EFFECT OF SPRAY DRYING CONDITIONS ON PHYSICAL AND CHEMICAL PROPERTIES OF DRIED GREEN TEA EXTRACT (Camellia sinensis var. Oolong No 12) MANUSCRIPT

EFFECT OF SPRAY DRYING CONDITIONS ON PHYSICAL AND CHEMICAL PROPERTIES OF DRIED GREEN TEA EXTRACT (Camellia sinensis var. Oolong No 12) MANUSCRIPT SARI WAHYUNI F24070130 FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY BOGOR AGRICULTURAL UNIVERSITY BOGOR 2011 i

EFFECT OF SPRAY DRYING CONDITIONS ON PHYSICAL AND CHEMICAL PROPERTIES OF DRIED GREEN TEA EXTRACT (Camellia sinensis var. Oolong No 12) Sari Wahyuni 1, Purwiyatno Hariyadi 1, Hanifah Nuryani Lioe 1, Natthawuddi Donlao 2 1 Departement of Food Science and Technology, Faculty of Agricultural Engineering Technology, Bogor Agricultural University, IPB Darmaga Campus, PO. BOX 220, Bogor, West Java, Indonesia 2 School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand ABSTRACT The effect of spray drying conditions on physical and chemical dried green tea (Camellia sinensis var. Oolong No 12) extract were observed. Tea extract was prepared from milled dried tea, dissolved in hot water at temperature 90 C and ratio of dried tea to water 1: 20 (w / v). The time of extraction used was 60 minutes, and ph value was 5.0. Then, tea extract was concentrated with ice maker machine until reached 3, 6 and 9% of total solid, and dried with spray drier with condition inlet air temperatures were 180, 200, 220 C, outlet air temperatures was controlled 75 C, blower speed was adjusted in 2500 rpm. The physical analysis, such as bulk density, color, solubility, hygroscopicity, and the chemical analysis, such as moisture content, total polyphenols content, antioxidant activity (DPPH assay), catechins, and caffeine content were determined. Results showed that different solid concentration of 3, 6, 9% in feed and inlet air temperatures of 180, 200, and 220 C affected the variation of physical and chemical properties of green tea powder. An increase inlet air temperature, resulted in a significant decrease (p<0.05) in bulk density, hygroscopicity, total polyphenols and antioxidant activity. An increase of solid concentration in feed gave an increase in tea powder solubility, L, a, b value, and antioxidant activity. However, the total polyphenols contents were not affected by the increase. The condition of feed concentrated to 3% and inlet temperature at 220 C was evaluated with the highest of physical, chemical results and the highest of kg water removal/hour by spray dryer. Keywords: Green tea, tea powder, freeze concentration, spray dryer ii

Sari Wahyuni. F24070130. Effect of Spray Drying Conditions on Physical and Chemical Properties of Dried Green Tea Extract (Camellia sinensis var. Oolong No 12). Supervised by Purwiyatno Hariyadi, Hanifah Nuryani Lioe, Natthawuddi Donlao. 2011. SUMMARY Tea is globally one of the most popular and lowest cost beverages, next only to water. Nowadays, tea powder is being developed because it has many advantages such as more practical, simple transport economics, and simply to prolong product s shelf-life. There are several method to produce tea powder, one of them is spray drying method. The objectives of this research are to investigate the effect of solid concentration in feeds and inlet air temperatures of spray dryer on physical and chemical properties of green tea powder. In the preliminary research, chemical composition of raw materials involved moisture content, total polyphenols, antioxidant activity, caffeine, and catechin content were determined. In experiment I, production of concentrated green tea was made from extract green tea and increase its concentration with ice cream maker. In experiment II, production of green tea powder with JMC-minilab spray dryer. The resulted green tea powder were then analyzed for its physical properties (such as bulk density, color, solubility, and hygroscopicity) and chemical properties (such as moisture content, total polyphenol content, antioxidant activity catechins, and caffeine contain). Results showed that dried green tea (Camellia sinensis var. Oolong No 12) contains moisture content 6.05 ± 0.06% w/w wb, total polyphenol 14.98 ± 0.42% db, antioxidant activity 141.5 ± 6.88 mmol Trolox/100 g db, caffeine 2.77 ± 0.23 g/100 g db, and catechin 12.04 ± 1.20 g/100 g db. There is a linear relationship between brix from refractometer and total solid of oven method with regression y = 0.842x + 0.142, R 2 = 0.997. Limited concentration of concentrated tea with ice cream maker machine until 11% total solid. Concentrated tea more than 11% total solid, it will deposit at the wall machine and decreased percentage of freeze concentration recovery. Therefore, parameter solid concentration in feed that used were 3,6, and 9%. The quality of product especially chemical quality is greatly influenced by production method. The effect of green tea powder production method such as extraction, freeze concentration, and spray drying on its chemical properties such as total polyphenols content, catechins, caffeine, and antioxidant activity have been studied. Amount of total polyphenols, catechins, caffeine, and antioxidant activity of sample decreases significantly (p<0.05) after it was treated by different process such as extraction, concentration, and spray drying. Base on the results, the powder with treatment solid concentration 3% in feed and inlet temperature 220 C has the highest chemical yield and powder with treatment total solid concentration 9% in feed and 180 C has the lowest one. The different solid concentration 3, 6, 9% in feed and inlet air temperatures of 180, 200, and 220 C affected physical and chemical properties of green tea powder. An increase inlet air temperature, resulted in a significant decrease (p<0.05) in bulk density, hygroscopicity, total polyphenols and antioxidant activity. An increase of solid concentration in feed gave an increase in tea powder solubility, L, a, b value, and antioxidant activity. However, the total polyphenols contents were not affected by the increase of solid concentration. The physical results of green tea powder are bulk density of the powder variated between 0.3933-0.5014 g/ml, L value variated between 68.07-74.41, a value variated between 3.50-5.79, b value variated between 30.08-37.37, solubility variated between 75.56-91.17%db, hygroscopicity 8.84-17.84%. The chemical results of green tea powder are moisture content variated between 2.33- iii

