Title of Invention	" A PROCESS FOR PRODUCING IMPROVED QUALITY OF TEA".
Abstract	A process for producing improved quality of tea comprising: treating tea leaves post plucking, with one or more senescence accelerating agents such as herein described, wither at a time or in parts at a concentration of 0.(3001-0.5% for a time period of 10 minutes to 24 hours and at a temperature range of 10-45 C, optionally processing the tea leaves to provide for black tea or instant tea.

Full Text

The invention relates to a method for obtaining improved composition of tea shoot and quality of final tea leaves. The invention particularly relates to a process of treating tea leaves post plucking to reduce the withering and processing time and improve the quality of tea. The invention more particularly relates to a process of treating tea leaves post plucking with senescence accelerating agents. Background and prior art: In the manufacture of black tea, young leaves usually 2-3 leaves and a bud are plucked and withered for about 12-24 hours to reduce moisture and to bring about desirable chemical/Biochemical changes. Withering process allows certain chemical and biochemical changes to occur and also the moisture content of the leaves is brought down from 90% to 50-70%. Biochemical/chemical changes taking place during withering, increase the yield of the volatile flavour compounds in tea. At the end of withering period, the tea leaves are macerated using a Cut-tear-and curl machine (CTC machine) or rolled using rollers (old method) and subjected to a process of fermentation and fired at a high temperature to stop the enzyme activity. Disruption of cellular integrity of leaf tissues by CTC machine allows oxidation of catechins by enzyme polyphenol oxidase which results in the development of colour pigments i.e. theaflavin and thearubigins. These important colour pigments of the tea are formed from 2 catechins which are formed in tea shoot during growth. Phenylalanine ammonia lyase(PAL) is the key enzyme responsible for the synthesis catechins and flavonal glycosides. The taste of black tea is a result of complex interaction, occurring during fermentation, between the polyphenols, caffeine, theaflavins and thearubigins and this interaction also contributes to the brightness, briskness, colour, thickness and body of black tea. Withering process is known to increase the flavour components and briskness of black tea liquors. However, catechin contents decreases during withering. Thus, maintaining a higher level of catechins at the end of the withering process will have a direct relation to yield of colour pigments during fermentation such as theaflavins and thearubigins, which contribute to the taste of tea, (Owuor,O.P. and Obanda, M. Tea 13(1) 1992, 50-61). Ethylene is a senescence inducing plant hormone that acts by increasing respiration rates and degradation of chlorophyll. Particularly it increases the activity of cell wall degrading enzymes. Commercially ethylene has been used in several agricultural operations such as defoliation, fruit/flower abscission, fruit ripening etc. 3 SU 3835049 refers to a two stage method of uniform drying of tea leaves wherein the first stage is carried out with air at 35-37°C for 70-80 minutes and the second stage air saturated with ethylene at 33-35°C for 32-34 minutes. This invention enables to improve the quality of the final product by ensuring the uniformity of dry curing of the raw material with various mechanical means. Definition of the invention: In the present invention it has been found that treatment of leaves soon after plucking prior to the withering with senescence inducing agents leads to reduction in the withering time and improved quality of tea. The present invention differs from the prior art in that there is no drying period prior to the treatment. Accordingly the present invention relates to a process of treating tea leaves post plucking to reduce the withering and processing time and improve the quality of tea. The invention particularly relates to a process of treating tea leaves post plucking with one or more of senescence accelerating agents, singly or in split doses, at a concentration of 0.0001-0.5% for 10 minutes to 24 hours at a temperature 10-45°C. The invention more particularly relates to a process of treating tea leaves post plucking with ethylene or ethylene generating compounds at a concentration of 0.001-0.1% for 1-8 hours at a temperature 15-30°C to improve composition of shoot and reduce withering and processing time and to improve the quality of tea. 4 Detailed description of the invention: The essentials of the present invention relates to treating the plucked tea leaves with ethylene or ethylene generating compounds prior to withering. The ethylene concentration is preferably 0.001-0.1%. Examples of senescence accelerating compounds are ethylene, ethrel, abscissic