lower (all less than 14% reduction). In the supplementary information data on the FOG content of nearly 500 teas – most of them from China – can be found. Not too many data are available for acylated glycosylated flavonols, which have been tentatively identified (Zhao, Chen, Lin, Harnly, Yu. & Li, 2011). When comparing data from the literature with each other it has to be clearly observed whether the authors have calculated the contents as glycosides or the respective aglycones. In case of quercetin-3-O-rutinoside it goes by a factor of around 0.5 (MR glycoside: 610, MR aglycone 302). Not many data are available for the flavone C-glycosides from tea. Data from the older literature indicate, that the concentration (sum of 8 compounds) is a roughly a factor 10 lower compared to the FOG (sum of 19). The content in tea is in the range from 0.05 to 1.4 g/kg (calculated as aglycones) for both green and black teas, however, it was just a few samples (Engelhardt, 2013). It can be anticipated that the FCG are even more stable than the O-glycosides, however, systematic studies are missing. FCG have been considered as a marker for tea content in hard treated cold water soluble tea powders and consequently as a marker for the amount of tea used in ice teas or similar products (Lakenbrink, 2000). 6. Amino acids Tea contains roughly 4% of free amino acids, depending on the origin and the type of tea. Theanine (γ-glutamylethylamide) often constitutes around 50% of the free amino acids in tea and can be more than 3% (Engelhardt, 2013). There is a number of papers dealing with the methodology of the determination of amino acids in tea, and also supplying compositional data. Amino acid determination and data have also been used for authentication purposes by discriminating between green, white, oolong, black and Pu-erh teas (Alcázar, A., Ballesteros, Jurado, Pablos, Martín, Vilches, & Navalón (2007). Currently a validated method is available only for theanine, the most abundant amino acid in tea, (ISO, 2017). This method is very 11

simple, it works without any derivatization and uses an isocratic HPLC system with a detection in the UV at 210 nm. An aqueous extraction is carried out as theanine is good water-soluble and a clean-up not mandatory. It is possible to get better chromatograms and a column protection by an (optional) polyamide column chromatography in the standard method which removes most of the phenolic compounds. This method is not capable to separate D- and L-Theanine, however, it is in the literature that only very small amounts of D-theanine are present (EkborgOtt, Taylor & Armstrong, 1997; Horanni & Engelhardt, 2015). Other approaches, such as HPLC with post column derivatization or HPLC-MS analysis (Desai & Armstrong, 2004) have also been employed. As regards the determination of free amino acids a number of HPLC-systems is available. A method for the quantification of nineteen amino acids after precolumn derivatization with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) has been published along with quantitative data for more than 50 teas of different types (Horranni & Engelhardt, 2013). More recently a HPLC method using 6-aminoquinolylN-hydroxysuccinimidyl carbamate reagent for derivatization was published also capable to separate 19 free amino acids (Li, Li, Tai, Gu, Song, Jiao, Ning, Wei, Gu, Ho, Hajano & Wan, 2018). Other approaches with different derivatization reagents or techniques (“postcolumn”) have also been employed. The quantitative data for the amino acids are certainly depending on the origin and the processing of the tea leaves. The results of several studies are in most cases in the same order of magnitude. For authenticity detection a database with origin teas and as much information on the clone, the area and the processing condition would be very helpful.

7. Aspects of authenticity Tea authenticity might have different aspects, e.g. the geographic origin, the type of tea or the age of tea, among others. Some aspects of authentication have been summarized on the occasion of earlier symposia (Engelhardt, 2007; Engelhardt, 2011), including the methods frequently used for that purpose. An example for frequently used methods is NIR spectroscopy, which was recently employed for the authentication of green teas (Zhuang, Wang, Chen, Wu & Fang, 2016) and Darjeeling teas (Firmani, De Luca, Bucci, Marini & Biancolillo, 2019), detection of 12