3.77%db, total polyphenol variated between 27.27-30.69%w/w db, antioxidant activity variated between 218,805-247,768 mmol Trolox/100g db, catechine varieated between 19.28-23.23, caffeine variated between 4.96-5.63 g/100g db. The single catechin GC, C, and EC in green tea powder were positively correlation with antioxidant activity but EGC, EGCG, GCG, ECG, and caffeine has negative correlation. The Catechin (C) and Epicatechin (EC) were highly significant (p< 0.05) with antioxidant activity. For spray drying process, an increase in inlet air temperature gave an increase in recovery and amount of water removal. On the other hand, it gave a shorter time of drying and a higher reduction of energy consumption. An increase in solid concentration of feed yielded a decrease in recovery, and resulted the variation in the amount of water removal. It also allowed a short time of drying and reduce of the energy. The powder which has the best physical and chemical properties is that produced by the application of 3% solid concentration in feed and inlet air temperature 220 C. iv

EFFECT OF SPRAY DRYING CONDITIONS ON PHYSICAL AND CHEMICAL PROPERTIES OF DRIED GREEN TEA EXTRACT (Camellia sinensis var. Oolong No 12) MANUSCRIPT In the partial fulfillment of the requirement for degree of SARJANA TEKNOLOGI PERTANIAN At the Departement of Food Science and Technology Faculty of Agricultural Engineering and Technology Bogor Agricultural University By: SARI WAHYUNI F24070130 FACULTY OF AGRICULTURAL ENGINEERING TECHNOLOGY BOGOR AGRICULTURAL UNIVERSITY BOGOR 2011 v

Title Name Student ID : Effect of Spray Drying Conditions on Physical and Chemical Properties of Dried Green Tea Extract (Camellia sinensis var. Oolong No 12). : Sari Wahyuni : F24070130 Approved by, Advisor I, Advisor II, (Prof. Dr. Ir. Purwiyatno Hariyadi, M.Sc) (Dr. Ir. Hanifah Nuryani Lioe, M.Si) NIP 19620309.198703.1.003 NIP 19680809.199702.2.001 Acknowledged by: Head of Departement of Food Science and Technology, (Dr. Ir. Feri Kusnandar, M.Sc) NIP 19680526.199303.1.004 Graduation date : November, 22 th 2011 vi

STATEMENT LETTER OF MANUSCRIPT AND SOURCES OF INFORMATION Hereby I genuinely stated that the manuscript entitled Effect of Spray Drying Conditions on Physical and Chemical Properties of Dried Green Tea Extract (Camellia sinensis var. Oolong No 12) is an authentic work of mine under supervision of academic counselor and never being presented in any forms and universities. All the information taken and quoted from published or unpublished works of other writers had been mentioned in the texts and attached in the bibliography at the end of this manuscript. Bogor, November 2011 The undersigned, Sari Wahyuni F24070130 vii

AUTHOR BIOGRAPHY Sari Wahyuni was born in Tangerang, Banten, on June, 27 th 1989. She was graduated from SD Slamet Riyadi Tangerang elementary school in 2001, SLTP Slamet riyadi II junior high school in 2004, SMA Stella Duce I Yogyakarta senior high school in 2007. Tn the same year, she joined Bogor Agricultural University through Seleksi Penerimaan Mahasiswa Baru (SPMB) and she was graduated her bachelor degree of major Food Science and Technology in 2011. She was much involved in student activities such as Himpunan Mahasiswa Ilmu dan Teknologi Pangan (HIMITEPA), Keluarga Mahasiswa Katolik IPB (KEMAKI), and Assistant of Catholic Religion (Tim Pendamping). Beside, she also was active as committee in Lomba Cepat Tepat Ilmu Pangan (2009-2010), BAUR (2009) dan PLASMA (2009). The autor got some scholarships and achievements during her study at Bogor Agricultural University. She got scholarship from KEMAKI in 2007-2008, Peningkatan Prestasi Akademik in 2008-2009, and schorlarship from Tanoto Foundation in 2009-2011. In 2010, she won as first winner UNIVATION of Padjajaran University (Food Innovation and Business Competition), Bandung. In 2011, she did oral Presentation entitled Functional Ready-to-Drink Beverage from the Mixture of Nutmeg Extract and Crushed Nata de Coco as Antiinsomnia and Dietary Fiber Source in AISC-2011 (The Second Annual Indonesian Scholars Conference in Taiwan), Asia University, Taichung-Taiwan. At last, in 2011, the author was selected to be the Indonesian delegates for the Credit Transfer MIT (Malaysia-Indonesia-Thailand) Student Exchange Program in Mae Fah Luang University, Thailand. Under such program, she did an undergraduated research project entitled Effect of Spray Drying Conditions on Physical and Chemical Properties of Dried Green Tea Extract (Camellia sinensis var. Oolong No 12) supervised by Aj. Natthawuddi Donlao, Prof. Dr. Ir. Purwiyatno Hariyadi, M.Sc., Dr. Ir. Hanifah Nuryani Lioe, M.Si. viii

