Bio-chemical and Technological investigations on tea

Borse, B. B. (2008) Bio-chemical and Technological investigations on tea. PhD thesis, University of Mysore.

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Abstract

Ch. 1. INTRODUCTION AND REVIEW OF LITERATURE In this Chapter a brief introduction is presented followed by the status of Indian tea industry, processing, tea quality, determination, isolation and identification of quality indicators and health benefits. The review of literature covers aspects relating to the chemical composition, quality co-relation and bioactivities in health benefits of teas as well as objectives and scope of the research work. Besides, the general pathway of biogenesis leading to the volatile aromatic compounds present in the tea have also been discussed. The brief account is given below. Sheng Nung the chinese emperor (2737 B.C.) was the first to recognize the stimulant effect of tea. Tea is one of the important agro-industrial plantation crops of India. Tea is the beverage with which most of the Indians start their day. The recent research findings indicative of several health benefits have further popularized tea as a beverage. During the year 2007, India produced 945 million kg of tea from 38,705 gardens spread over an area of 4, 35, 057 ha. Out of this, domestic consumption accounts for 76 per cent and exports accounts for 24 per cent. Tea plant belongs to the Camellia species of Theaceae family. The two basic varieties are recognised namely chinese variety Sinensis and Assamese variety - Assamica. The commercially grown tea plant is highly heterogenous. Tea flush contains polyphenols, amino acids, organic acids, polysaccharides, lipids, carotenoids, caffeine, chlorophylls, minerals and volatiles. The polyphenols which includes catechins constitute 25-30% of the fresh flush on dry 2 weight basis. These are converted to theaflavins, thearubigins, theaflavic acids and bisflavanols during the manufacture of black teas and these are responsible for colour, briskness, brightness and astringency. Theaflavins are determined qualitatively and quantitatively whereas quantitative determination of thearubigins has been possible tentatively but their structures are yet to be explored exhaustively. Caffeine is the major alkaloid present in tea and it is responsible for stimulating action. Highly efficient HPLC method to determine soluble caffeine is reported from this laboratory. Carbohydrates play an important role in the formation of tea aroma. Lipid concentration increases with the maturity of the leaves and is responsible for the formation of C6 volatiles during the manufacture of black tea. Three types of organic acids are present in tea viz., dicarboxylic acids, fatty acids and monocyclic acids. Monocyclic acids (eg. Quinic and Shikimic) are the precurors of polyphenols. Chlorophyll a and b are reported to be present in the tea and are converted to phaeophytins which are responsible for black colour of commercial tea. ß-carotene is the major compound among the carotenoids and degrades to character impact volaitle compounds such as theaspirone, ß-ionone and related compounds. Although K (Potassium) is the major mineral found the Cu (Copper) and Al (Aluminium), are important for the colour and taste of brewed teas. Theanine is the most abundant amino acid and accounts for 50% of the total amino acids and 1% of the dry weight of tea. Theanine is a constituent of the "thearabigin" fraction while glutamic acid and ethylamine are its precursors. Amino acids and glucose interact with tea polyphenols during thermal processing and yield coloured moieties and Amadori products, which improve the flavour of tea. Catechins, theaflavins and thearubigins contribute to the bitterness, astringency, brightness and total colour of black tea infusion. Further thearubigins are responsible for body and richness of the tea brew. Theaflavin digallate is having lowest threshold value for the astringency. Caffeine contributes towards the bitter taste in tea. Characteristic umami or brothy taste of 3 black teas is due to the presence of amino acids. The ionone related aroma compounds such as theaspiranes are formed from carotenoids and they are found to have different odour properties. The aroma quality of tea with respect to theaspiranes is yet to be exploited. Volatile flavour compounds (VFC) play a major role in detemining the unique flavor of tea. Although >600 compounds are reported but the unique composition for character impact aroma of black tea is not yet established. The aroma quality of black teas with respect to the VFCs is measured by different ratios/indices viz., Terpene index, Wickremasinghe-Yamanishi ratio, Mahanta ratio, Yamanishi-Botheju ratio. Wickremasinghe-Yamanishi ratio is the ratio of sum of the peak areas of compounds eluting before linalool to the sum of the peak areas linalool plus all compound that elute after