geographic origin (Hong, Fu, Wang, Zhang, Yu, & Ye, 2019) and also for other purposes such as determination of fresh tea leaf (Hazarika, Chanda, Sabhapondit, Sanyal, Tamuly, Tasrin, Sing, Tudu, & Bandyopadhyay, 2018) or matcha quality (Wang, Zareef, He, Sun, Chen, Li, Ouyang, Guo, Zhang & Xu, 2019).A new aspect is here the age of teas. For Chinese white teas the price is dependent on the age of the white tea (Xie, Dai, Lu, Tan, Zhang, Chen & Lin, 2019). Recently a group of marker compounds were identified in a non-targeted metabolomics study using UHPLC−QTOF/MS (ultrahigh-performance liquid chromatography-quadrupole timeof-flight mass spectrometry). The so-called EPSF (8-C N-ethyl-2-pyrrolidinonesubstituted flavan-3-ols, see figure 5) are formed from theanine after cyclisation and flavanols during storage in a time dependent manner (Dai, Tan, Lu, Zhu, Li, Peng, Guo, Zhang, Xie, Hu. & Lin, 2018). Originally those compounds were identified as marker compounds for Chinese post fermented teas (Wang, Zhang, Wang, Shi, Jiang, Li & Tu, 2014) and later in white teas (Li, Liu, Zhang, Zhou, Ling, Wan & Bao, 2018). The behavior and the formation of those reaction products are not quite clear. In a study with yellow tea it was stated, that heat plays an important role in the formation of N-ethyl-2-pyrrolidinone-substituted flavan-3-ols and the content is increasing with temperature (Zhou, Wu, Long, Ho, Wang, Kan, Cao, Zhang & Wan, 2019). It is not completely understood whether or not the compounds are also present in other types of tea of a certain age or treatment, but this merits more in-depth studies. There are probably also new reaction products from other precursors to be identified. Those should be evaluated in terms of authentication as we need more specific tools for that purpose. Another example for authentication issues is Matcha, which was not consumed in the western world to a huge extent before 2010. Matcha is a traditional Japanese and Chinese tea. Clearly, Matcha is more expensive than a normal finely ground green tea and consequently a possible victim for food fraud. Japanese Matcha is defined by a certain type of shading before plucking, a special steaming process and milling in a stone mill (ISO, 2017a; Horie, Ema, Nishikawa, & Nakamura, 2018). For the time being the detection of shading seems to be the one which can be easier detected than the others. As shading modifies the biosynthesis, it is possible to detect a difference between shaded and non-shaded tea by chemical analysis. It was found 13

that shaded teas have higher contents of theanine and lower contents of phenolic compounds. The chlorophyll content was also higher in original matcha compared to ground green tea by a factor of 2 (Engelhardt, Kaiser & Maiwald, 2016). At the ISO level a technical report for Matcha has been proposed. The effect of shading on the metabolites were studied using a 1H-NMR spectroscopy. The levels of amino acids increased, while some of the catechins decreased as did the glucose level. The authors conclude that there might be a proteolysis the low glucose level (Ji, Lee, Lee, Hwang, Park, Kim, Park, & Hong, 2018). One aspect of authenticity or detection of food fraud may not be forgotten: mixtures. The query is how to detect 10 to 20% of a finely ground non-shaded green tea in an authentic Matcha. This is probably not too easy using just compositional data. The same problem will probably come up with a detection of geographic origin. There are a number of teas on the list for Protected Geographic Origin (PGI) in the European Union (European Union, 2019). It will be quite a task to authenticate those PGI teas. On the other hand: what does a PGI help if no one could detect a potential fraud? Not much information is available on tea low molecular carbohydrates. A recently published paper employs HILIC (hydrophilic interaction liquid chromatography) coupled to MS to quantify low molecular weight carbohydrates from tea (MegíasPérez, Shevchuk, Zemedie, & Kuhnert, 2019). This work included also data for minor compounds like myo-inositol, maltose and stachyose. The carbohydrates could be included in authentication concept in the future. For geographic traceability often stable isotope ratio combined with statistical procedures like PCA or LDA have often been employed. A recently published paper to detect the origin of green teas from China may serve as an example (Liu, Yuan, Zhang, Shi, Hu, Zhu & Rogers, 2019). Authentication will be an issue in the future. There are a number of methods and tools we can use. The following should be kept in mind: We need to join forces. It is scientifically a good thing to publish a paper with e.g. 50 samples and a certain methodology, however, in most cases it will be cited by other authors and not at all be of a relevance for the trade. It would push things forward if an obviously valid approach could be tested and used by a consortium of 4-5 labs worldwide to generate a database to be used for authentication. 8. Recent progress in methodology – Use of high sophisticated equipment 14