FOREWORD The author would like to thank God for His blessings, guidance and protection upon the writer throughout the whole research work and manuscript preparation. The research project entitled Effect of Spray Drying Conditions on Physical and Chemical Properties of Dried Green Tea Extract was conducted at the School of Agro-Industry, Mae Fah Luang (MFU, Thailand) under Credit Transfer-MIT Program. The thanks and extensive gratitude of the author to: 1. My lovely Mom (Sannie), Dad (Alm. Soediarko Halim) and my sisters (Natasya, Lidya, and Clara Stephanie), Mama Ketty, Ako Erna, Ci Lisa, my big family and Yohanes Bagus Christiant, who have always support me, for their love and their understanding in my life. 2. My academic advisor Prof. Dr. Ir. Purwiyatno Hariyadi, MSc. and my co-advisor, Dr. Ir. Hanifah Nuryani Lioe, M.Si, thanks for the considerable advise. 3..Dr. Eko Hari Purnomo, S.T.P., M.Sc., Mrs.Antung Sima Feriliyanti, S.TP., M. Sc and Mrs. Dias Indrasti, S.TP, M.Sc as the commitee of MIT exchange program in Departement of Food Science and Technology, Bogor Agricultural University. 4. All of faculty member of the Department of Food Science and Technology, Bogor Agricultural University, for making the knowledge available. 5. Ajarn Natthawuddi Donlao, my kind advisor in MFU and has guided me during my research with his patience and knowledge. 6. The commitee in School of Agroindustry who have evaluated my research during presentation. 7. Directorate General of Higher Education- Ministry of National Education of the Republic Indonesia, which facilicated a student mobility program and for full financial support during MIT program. 8. Tanoto Foundation for scholarship during the author studied at Bogor Agricultural University 9. All my classmates, especially for Adi, Atika, Lailya, Vita, Daniel, Arief, Hana Sutsuga, Punjung, thanks for the sweet friendship. 10. My lovely friends, Mita, Ume, Disil, Anti, Densus 08 (Lisa, Ulin, Eny, Luci, Brury, Anton, Adian, Anti, Bambang, Manta, Rio, Ella, Chissy, Ayu, Dika), Ka Nobo, Ka Rico, MIT Students 2011 (Atika, Lay, Alyn, Seri, As, Wani, Ana, Tqmee, Aida, Meegie, Chong, and Tee), friends in MFU food technology major (Beam, Pom, Aom, Ben, Pang, Cindy) 11. My family at KEMAKI IPB and Tim Pendamping IPB, who gave me color and cheersfull in my life, thanks for the Christ-like servanthood. 12. Laboratory assistants in both MFU and IPB, for the technical assistance and the knowledge sharing. Finally, I wish this manuscript will useful for anyone who read. Bogor, November 2011 Sari Wahyuni ix

LIST OF CONTENT Page FOREWORD... ix LIST OF CONTENT... x LIST OF TABLES... xii LIST OF FIGURES... xiii LIST OF APPENDIXS... xvi I. INTRODUCTION... 1 A. BACKGROUND... 1 B. OBJECTIVES... 2 II. LITERATURE REVIEW... 3 A. TEA AND GREEN TEA... 3 B. FOOD POWDER... 7 C. FREEZE CONCENTRATION... 8 D.SPRAY DRYING... 8 III. RESEARCH METHODOLOGY... 11 A. MATERIALS AND INSTRUMENTS... 11 1. Materials... 11 2. Instruments... 11 B. EXPERIMENTAL DESIGN... 11 1. Production of Concentrated Green Tea... 11 2. Production of Green Tea Powder... 12 3. Method Analysis... 13 3.1 Bulk density... 13 3.2 Color... 13 3.3 Solubility... 13 3.4 Hygroscopicity... 13 3.5 Moisture content... 14 3.6 Total polyphenols content... 14 3.7 Antioxidant activity DPPH... 15 3.8 Caffeine and catechin content... 16 3.9 Product recovery... 16 3.10 Energy consumption... 16 3.11 Statistical analysis... 16 IV. RESULTS AND DISCUSSIONS... 17 A. CHEMICAL COMPOSITION OF DRIED GREEN TEA... 17 B. EXTRACTION... 18 C. FREEZE CONCENTRATION... 19 D. CONCENTRATED GREEN TEA... 21 E. PRODUCTION OF TEA POWDER... 22 1. Physical Analysis Results... 23 1.1 Bulk density... 24 1.2 Color... 24 1.3 Solubility... 25 1.4 Hygroscopicity... 25 2. Chemical Analysis Results... 26 2.1 Moisture content... 26 2.2 Total polyphenols content... 27 2.3 Antioxidant activity... 27 2.4 Cathecins and caffeine content... 27 2.5Correlation coefficients (r) for association between Total Polyphenol and antioxidant activity in green tea powder... 29 3. Recovery of spray drying, water removal, and energy consumption... 30 F. EFFECT OF EXTRACTION, FREEZE CONCENTRATION, AND SPRAY DRYING ON CHEMICAL PROPERTIES.. 31 V. CONCLUSIONS AND RECOMMENDATIONS... 37 A. CONCLUSIONS... 37 x