acid, auxins, succinic acid-2, 2-dimethyl hydrazide, Jasmonic acid, methyl jasmonates etc. The application of the senescence accelerating compounds is post plucking and prior to drying and preferably the compounds are in the form of a solution or gas. The application of the senescence accelerating compounds is with one or more of these compounds either singly or in split doses. The treatment is given in the form of a spray, dip or gas and preferably in a closed container. The concentration of the compounds is in the range 0.0001-0.5% and preferably in the range 0.001-0.1%. The duration of the treatment is for 10 minutes to 24 hours and preferably 1-8 hours. The temperature during the treatment is maintained at 10-45°C and 5 preferably at 15-30°C The invention will now be demonstrated by the following non limiting examples After treatment with the senescence accelerating agent, the shoots are withered following the normal procedure. However due to the treatment a shorter withering time is possible while obtaining the same quality as when using standard withering time (i.e. 12-24 hours particularly 16-20 hours). Thus the procedure could reduce this withering time to 2-12 hours particularly 4-10 hours, without affecting the quality of the tea. Alternatively, the normal withering time could be maintained, in which case an improvement of the quality of the tea is obtained particularly relating to higher content of catechin and phenylalanine ammonia lyase after withering. Any desired combination of the two possibilities is possible as well. After withering the tea is further processed according to conventional procedures, i.e. fermented, fired and then either packaged for use by the consumer or further processed to be converted into instant tea. 6 The invention now will be illustrated with reference to non-limiting examples. Examples: Example 1 Process of withering Freshly harvested green leaves (3 Kg) were sprayed with 100 ml aqueous solution of Ethrel which contains 2-chloro ethyl phosphonic acid as the active ingredient obtained from Leo Chem, Bangalore, at a final concentration of 0.08% of the active ingredient, at a temperature of -25°C for two hours in an air tight compartment. Leaves with and without the ethrel treatment were withered for 18 hours under ambient conditions. Leaves without the ethrel treatment were used as control. Example 2: Improved composition of the withered leaves. The withered leaves both control and treated were analysed for catechin content, Flavonol glycosides and the activity of the enzymes phenylalanine ammonia lyase (PAL) which is a key enzyme in the synthesis of catechins by the following the procedure. Enzyme Assays: For assaying enzyme activity acetone powder from the test sample was used. The acetone powder was prepared by grinding the leaves in 7 chilled acetone (-20°C) in a pestle and mortar. The sample was filtered through Whatman No 541 and subsequently washed and ground in acetone:water (80:20) till it was colourless. The powder thus made was dried in vacuo and stored in a deep freezer at -12°C. Phenylalanine Ammonia Lyase (PAL): PAL activity was assayed by extracting the enzyme in phosphate buffer (0.1M, pH 6) from the acetone powder in a pestle and mortar, incubated at 4°C for 60 min and centrifuged at 6000 rpm for 10 minutes. The supernatant was used for the assay. The reaction mixture contained Borate buffer (0.05M, pH 8.55), 0.5mM L-phenyl alanine and crude enzyme in a total volume of 5 ml. The reaction was allowed to proceed for 60 min at 35°C and was stopped by the addition of 1ml, 5N HCl. The reaction mixture was then extracted with 10ml ether which was then dried in Nitrogen atmosphere. The residue was dissolved in 10 ml 45% ethanol. The A273 of the cinnamic acid recovered was measured against the blank of ethanol. An unit of PAL activity is defined as 1uM of cinnamic acid formed per g acetone powder per hr under stipulated conditions. Analysis of catechin The method is based on Hoeffler and Coggons analysis of catechins using a reverse phase column (Hoeffler A.C. and Coggons, P. 1976. 8 J of Chroraatography 129:460-463). The method of the tea preparation was as follows: Black tea, 2.4 g, was infused in 100 ml of boiling distilled water and kept at 90°C for twenty minutes. The infusion was rapidly filtered through a glass funnel into a 100 ml volumetric flask & made up to volume with water. The infusion was rapidly cooled to room temperature to avoid cream formation. Moisture content of samples were measured by a Mettier LJ16 moisture analyser and corrected for data interpretation. The method of catechin estimation involves a direct injection of 20 |il of aqueous tea solutions after filtration through a membrane (cellulose. 0.22 p.) to HPLC machine. Instrument : Waters HPLC system was used which 501 pump, Waters 486 U.V. detector and Millinium software for data analysis. Column : Bonda Pak C-18, 60A, 10mm, 3.9x300mm Mobile Phase : 20% DMF 0 . 