Recent tea papers – most of them from China – make use of high-end methods like UHPLC-HR-MS techniques. Most of the approaches are not-targeted, e.g. Qi, Li, Qiao, Lu, Chen, Miao, Guo & Ma (2019). The typical workflow of targeted and nontargeted work can be found in Ribbenstedt, Ziarrusta & Benskin (2018). In those papers always a number of compounds is identified via high-resolution mass spectrometry out. Using that tool in combination with modern statistical approaches often a differentiation of the teas analyzed with respect to geographical origin, type of tea or processing can be achieved. Work of that kind has been published e.g. with respect to the seasonal variations of compounds affecting the taste of the beverages (Dai, Qi, Yang, Lv, Guo, Zhang, Zhu, Peng, Xie, Tan & Lin, 2015). Similar approaches have been used in the authentication of black tea infusions using LC-MS measurements and multivariate statistics (Shevchuk, Jayasinghe & Kuhnert, 2018). Some other studies using similar techniques have been already cited in section 7. A comparison of targeted foremost chromatographic methods and (non-targeted) NMR approaches with special reference to the analysis of tea has been reviewed showing the advantages of a combined work (Daglia, Antiochia, Sobolev & Mannina, 2014). 9. Conclusions New concepts have to be developed which make use of both targeted and nontargeted approaches for definition and authenticity purposes. As regards the targeted work it should be tried to combine methods were possible to get more data by less work, e.g. flavanols, theaflavins and flavonol glycosides. Faster methods, such as UHPLC will certainly have to be used to a higher extent in the future and existing HPLC methods have to be modified for UHPLC and validated. Researchers should go for reaction products which might be correlated to a certain step in manufacture, e.g. able to discriminate between steaming and pan-firing. There still is a need to make use of classical targeted analysis, and more validated methods need to be established. In earlier times foremost the major compounds could be identified and quantified. Due to must more sensitive and selective analytical methods nowadays also minor products of the reactions during processing can be identified. In terms of structural elucidation, the gold standard was an isolation of the pure compounds and characterization by spectroscopic method, foremost NMR techniques and mass spectrometry. In case it is possible it still should be aimed 15

for that, however, it is certainly laborious and time-consuming. Researchers tend to use hyphenated methods with a high-resolution mass spectrometer nowadays often as the only method. With non-resolvable compounds, such as thearubigin fractions, this is the only way to get any structural information. A number of recently published state-of-the-art studies was carried out in China, sometimes with a focus on special problems in Chinese origins. This is very good and will hopefully encourage researchers from other countries to contribute so we can get the full picture.

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Figure 1: Definitions and analytical approaches: Overview of items depending on each other.

Figure 2: Natural tea constituents – A catechins or flavanols: R1=R2=H: epicatechin (EC), R1=H, R2=OH: epigallocatechin (EGC), R1=H, R2=galloyl: epicatechin gallate (ECG), R1=OH, R2= galloyl: epigallocatechin gallate (EGCG). A1: catechin, gallocatechin and so on. B: theogallin (TG: 5-galloyl quinic acid); C: Chlorogenic acid = 5-caffeoyl quinic acid, 5-CQA; D: principle formula of a 4-8 proanthocyanidin: shown here is EGCG-4-8-EGCG; E: Flavonol-3-O-glycosides: Aglycone R1=R2=H: Kaempferol, R1=OH, R2=H: quercetin, R1=R2=OH: myricetin – the sugar attached at C3 is galactose F: A flavone-C-glycoside (shown is vitexin: apigenin-8-C-glycoside). R=OH: aglycone is luteolin

Figure 3: UPLC chromatogram of a tea powder. Abbreviations see figure 2 except Caf=caffeine, GA=gallic acid, TB=theobromine.

Figure 4: Products of enzymatic conversion. A: theaflavins; R1=R2=H: theaflavin (TF), R1=galloyl, R2=H: theaflavin-3-gallate(TF-3-g), R1=H, R2=galloyl: theaflavin3’-gallate (TF-3’-g), R1=R2=galloyl: theaflavin-3,3- gallate (TF-3,3’-g) B: theacitrin C: theanaphthoquinones; D: bisflavanols E: a theaflavin after oxidation during the oxidative cascade

Figure 5: Recently identified reaction products. A1/2: Structures of 8-C N-ethyl-2pyrrolidinone-substituted flavan-3-ols (EPSFs) A1: R1=OH, R2=galloyl S-EGCGcThea; A2; R1=OH, R2=galloyl R-EGCG-cThea; B: Reaction products from ECG and phenolic acids from Zijuan tea. C: theaflavin-like products from CQA and EGC (R=H) or EGCG (R=galloyl): EGCG-CGA and EGC-CGA

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Biochemical analysis

Analytical methods (chemical)

Definitions

Constituents (quant data)

Authentication

Human studies

Health effects

Epidemiology Non-targeted analysis

on pack claims

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Credit Author statement Ulrich H. Engelhardt: Conceptualization, Writing - original draft; Writing - review & editing

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Highlights

Camellia sinensis; definitions; flavonoids; thearubigins; non-targeted analysis; authentication; chromatography; mass spectrometry.

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