B. RECOMMENDATIONS... 37 REFERENCES... 38 APPENDICES... 41 xi

LIST OF TABLES Page Table 1. The classify the taxonomy of tea....... 3 Table 2. Composition (%) of green tea, black tea, infusion... 4 Table 3. The Percentage of Major Polyphenols in tea...... 5 Table 4. The chemical composition of dried green tea...... 18 Table 5. The chemical composition of green tea extract... 18 Table 6. The chemical composition of concentrated green tea.. 21 Table 7. The physical analysis results of green tea powder... 23 Table 8. The chemical analysis results of green tea powder... 26 Table 9. Catechins and caffeine content (µmol Trolox/100g db) of green tea powder 28 Table 10. Table 11. Table 12. Correlation coefficients (r) between single catechin and antioxidant activity in green tea powder... 30 The percentage of recovery, amount of water removal, and energy consumption of spray drying process.... 31 The chemical properties on dried tea, extract tea, concentrated tea, and tea 32 powder.. Table 13. The single catechin compound in dried, extract, and concentrated tea... 34 Table 14. Table 15. Table 16. The single catechins content in powder green tea with treatment 3% solid concentration in feed... 35 The single catechins content in powder green tea with treatment 6% solid concentration in feed... 35 The single catechins content in powder green tea with treatment 9% solid concentration in feed. 35 xii

LIST OF FIGURES Page Figure 1. Tea plant (Camellia sinensis)... 3 Figure 2. The manufacturing process of tea: (A) white tea, (B) green tea, (C) oolong tea, and (D) black tea... 4 Figure 3. Structures of the major catechin and caffeine in tea... 5 Figure 4. Schematic of a basic freeze-concentration process... 8 Figure 5. Spray dryer... 9 Figure 6. Flow chart of green tea concentrated... 12 Figure 7. Flow chart of spray drying... 12 Figure 8. The equipment of hygroscopicity... 14 Figure 9. Dried green tea (Camellia sinensis var. Oolong No 12)... 17 Figure 10. Ice cream maker machine... 19 Figure 11. Centrifuge machine. 19 Figure 12. The Relationship between of Brix and Total Dissolved Solid... 20 Figure 13. Recovery of concentrated tea from freeze concentration method that used ice cream maker machine (% solid/solid). 21 Figure 14. JMC-minilab spray dryer... 22 Figure 15. Green tea powder... 23 Figure 16. The correlation between total polyphenols and antioxidant activity in green tea powder. 29 Figure 17. Antioxidant activity in dried tea, extract tea, concentrated tea, and tea powder (mol/3000 g dried tea). 32 xiii

LIST OF APPENDICES Appendix 1. Condition of Spray Drying Process...... 42 Appendix 2. Page Clasification of Hygrospocity Type of Powder Product (GEA Niro Research Laboratory)...... 43 Appendix 3. Data and Calibration Curve of Gallic Acid Standard by Spectrophotometer.. 44 Appendix 4. Data of Trolox and DPPH preparation; Data of standard Trolox; Standard Trolox calibration curve by Spectrophotometer... 45 Appendix 5. Data of Concentration Caffeine and Catechin in Mix Standard... 46 Appendix 6. Data and Curve of Gallocatechin standard... 47 Appendix 7. Data and Curve of Epigallocatechin Standard... 48 Appendix 8. Data and Curve of Catechin Standard... 49 Appendix 9. Data and Curve of Epicatechin Standard... 50 Appendix 10. Data and Curve of Epigallocatechin gallate Standard... 51 Appendix 11. Data and Curve of Gallocatechine gallate Standard... 52 Appendix 12. Data and Curve of Epicatechin gallate Standard... 53 Appendix 13. Data and Curve of Catechin Gallate Standard... 54 Appendix 14. Data and Curve of Caffeine Standard... 55 Appendix 15. Calculation of Total Polyphenols in Tea Powder... 56 Appendix 16. Calculation of Antioxidant Activity in Tea Powder... 57 Appendix 17. Chromatogram of polyphenols in dried tea analyzed by HPLC-UV... 58 Appendix 18. Calculation of caffeine (CF) and catechin content in dried tea... 59 Appendix 19. Calculation of caffeine (CF) and catechin content in extract tea. concentrated tea 3.6.9% of total solid... 60 Appendix 20. Calculation of caffeine (CF) and catechin content in green tea powder 3% solid concentration in feed and inlet air temp. 220 C... 62 Appendix 21. SAS output. 63 Appendix 22 Duncan s test results for physical properties in green tea powders... 64 Appendix 23 Duncan s test results for chemical properties in green tea powders... 67 Appendix 24 Duncan s test results for single catechins in green tea powders... 70 xiv