linalool. Smaller the ratio better is the quality. Mahanta ratio is the sum of the peak areas of terpenoids to non-terpenoids. Yamanishi-Botheju ratio is the ratio of peak area of linalool to E-2-hexenal. All the three ratios mentioned above have limitations for their applicability. Another aroma quality indicator called flavour index (F.I.), the ratio of VFC II to VFC I is reported for kenyan clonal black teas and F.I. is positively correlated to tasters evaluations. This confirms that F.I. is a good aroma quality indicator for Kenyan black teas. However, it should only be used qualitatively since the olfactory perception limits of individual VFC are different. A suitable ratio for Indian black teas with reference to aroma and quality is yet to be explored and the limitation is vide variation in weather. A new approach in terms of novel quality index for tea through the present work has been innovated (chapter 2). Tea is a good source of flavanoid antioxidants which has a role in prevention of cancer and coronary heart diseases. Tea is known to improve blood flow, eliminate alcoholic toxins, relieve joint pains and acts as a diuretic and improves resistance to diseases. Flavonoids present in tea can effectively stabilize free electrons through several mechanisms viz., delocalisation of electrons, formation of intramolecular hydrogen bonds and rearrangement of their molecular structure. This may be 4 the reason for their antioxidant property. The catechins ranked depending on their antioxidant potential as ECG > EGCG > EC > GC > EGC > C. Theaflavins and thearubigens inhibited the formation of TBARS and these are more effective than vitamin E, glutathione, vitamin C and synthetic phenolic antioxidants. Catechins were also found to be the scavengers of peroxynitrites which are capable of oxidising LDL. Theaflavins and catechin gallates are more effective scavengers of aqueous and lypophilic stable radicals than many other flavonoids and many antioxidant vitamins. The inhibition mechanism of tea flavonoids is independent of metal ion chelation properties. Tea flavonoids were found to reduce oxidative damage in animals from radiation, chemical oxidants, diet stress. Drinking of tea beverage was shown to reduce oxidative biomarkers in chronic smokers. Tea was found to reduce the metabolism of compounds to known carcinogens and enhance their detoxification. Thus it is claimed to inhibit variety of cancers such as oesophagal, gastrointestinal, lung and skin cancers. A cup of black tea is reported to be three times and two times more effective than one serving of common vegetables and one serving of common fruits respectively. Ch. 2. PROFILING OF INDIAN BLACK TEAS This chapter describes analytical determination of volatiles and non-volatiles by different methods for profiling Indian black teas. Chromatographic techniques for separation and their determination using spectroscopic techniques besides flavour indices have also been discussed and the quality co-relation on the scientific basis is discussed. Indian teas especially Darjeeling, Assam and Nilgiris are valued world over for their superior aroma and taste. In order to improve our scientific understanding on objective tea quality and thereby to help retain supremacy in the world tea trade, it was proposed to 5 make an in depth study to generate fingerprint profile of teas grown in different regions of India which ultimately may result in a database with respect to volatile flavour compounds (VFC) as well as non volatile flavour compounds (NVFC) which are responsible for aroma, taste and quality of tea. Novel approach for overall quality based on Seasonal, regional variations and bio-chemical quality fingerprint A study was carried on tea samples collected from nine regions spread over four seasons. Profiling of the black tea samples from four seasons (S1=April-June), (S2=July-Sept.), (S3=Oct.-Dec.) and (S4=Jan.-March) based on bio-chemical fingerprint was accomplished. The brief of the research findings is given below: Codes for Region / Grade / Garden Region / Grade / Garden Code Tamilnadu, Parajulie A Tamilnadu, Pandiar B Darjeeling Medium C Darjeeling Premium D Assam AFTL E Assam Magor F Nilgiris HG G Dooars, Aibheel H Dooars, Chinchula I Palampur G1 J Nilgiris HG-CTC K Dibrugarh, Rose kandy L Palampu G2 M Assam, Cachar best N Assam, Cacher Med. O Darjeeling, Kurti P Assam BOP Q Nilgiris Waynad R Annamalai S Assam OP T 6 Seasonal variation of TF/TR ratio over tea producing region/grade and quality The TF content of a tea or the ratio TF/TR is considered to be a good quality indicator of tea. Accordingly seasonal variations of TF/TR ratios over the coded tea producing regions / grades in all the four seasons (s1, s2, s3, s4) were studied. The teas having TF/TR ratios up to 0.04, >0.04-0.08 and >0.08 can be considered to be a good, better and best quality indicator of tea quality respectively. Teas from