5% Acetic acid 1% methanol 78.5% water Gradient - 92% A to 69% A over 50 mins Flow rate = 2 ml/min. Detector - U.V. visible, 1 max - 280 nm Column Temperature - 30°C 9 Flavonol Glycosides analysis by HPLC The method used was similar to that developed by Bailey et al (Bailey et al, 1990. J Sci Food & Agric 52:509-525). Peaks were identified with authentic samples. Instrument : Waters HPLC system was used which consists of Waters 510 and 501 pumps, Waters 486 U.V. detector and Millinium software for data analysis. Column : Nova Pak C-18, 60A, 4 mm, 3.9xl50mm Mobile Phase : A- 2% Acetic Acid B- Acetonitrile Gradient : 92 % A to 69 % A over 50 mins Flow rate = 1 ml/min. Detector - U.V. visible, 1 max = 380 run Column Temperature - 40°C Sample Preparation : Sample preparation was same as described for estimation of catechins. The solution was then filtered through 0,. 22 u Millipore filter paper, 20 ul of the sample was injected & the chromatogram was compared with the chromatogram of standard theaflavins. 10 Table 1 Treatment % Total PAL 1uM of Total Flavonol catechin cinnamic acid Glycosides (Peak formed/g areas) acetone powder /hr Control 16.87 32.13 1124305 Ethrel treated 20.23 74.98 2316950 Data shows that even at the end of 18 hours of withering time the catechin levels and flavonol glycosides were significantly higher than the control which is also supported by the corresponding increases in the activity of PAL, the enzyme responsible for the synthesis of these substances. Example 3 Withering time: Leaves were withered with and without ethrel treatment as described in Example 1. The levels of catechin and the activity of PAL were monitored by the procedures described in Example 2 in fresh leaves and after 6h, 12h and 18h withering time. The data presented in Fig 1 shows that the catechin level decreases in the untreated sample during withering whereas in the treated sample there was an increase upto 12h and then level goes down. However, the level of catechin is higher in the treated sample at the end of withering. The activity of the enzyme PAL and catechin 11 levels are correlated. Tea made from shoots subjected to different withering time can be efficiently reduced to 12 hours as against standard 18 hours. Example 4 Quality of made tea: Preparation of made tea: After 18 hours of withering the leaves were processed using a mini-manufacturing facility at Daverashola (Tea Estate India Ltd., Daverashola, The Nilgiris, Tamil Nadu) factory. The mini-line consisted of CTC (cut-tear and -curl), fermentor (controlled temperature and humidity chamber) and fluid bed drier. The leaves were cut four times using the CTC machine and fermented for 60 minutes at 25°C at 90% RH. The fermented dhool (macerated leaf) was fired for 20 minutes using a fluid bed drier. Chemical analysis of black tea 2.4g of Black tea obtained from control and ethrel treated (during withering) was infused in 100 ml of boiling water and maintained at 90°C for twenty minutes. The infusion was filtered and the volume was made upto 100 ml. This was cooled rapidly to 20-25°C. The theaflavins (TF), thearubigins (TR), brightness, caffeine and total soluble solids were measured using the black tea infusion and the data is presented in Table 2. 12 Analysis of theaflavins and thearubigins and % brightness The levels of theaflavins and thearubigins were measured spectrophotometrically following the method described by Roberts and Smith 1963 (J. Sci. Food Agric, 14: 689-699). The absorbance was measured at the different wavelengths and the levels of theaflavins and thearubigins and % brightness was calculated as follows. CALCULATION OF RESULTS: A1 = Absorbance of the solution A at 380nm. B1 = Absorbance of the solution B at 380nm. C1 = Absorbance of the solution C at 380nm D1 = Absorbance of the solution D at 380nm A2 = Absorbance of the solution A at 460nm. B2 = Absorbance of the solution B at 4 60nm. C2 = Absorbance of the solution C at 460nm D2 = Absorbance of the solution D at 460nm SOLUTION A: Theaflavins + some Thearubigins SOLUTION B: Thearubigins at the pH of infusion SOLUTION C: Theaflavins SOLUTION D: Thearubigins at low pH (oxalic acid present) %TF = C2 *6.69 %TR = 7.06*(2D1+A1-C1) Check that C1\C2 = 3 (approximately) If this ratio is >3, then not all of the thearubigins were removed from the solution C during the bicarbonate wash. A380YA460 Ratio for TR (2D1+A1-C1) \ (2D2+A2-C2) % BRIGHTNESS 100 C2\2B2+A2 13 Brightness analysis The % brightness of the black tea infusion obtained by the procedure described above. Analysis of caffeine The caffeine content of the black tea infusion obtained by the procedure described above was measured by the procedure described by Hoeffler, A..C. and Coggons, P., (1976), J of Chromatography 129:460-463. The method of tea preparation