I. INTRODUCTION A. BACKGROUND Tea is globally one of the most popular and lowest cost beverages, next only to water. Tea is consumed by a wide range of age groups in all levels of society. The tea plant (Camellia sinensis) has been widely used for over 5000 years for its specific aroma, taste, and putative positive physiological functions. According to statistics from the Food and Agricultural Organization (FAO) of United Nations 2008, production and consumption of tea are steadily increasing. The worldwide production of tea in 2006 reached up to 3.60 million ton and the worldwide consumption reached up to 3.64 million ton. Over past decade, world tea consumption has increased by 2.7% annually. The main tea-producing countries are China, India, Sri Lanka, Kenya, Turkey, Indonesia, and Vietnam, which accounted for 28.73, 25.93, 8.60, 8.59, 5.49, 5.15, and 3.65%, respectively, of the 2006 output of total global tea production (Hicks, 2008). Freshly harvested tea leaves are processed differently to produce specific types of tea such as green, oolong, and black tea. Of all the tea consumed in the world, 78% is black tea, 20% is green tea, and 2% is oolong tea. The green tea consumption in Indonesia was 3.13 thousand tons in 2005, while black tea consumption was more than green tea consumption, 67.9 thousand tons in 2005. FAO projected that world green tea production would grow at a faster rate than black tea by 2.0% annually, to reach 1097.7 thousand tons by 2016 (Ho et al, 2005). Green tea is heated and dried to avoid enzymatic oxidation. Green tea contains polyphenols, and most of the green tea polyphenols (GTPs) are flavonols, commonly known as catechins. Tea polyphenols have been known for their antioxidant activity and antimutagenic and anticarcinogenic properties (Yang et al 2007). Traditionally, tea is prepared from its dried young leaves and leaf buds, made into a beverage by steeping the leaves in boiling water. But today, tea powder is being developed because it has many advantages such as more practical, simple transport economics, and simply to prolong product s shelf-life. Tea powder could be applied as functional food and as non food product, like handbody, shampoo, and toothpaste. Food products being developed are tea-rice, tea-noodles, tea-cake, tea-biscuits, tea-wine, tea-candy, tea-ice cream (Hicks 1998). There are several method for produce tea powder, one of them is spray drying method. Spray drying is one-step continuous processing operation that can transform feed from a fluid state into a dried form by spraying the feed into a hot drying medium (Okos et al, 2007). A short processing time, usually between three and thirty seconds and controlled operational conditions make the spray drying as an effective and unique method for various products, especially heat sensitive products and its retaining the high quality properties such as color, flavor, and nutrients. The quality of a food powder is judged by the amount of physical and chemical degradation occuring during the dehydration process. There are many researchs were did by researchers to know the effect of spray drying on powder characteristic. At this research, different of feed concentrations and spray dryer inlet air temperatures were used to evaluate its effects on physicochemical properties of the spray-dried green tea extract. This information 1

however is necessary to establish processing conditions to produce value-added powder green tea as there is an increasing demand for herbal tea products in the market. B. OBJECTIVES The objectives of this research were to investigate the effect of total solid concentration in feed and inlet air temperatures on physical and chemical properties of green tea powder which was produced by spray dryer. 2

II. LITERATURE REVIEW A. TEA Tea, one of the most popular beverages consumed worldwide, is a processed product from the leaves of tea plant (Camellia sinensis). The taxonomy of tea is shown in Table 1 and the figure of tea plant shown in Figure 1. Table 1. The classify the taxonomy of tea Common name: Kingdom: Division: Subdivision: Class: Ordo: Family: Genus: Species: Tea Plantae Spermatophyta Angiospermae Dicolyledone Guttiferales Theaceae Camellia Camellia sinensis Figure 1. Tea plant (Camellia sinensis) The worldwide production of tea in 2006 reached up to 3.60 million ton and the worldwide consumption reached up to 3.64 million ton. Over past decade, world tea consumption has increased by 2.7% annually. Tea Production of Indonesia in 2006 reached up to 187.9 thousand of tonnes. (Hicks, 2008). Indonesia, a country with more than 222 million people, produces more than 150,000 tons of tea per year, exporting 80% of it, with the balance consumed by domestic people. The large population provides a ready workforce, as well as a promising market for tea consumption. Tea products are usually classified as white tea, green tea, oolong tea, and black tea, categorized by manufacturing process as shown in Figure 2. 3

(A) (B) (C) (D) Fresh leaves Fresh leaves Fresh leaves Fresh leaves Steaming Solar withering Withering Withering Drying Primary dryingrolling Rolling Indoor withering and rolling Pan firing Rolling by tea roller, rotor vane or CTC Secondary drying-rolling Final dryingrolling Rolling Mass breaking Drying Fermenting Drying Drying Figure 2. The manufacturing process of tea: (A) white tea, (B) green tea, (C) oolong tea, and (D) black tea (Hara, 2000) Tea is consumed in different parts of the world as white, green, black, or oolong tea. White and green tea are known as unfermented tea. The polyphenol oxidase enzyme of green tea is inactivated by steaming. Oolong tea is produced by withering and half fermenting the leaves. Thus oolong tea is called semi-fermented tea. Black tea is known as fermented tea because the leaves are fermented, allowing enzymic oxidation of the polyphenols. Processing tea differently results in variation of chemical component in tea (Hara, 2000). The chemical component of tea are presented in Table 2. Table 2. Composition (%) of green tea, black tea, infusion Compound Green Tea* Black Tea* Infusion* Protein 15 15 Trace Amino Acids 4 4 3.5 Fiber 26 26 0 Other carbohydrates 7 7 4 Lipids 7 7 Trace Pigments 2 2 Trace Mineral 5 5 4.5 Phenolic compounds 30 5 4.5 Oxidixed phenolic compounds 0 25 4.5 *Data refer to dry weight of tea leaves (Chako et al, 2010) 4