the region/grade A-I are the better (TF/TR ratios >0.04-0.08) to best (TF/TR ratios >0.08) quality teas over all the four seasons except for the teas from region A, C, D (s1), which fall under good quality category considering their TF/TR ratios (upto 0.04). Also the teas from region/grade K-L (s2), N,O (s1), PQ (s3) and RS (s4) are the better (TF/TR ratios >0.04-0.08) quality teas except for the teas from region J (s2), M(s3) and T(s4) teas, which fall under good quality category considering their TF/TR ratios (upto 0.04). The teas from region/grade JKL (s1, s3), M –T (s2), ORST (s3), MP (s1), Q (s4) are also falling under good quality category teas, considering their TF/TR ratios (upto 0.04). Seasonal variation of sum of Yamanishi-Botheju and Mahantha ratio over tea producing region/grade and tea quality The VFC (Volatile Flavour Compounds) content of a tea or the sum of the VFC ratios (Yamanishi-Botheju ratio and Mahanta ratio) is considered to be a good quality indicator of tea. Accordingly seasonal variations of or the sum of the VFC ratios (Yamanishi-Botheju ratio and Mahanta ratio) over the coded tea producing regions / grades in all the four seasons (s1, s2, s3, s4) were studied. Accordngly based on the sum of the VFC ratios (i.e.Yamanishi-Botheju ratio and Mahanta ratio) the teas can be categorized as a good (upto 1), better (>1-4) and best (>4) quality indicator of tea respectively. The teas from regions/grade A-J (all seasons) have better (>1-4) to best (>4) quality as indicated by sum of the VFC ratios (i.e.Yamanishi-Botheju ratio and Mahanta ratio) except for teas from regions/grade AEFHIJ(s2),K-T (s2), 7 BDFHIJ(s1), BIJ(s3) which are good (upto 1) quality teas, as indicated by sum of the two VFC ratios. Also the teas from regions/grade M (s3), O (s1), P (s3), RST (s4) are good (upto 1) quality teas as indicated by sum of the two VFC ratios. Seasonal variation of Borse-Rao quality index over tea producing region/grade and tea quality A new approach in terms of novel quality index for tea has been innovated through present work and the results are presented. The sum of TF/TR ratios of tea and the sum of the VFC ratios (Yamanishi-Botheju ratio and Mahanta ratio) added together is proposed for the first time as a new and novel quality index, hence forth referred to as Borse-Rao quality index, considered to be an overall quality indicator of tea as both the non-volatiles/volatiles are given due consideration in this quality index. Accordingly seasonal variations of the Borse-Rao quality index over the coded tea producing regions / grades in all the four seasons (s1, s2, s3, s4) were studied. Based on the the Borse-Rao quality index teas can be categorized as a good (upto 1), better (>1-4) and best (>4) quality tea respectively. The teas from regions/grade having Borse-Rao quality index more than four are C (s2, s4), D (s3, s4), G (s1, s2, s3), H (s3) and I (s4) are the best (>4) quality teas. The teas from regions/grade having Borse-Rao quality index ranging from one to four are A (s1, s2, s3, s4), B (s2, s3, s4), C (s1, s3), E( s3, s4), F( s3, s4), H (s2, s4), I (s3, s4), J(s2), M (s3) and RS (s4) and indicate that these are better (>1-4) quality teas. The rest of the teas from regions/grade having Borse-Rao quality index upto one are good quality teas which are BDFH (s1), EF (s2), I (s1, s2), JKL (s3), K-T (s2) and P-T (s3). The profile of Indian black teas in terms of a bio-chemical fingerprint is carried out in present study which will not only help in understanding the intrinsic 8 quality objectively but also help in tracing the origin of the teas based on the markers identified. For the first time a novel approach has been evolved to mark teas on the basis of TF/TR ratio, VFC ratios and a novel tea quality index (Borse-Rao quality index) is proposed which takes both volatiles and non-volatiles into account. Ch. 3. FUNCTIONAL INGREDIENTS FROM UNUSED GREEN TEA LEAVES: ACTIVITY AND APPLICATIONS Despite several reports on the radical scavenging activity of green tea from two leaves and a bud, the radical scavenging activity of green tea from coarse and pruned leaves in particular is not studied. The pruned and coarse tea leaves are tea plantation waste, India is one of the largest producers of tea. Therefore, testing of its radical scavenging properties is of interest primarily in order to find new promising sources for natural antioxidants. In this chapter novel approach for preparation of the green teas from the pruned or coarse tea leaves and optimization of extraction conditions to obtain catechin rich radical scavenging conserve and its application with the following two objectives is presented. 1. To isolate active conserves from coarse and pruned green tea leaves. 