was same as in Example 2. It involves a direct injection of 20 ul of aqueous tea solution after filtration through a membrane (Cellulose 0.22 u) Total soluble solids The total soluble solids (TSS) of the black tea infusion was determined by brewing 2 g tea on a hot plate filtering the infusion on Whatman No 541 paper, taking 50ml of the infusion and measuring the weight loss when the samples were dried on a water bath set at 90°C for 16h followed by keeping it overnight on an oven set at 100°C. Table 2 Treatment % TF % TR %Brightness % Caffeine % TSS Control 1.16 15.6 21.2 3.19 37.6 Ethrel 1.2 6 14.4 23.8 3.33 39.2 0.008% Ethrel 1.40 13.6 27.5 3.57 38.4 0.08% 14 The data presented in Table 2 shows that the levels of the different chemicals responsible for the colour of the tea and the brightness was linearly correlated with the ethrel concentration. These parameters show that ethrel treatment at the withering stage had positive effect on the final tea. The lowering of thearubigins affects the brightness as the TF:TR ratio increases. Example 5 Sensory characters Quantitative descriptive analysis (QDA) of made tea QDA Methodology The panel consisted of 10-15 numbers of housewives who have been trained for leaf and end cup evaluation. The panelists score for 7 end cup attributes (includes visual, in-mouth and aroma components) on a ten point scale. The method of tea preparation used in the QDA study was based on "Tea preparation observation studies" carried out by Indian Market Research Bureau (IMRB). The standardised method is as follows : For 150 ml end cup of tea 1. Bring 82 ml of water to boil 2. Add 3 grams of tea and boil for 1 minute 3. Add 68 ml of preboiled milk and 6 grams (4%) sugar. 4. Boil till one rise 5. Serve at 70-75°C Using the black tea obtained from control and ethrel/ethylene 15 treated (during withering) the QDA was carried out by a trained panel for evaluating the effects on various tea attributes and analysed statistically and the data on QDA in table 3. Table 3 QDA attributes Treated Control Colour 5.38 6.42* Brightness 5.81 5.11* Tea flavour 3.65 3.31* Body 4.73 4.62 Bitter taste 1.54 2.54* Bitter after taste 1.92 2.61* Astringent after 4.07 4.62* taste * Significant over control The QDA analysis suggested that the treated samples were brighter and had less bitter taste and bitter after taste which results in enhanced acceptability by the consumer. Example 6: Effects of Abscissic acid (ABA): Freshly harvested leaves (1 Kg) were treated with 100ml solution of ABA at concentrations of 0.003-0.012%, at a temperature of 25°C for 18 hours. Leaves without ABA treatment were used as a control. 16 The withered leaves, both control and treated were analysed for catechin, caffeine and the made tea infusions were analysed for theaflavin, flavonol glycosides by HPLC and Total Soluble Solids (method described in Example 2 and-Example 4). The data presented in Table 4a and 4b show that the chemicals responsible for colour and brightness in tea infusions were higher in the treated samples compared to control. Table 4a Treatment %Total catechin % Caffeine Control 15.49 5.11 ABA 0.003% 16.87 5.27 ABA 0.012% 17.32 5.46 Table 4b Treatment Total Total Flavonol % Total Theaflavin glycosides Soluble Solids (Peak Areas in (Peak Areas in HPLC) HPLC) Control 1170174 13 638 60 4 6.31 ABA 0.002% 1408329 2396105 47.32 ABA 0.008% 1569217 2449438 46.60 Thus the present invention is directed to provide a method of obtaining tea leaves with higher catechins, flavonol glycosides and with improved quality of tea. 17 2. A process as claimed in claim 1 wherein the senescence accele rating agent is selected from ethylene, ethrel, abscissic acid, auxins, succinin acid-2,3-dimethyl hydrazide, Jasmonic acid and methyl jasmonates. 3. A process as claimed in claim i wherein the time required is 1-8 hours. 4. A process as claimed in claim 1 wherein the temperature required is in the range of 15-30°C. 5. A process as claimed in claim 1 wherein the concentration of the accelerating agent is 0.001-0.1%. 6. A process as claimed in claim 1 substantially as herein described. A process for producing improved quality of tea comprising: treating tea leaves post plucking, with one or more senescence accelerating agents such as herein described, wither at a time or in parts at a concentration of 0.(3001-0.5% for a time period of 10 minutes to 24 hours and at a temperature range of 10-45 C, optionally processing the tea leaves to provide for black tea or instant tea.

Documents:

00665-cal-1996 abstract.pdf

00665-cal-1996 claims.pdf

00665-cal-1996 correspondence.pdf

00665-cal-1996 description(complete).pdf

00665-cal-1996 description(provisional).pdf

00665-cal-1996 drawings.pdf

00665-cal-1996 form-1.pdf

00665-cal-1996 form-2.pdf

00665-cal-1996 form-3.pdf

00665-cal-1996 form-5.pdf

00665-cal-1996 letters patent.pdf

00665-cal-1996 p.a.pdf