In process of green tea production, tea leaves are steamed immediately after harvesting and the enzymes are inactivated at the initial stage. Therefore, the composition of green tea is simple and similar to that in the fresh tea leaves. Green tea contains polyphenols, which include flavanols, flavandiols, flavonoids, and phenolic acids; these compounds may account for up to 30% of the dry weight. Most of the green tea polyphenols (GTPs) are flavonols, commonly known as catechins accounting for up to 30% of the dry weight of the leaves,. which are composed of eight kinds of catechins and their derivatives slightly deviates depending on the species of tea plant and the season of harvesting. There are eight major catechins in green tea: (+)-catechin (C), (-)-epicatechin (EC), (-)- gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG). The major Epigallocatechin gallate (EGCG) is the major component of the polyphenolic fraction of green tea, it makes up about 10-50% of total green tea catechins. EGCG is also most potent antioxidant of polyphenol type of tea, is at least 100 times more effective than vitamin C and 25 times more effective than vitamin E. The antioxidant activity increase in the following order: EC<ECG<EGC<EGCG (Meterc et al, 2007). The percentage of major polyphenols in tea are shown in Table 3. Table 3. The percentage of major polyphenols in tea Compound Green Tea Oolong Tea Black Tea EC 0.74 1.00 0.21 0.33 ECG 1.67 2.47 0.99 1.66 0.29 0.42 EGC 2.60 3.36 0.92 1.08 EGCG 7.00 7.53 2.93 3.75 0.39 0.60 (Yamanishi, 1995) Caffeine (1,3,7-trimetylxantine, C8H10N4O2) is a plant alkaloid, one of the few plant products which the general public is readily familiar because of its occurence in beverages such as coffee and tea, as well as various soft drinks. Caffeine extract from tea is added to some painrelief medicines. Caffeine compound is well known for its stimulant effect and is present at 2-4% of dried tea leaf weight, depending on the types and quality of teas (Yoshida et al, 1999). Studies using animal models show that green tea catechins provide some protection against degenerative diseases. Green tea catechins could also act as antitumorigenic agents (Roomi et al, 2007) and as immune modulators in immunodysfunction caused by transplanted tumors or by carcinogen treatment. Green tea consumption has also been linked to the prevention of many types of cancer, including lung colon, esophagus, mouth, stomach, small intestine, kidney, pancreas, and mammary glands (Koo, 2004). This beneficial effect has been attributed to the presence of high amounts of polyphenols, which are potent antioxidants. In particular, green tea may lower blood pressure and thus reduce the risk of stroke and coronary heart disease. Some animal s studies suggested that green tea might protect against the development of coronary heart disease by reducing blood glucose levels and body weight (Tsuneki et al, 2004). 5

(-)- Epicatechin gallate (ECG) (-)- Epigallocatechin (EGC) (-)- Epigallocatechin gallate (EGCG) (-)- Catechin gallate (CG) (-)- Catechin (C) (-)- Epicatechin (EC) Caffeine (CAF) Figure 3. Structures of the major catechin and caffeine in tea (Zuo et al, 2002) The stability of a functional ingredient is fundamental to elaborate a nutraceutical product ecause changes in the ingredient may affect its nutritional value (e.g. antioxidant capacity, composition, and bioavailability). The stability of the grape seed extract (GSE) was evaluated based on changes in their main individual phenolic compounds, as well as changes in their antioxidant activity and browning. ph affects the stability of polyphenolic compounds and that a between 4 and 5 confers more stability to catechins and their isomers and polymers than more alkaline or acidic values(tabart, 2009). The concentration of catechins was more stable than the concentration of the rest of compounds. According Pardo et al 2011, the catechins and antioxidant activity in grape seed extract generally showed decreases after the thermal. The 6

decrease in ECG and EGCG may have affected the antioxidant activity of the extracts because these phenolic compounds are known to have more scavenging power than the flavan-3-ols that clearly increased: gallic acid, gallocatequin, and catechin), due to their stearic conformation and the presence of the gallate group joined to the C ring treatments,but they were not always significant (p<0.05). B. FOOD POWDER A major reason for production in powder form is simply to prolong shelf-life of the ingredient by reducing water content; otherwise the ingredient would be degraded in its natural biological environment. Another important reason is simple transport economics, because reducing water content reduces mass and costs of the ingredient to be transported (Gustavo et al, 2010). Food ingredient powders must possess a number of functionalities which can be broadly classified as: powder handling capability; reconstitution/ recombination ability and ingredient functionality in the food product to be consumed. Poor handling during manufacture, storage and transport causes many problems which are quite common, such as no or irregular flow out of hoppers and silos and problems associated with stickiness and caking of powders. Production and processing will determine the properties of particles and powder, such as particle size distribution, shape, surface properties and moisture content. They will also influence ingredient functionality, for example, higher temperatures may cause denaturation of proteins and coating may prevent the ingredient functionality from being destroyed by oxidation (Aguilera et al, 2008). It is well known that ingredient functionality in powder form may degrade over time between manufacture and final application. This depends on the sensitivity of the individual ingredient and its exposure over time to temperature, moisture and oxygen in the air. Some ingredients are encapsulated and some powders are coated in an effort to prevent its degradation and protect its functionality outlinedsome of the functional properties of food powders and particulates (Lillford, 2002). Powders are important ingredients in a large variety of food formulations and they are responsible for the development important product characteristics such as texture, flavour, colour and nutritional value. Most of the powders will be used in some sort of wet formulation and therefore their functionality will depend on their interaction with water. Because the influence of drying parameters is not the same for all materials, optimal drying conditions vary depending on the final objective: volatile retention, preservation of enzymatic activity and avoidance of protein denaturation, fat oxidation or crystallisation. Usually, the resulting powder is made of dry particles with an average size of 30 microns and mean water activity around 0.2. The powder outlet temperature is typically less than 100 C and the residence time is of seconds (Huntington, 2004). All these characteristics will have some effect on handling properties of powders such as: bulk and tapped densities, particle density, mixing with other powders, storage; wettability and solubility, porosity, specific area (rehydration, instantisation); flowability (size, surface asperities), friability and creation/existence of dust, stability in specific atmosphere and medium (oxidation, humidification, active component release) (Huntington, 2004). Study on quality evaluation instant green tea powder showed that the important quality attributes for a green tea sample was rated as taste > flavor > color > strength. Among the quality 7