2. To use the active conserves for food applications Processes for utilization of pruned/coarse green tea leaves (a plantation waste) have been worked out. A novel process for green tea preparation has been standardized and patented. Processes for isolation, fractionation and enrichment (50-70 % catechin) and separation of radical scavenging conserve (90-94% RSA @ 10-15 ppm) from pruned/coarse green tea leaves have been standardized and patented. Application of this catechin-rich radical scavenging conserve in nutraceutical ice-cream and cookies was worked out and patented. The details of the study are presented in the following paragraphs: 9 Normal, coarse and pruned fresh tea leaves (low grade) were procured and subjected to enzyme inactivation using cross flow dryer (80-120oC, 4 – 8h), and continuous infra red dryer (70-120oC, 0.5-1.5h) at different temperature and time intervals. The chemical parameters are found to be in the following range, Caffeine (1.30-3.20%); Total polyphenols (11.5-15.5%). Sensory characteristics were found to be similar to that of the commercial green tea samples. Forty four compounds have been identified from the volatiles of green teas by comparing the mass spectra as well as retention indices reported in literature, followed by retention times of the GC peaks with those of reference compounds run under identical conditions where ever possible. The broad classification of the compounds identified includes ten terpenoids, three aromatic compounds, eight alcohols, seven aldehydes, four acids, eight esters and four compounds derived from carotenoids. Ethyl hex-(2E)-enoate and dihydroactinidiolide are exclusively present in the green teas derived from coarse leaves. In general, coarse green teas irrespective of the method of processing contained more number of the volatile constituents, whereas the normal green teas irrespective of the method of processing contained less number of volatile constituents eluting before linalool. This can be attributed to the normal leaf quality, which contributes less volatiles from the group of constituents (Gr. I) which are undesirable, which is the prevalent practice in the industry as well. It is also evident from the results that normal as well as a commercial sample of green tea contained almost half the number of identified volatile constituents in the present investigation as compared to the coarse green teas contained. Dihydroactinidolide and ethyl hexenoate were found to be important markers and both were present in coarse green teas, whereas both were not found in the normal green teas including the commercial one. It can be concluded that the commercial green tea (Nilgiris green tea) is also prepared from the normal tea leaves. This can be very well used in spotting the normal / coarse green tea or the admixture of the coarse green tea leaves with the normal green tea leaves. Other important marker volatiles found were the both heptadienals [(E,Z)-2, 4 / 10 (E,E)-2,4]. Coarse green tea contained both the heptadienals, whereas normal green teas did not contain any of the identified heptadienals in the present investigation. cis-3-hexenyl-n-hexanoate, ∝-ionone, cis-geranylacetone and β-ionone-5,6-epoxide were not found in a normal green tea. This can be attributed to the method of processing (CFD) and higher temperature (110°C) used. Green tea extract The extraction of green tea samples at lab scale, using different solvents (viz., Ethyl acetate, acetone, ethyl alcohol, methyl alcohol and their aqueous mixtures) was carried out. The radical scavenging activity (RSA) of these extractives at 50 and 100 ppm concentrations were evaluated using the DPPH model system. The order of activity and extractability are as follows: Methanol > Ethanol> Acetone > Ethyl acetate The aqueous alcoholic mixtures showed higher activity and polyphenol extractability than the respective single solvents. It was found that the yields of the extractives from green teas of coarse leaves are relatively low on the expected lines and the radical scavenging activities of the extractives of green teas from coarse leaves are marginally low at different concentrations. This observation indicated that the green teas from coarse leaves could be used for the preparation of radical scavenging conserves, by separating / enriching the active components using suitable technique. Fractionation of the green tea extract The extractives were subjected to liquid-liquid extraction using water and low molecular weight ester to fractionate the catechins into the solvent fraction. These extracts were analyzed for total polyphenol content and evaluated for radical scavenging activity. The polyphenol content of the solvent extracts found to be 30±2.3% as gallic acid equivalents for coarse leaves, while, polyphenol content of the solvent extract of normal leaves is found to be 31±2.4% as gallic acid equivalents. The total polyphenol content in the aqueous portion of these extracts is 23±2.1% as gallic acid equivalents for normal leaves, while that for coarse leaves extracts is found to be 18±3.0% as gallic