attributes, taste was the strongest attribute for both instant tea and green tea granules produced, and strength was the weakest attribute. (Sinija, 2011). C. FREEZE CONCENTRATION Concentration of fluid foods by freezing involves lowering the temperature of the product in a sufficiently controlled manner to partially freeze the product, resulting in a slurry of ice crystals in a fluid concentrate. If formed under the appropriate conditions, these ice crystals will be very pure. That is, very little product will be incorporated within the ice crystals. The ice crystals are then removed in some way with a minimum of liquid carryover, resulting in a concentrated product. The basic components of a freeze concentration system, as shown in Figure 4. Feed Crystal Nucleation Crystal Growth Crystal Slurry Separation Concentrate Ice Figure 4. Schematic of a basic freeze-concentration process (Hartel, 1992) Freeze concentration is appliable to many food concentration, such as citrus fruit juices, vinegar, coffee, tea, sugar syrups, dairy product, and aroma extract. The major advantage of using a freeze concentration process as opposed to evaporation or reverse osmosis are related to the low temperature operation suitable for sensitive food products without the loss of product quality. In addition, the solid-liquid separation in freeze concentration results in no losses of the more volatile flavors and aromas, as occur in evaporation. The disadvantages of freeze concentration compared to evaporation and reverse osmosis have include higher capital cost, higher operating costs, and excessive loss of product during the ice separation (Hartel, 1992). D. SPRAY DRYING Spray drying is one-step continuous processing operation that can transform feed from a fluid state into a dried form by spraying the feed into a hot drying medium. The product can be a single particle or agglomerates. The feed can be a solution, paste, or a suspension. This process has become one of the most important methods for drying liquid foods to powder form. The principal of spray drying as shown in Figure 5. 8

Figure 5. Spray Dryer. 1, feed reservoir; 2, feed pump; 3, product feed pipeline; 4, atomizer; 5, drying chamber; 6, air fan; 7, air heater; 8, hot air duct; 9, a mixture of dried product and aircarrying duct; 10, cyclone separator; 11, heavy powder falling down; 12, product tank; 13,exhaust air (Sharma et al, 2000). The main advantages of spray drying are the following: Product properties and quality are more effectively controlled Heat-sensitive foods, biologic products, and pharmaceuticals can be dried at atmospheric pressure and low temperatures. Sometimes inert atmosphere is employed. Spray drying permits high tonnage production in continuous operation and relatively simple equipment The product comes into contact with the equipment surfaces in an anhydrous condition, thus simplifying corrosion problems and selection of material of construction Spray drying produces relatively uniform, spherical particles with nearly the same proportion of nonvolatile compounds as in the liqiud feed. The principal disadvantages of spray drying are as follows: Spray drying generally fails if a high bulk density product is required In general it is not flexible. A unit designed for fine atomization may not be able to produce a coarse product, and vice versa. For given capacity, evaporation rates larger than other types of dryers are generally required due to high liquid content requirement. The feed must be pumpable. Pumping power requirement is high There is a high initial investment compared to other types of continuous dryers. Product recovery and dust collection increases the cost of drying (Xin and Mujumdar, 2010) Spray drying consist of four process stages: 1. Atomization of feed into a spray The formation of spray and the contacting of the spray with air, are the characteristic features of spray drying. The selection and operation of the atomizer is of supreme importance in achieving economic production of top quality products. The selection of the atomizer type depend upon the nature of the feed and desire characteristics of the dried product. In all atomizer types, increased amounts of energy available for liquid atomization result in sprays having samller droplet sizes. If, the available atomization energy is held constant but the feed rate is increased, sprays having 9

larger droplet sizes will result. Rotary atomizers are used to produce a fine to medium coarse product (mean size 30-130 µm), while nozzle atomizers are used to produce a coarse product (mean size 120-250 µm). 2. Spray-air contact (mixing and flow) Product and air pass through the dryer in co-current flow, they pass through the dryer in the same direction. This arrangement is widely used, especially if heat-sensitive products are involved. Spray evaporation is rapid, the drying air cools accordingly, and evaporation times are short. The product is not subject to heat degradation. 3. Drying of spray (moisture/ volatiles evaporation) As soon as droplet of the spray come into contact with the drying air, evaporation takes place from saturated vapour film which is quickly established at the droplet surface. The temperature at the droplet surface approximate to the wet-bulb temperature of the drying air. A substantial part of the droplet evaporation takes place when the droplet surfaces are saturated and cool. Drying chamber design and air flow rate provide a droplet residence time in the chamber, so that the desired droplet moisture removal is completed and product removed from dryer before product temperatures can rise to the outlet drying air temperature of the chamber. Hence, there is little likehood of heat damage to the product. 4. Separation of dried product from the air Total recovery of dried product takes place in the separation equipment. This system places great importance on the separation efficiency of the equipment. Separation of dried product from the air influences powder properties by virtue of mechanical handling involved during the separation stage. Axcessive mechanical handling can produce powders having a high percentage of fines. (Master, 1991) There are many variables in spray dryer that give an effect on powder product, such as inlet temperature, feed solid content, drying temperature difference, and feed temperature. Increase of inlet temperature can decrease the heat requirement of the dryer for producing a given product rate because product dried quickly. Increase in feed solids (for a given production rate) from 50% to 60% reduces the heat load by nearly 50%. Spray drying is an expensive method of evaporating volatiles and thus to obtain optimum heat utilization condition the spray dryer should always fed with the maximum solids feedstock possible. The higher the temperature difference (ie. Inlet drying air temperature minus outlet drying air temperature), the lower the heat requirement to produce a unit weight of product of constant residual moisture content from a constant solid feedstock. Feed temperature, particularly in existing plants, can also be optimized. Increasing feed temperature reduces the heat required to produce a unit weight of dried product. Preheating of feed is normally carried out to reduce feed viscosities, thereby improving atomization performance and to present feed crystallization that can cause atomizer blokage (Master, 1991). 10