acid equivalents. The 11 yields of the solvent extracts are found to be 15±0.8% for coarse leaves and for normal leaves the yield of solvent extract is found to be 17±0.8%. The yield of the aqueous extract is 17±0.9 % for coarse leaves and for normal leaves the yield of solvent extract is found to be 19 ±1.0 %. However, the radical scavenging activity of the solvent extracts from both normal and coarse leaves is found to be same (92±1% at 15 ppm). The RSA of the aqueous extracts is found to be lower. Hence, it may be concluded that the solvent used separated the compounds responsible for the radical scavenging activity. The data obtained reveal that the green tea extracts / conserve is free radical inhibitor and primary antioxidant that react with DPPH radical, which may be attributed to its hydrogen donating ability. HPLC profiling of green tea extractives, chemical composition and quantification The total catechin content in the green tea extract based on the comparison of peak areas of each peak with that of authentic samples and from calibration curves was found to be in the range of 20–30 %. After fractionation the solvent extract is enriched with catechin and the total catechin content is found to be in the range of 55-85 %, while the HPLC profile of aqueous extracts showed only the presence of gallic acid and caffeine. Extracts from unused fresh green tea leaves have the potential for large-scale application as natural antioxidants. Extracts of the green tea are becoming increasingly important as functional ingredients in the diet and are being added to a range of foods and beverages. Improved method for the active conserve To minimize the processing cost and to control the epimerisation of catechins during processing, alternate methods were explored. Green tea sample from fresh batch was subjected to aqueous alcoholic extraction. The extract was concentrated to remove the alcohol to the extent possible. The 12 obtained miscella was cooled to 10oC and kept over night at that temperature. The separated solids were filtered and dissolved in low molecular weight ester and the filtrate was also treated with the same ester. Solvent was removed from the combined portion and the yield was found to be 12±2%. Both aqueous and ester portions were subjected to HPLC analyses. Ester soluble portion was found to contain most of the catechins. The aqueous portion (filtrate) was freeze dried and the solid yield was ~12±2%. Total polyphenol content and Radical scavenging activity (RSA) of the aqueous portion and ester portions were evaluated. RSA of the aqueous portion was found to be in the range of 70-85% at 40-50 ppm concentration, while that of ester portion was in the range of 85-90% at 10-15 ppm concentrations. The total polyphenol content in the aqueous portion was found to be 23-25%, while that of in the ester portion are in the range of 27-32%. The project economics of the process for catechin conserve is also presented. Catechin-rich nutraceutical ice-cream Ice-cream was prepared using the food ingredients along with the polyphenol conserve. The concentration of tea catechin conserve was tried in the range of 5-200 ppm. The optimum range of tea catechin concentration was found to be in the range of 20-30 ppm. The results of the sensory analysis for ice-cream with tea antioxidant extract show that , 15% of the respondents rated under like very much (LVM), 45% of the respondents under like moderately (LM) and another 40% under Like Slightly (LS). However the ice-cream samples are acceptable as the scores are falling on ‘Like’ category. Catechin-rich nutraceutical cookies The cookies were prepared using the required food ingredients along with green tea catechin conserve. The concentration of green tea catechin conserve was tried in the range of 10-400 ppm. The optimum concentration was found to be in the range of 20-50 ppm by sensory evaluation. 13 14 The results of the sensory analysis indicated that 53% of the respondents rated cookies as LVM, 32% of the respondents rated it as LM and 15% of the respondents rated it as LS indicating the product is acceptable. As the results are falling on the ‘Like’ category, the product is acceptable. Ch. 4. SUMMARY AND CONCLUSION The significant findings of the complete study have been presented in a comprehensive way under Summary and Conclusions.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	tea, chemical composition, extractives, Camellia, health benefits
Subjects:	600 Technology > 07 Beverage Technology > 08 Tea 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 04 Analytical Chemistry 600 Technology > 08 Food technology > 05 Processing and Engineering
Divisions:	Plantation Products Spices and Flavour Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	01 Mar 2011 04:38
Last Modified:	08 May 2012 06:54
URI:	http://ir.cftri.res.in/id/eprint/9938

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