III. RESEARCH METHODOLOGY A. MATERIALS AND INSTRUMENTS 1. Materials Dried green tea (var. Oolong No 12) was supplied by Boonrod Tea Factory (Thailand). Chemical reagents with analytical grade such as folin-ciocalteu (10% v/v) and gallic acid were supplied by Fluka (Buchs, Switzerland), anhydrous sodium carbonate and potassium hexacyanoferrate [K3Fe(CN)6] were purchased from Merck (Darmstadt, Germany), standard HPLC of caffeine and catechins were purchased from Sigma-Aldrich (St. Louis, Missouri, USA), acetonitrile, trifluoroacetic acid (TFA) and methanol (HPLC-grade) were purchased from Fluka (Buchs, Switzerland, trolox ((±)-6-Hydroxy-2,5,7,8- tetramethylchromane-2-carboxylic acid) and DPPH (2,2-diphenyl-1-picryhydrazyl) were purchased from Aldrich (Steinheim, Germany). monosodium phosphate monohydrate, Disodium phosphate heptahydrate and trichloroacetic acid (TFA) were purchased from Fluka (Buchs, Switzerland), citric acid (food grade), potassium mitrute salt (food grade). Then, distilled water, filter paper No 1 and No 4. 2. Instruments The main instruments were JMC-miniLAB spray dryer ( Euro Best Technology, ltd, Thailand), ice cream maker and centrifuge (March Cool Industry Co.ltd, Thailand), hydrolic press (Owner Food Machinery Co.ltd, Thailand), hand refractometer (1-32 Brix ATAGO Model N-2E, Japan), color analyzer (Colorquest XE HunterLab, Hunter Associates Laboratory, Inc, Virginia-USA), a spectrophotometer (UV Vis. Biochrom/Libra S22, England), HPLC C18, oven, disc mill, analytical balance, ph meter, vacuum pump. B. EXPERIMENTAL DESIGN This research was divided into two parts. The preliminary research were investigation on the chemical properties of raw material (dried green tea leaves) involved moisture content, total polyphenol content, antioxidant activity, catechins, and caffeine. In experiment I (Figure 6), production of concentrated green tea was made from extract green tea and increased its concentration with ice cream maker, and then determine its chemical properties. In experiment II (Figure 7), production of green tea powder was made from concentrated green tea which dried with spray dryer, and then determine its physical and chemical properties. 1. Production of Concentrated Green Tea Dried green tea was milled with a disc mill into the small size of green tea. Milled green tea was extracted dissolved in the temperature of the hot water: 90 C and with regarding water: 1: 20 (w / v). The time of extraction used was 60 minutes, and ph value was 5,0 (Butsoongnern, 2006). Tea that has been extracted was filtered by clothes sheet and pressed by press machine to obtain the pure extract of green tea. Green tea extracts were analyzed TDS (total dissolved solids) with Refractometer and oven method. After that, green tea extract was concentrated with Freeze concentration method and it used ice maker 11

machine. The way of that machine working is turn on the power, paddle, and compressor buttons. After 10 minutes, compressor was turned off and wait the ice was released from the wall. Repeat this step for several times and it will resulting in a slurry of ice crystals in a fluid concentrate. The ice crystals were then removed in some way, in this study it used centrifuge machine for separate the ice crystals and a concentrated product. This step was continued until concentrated tea contain of total solid about 3, 6, and 9%, which was measured with refractometer and confirmated with oven method. Dried green tea leaves Milling Chemical Analysis: Moisture content, Total polyphenol content, antioxidant activity, catechins, and caffeine Extraction Filtering Green tea Extract Freeze Concentration Concentrated Green Tea Extract (3,6,9%) Figure 6. Flow chart of green tea concentrated making 2. Production of Green Tea Powder Production of green tea powder with spray drying method is shown at Figure 7. Green tea extract concentrated (3,6,9%) Spray Drying inlet air temperatures: 180, 200, 220 C Green tea powder Characteristic of Final product Physical: bulk density, color, solubility, hygroscopicity Chemical: Moisture content, Total polyphenol content, antioxidant activity, catechins, and caffeine Figure 7. Flow chart of spray drying process 12

Green tea extract that was concentrated by ice maker until its concentration reached 3, 6 and 9%, were dried with spray drier. The operational conditions of the spray drying were as follows: inlet air temperatures were 180, 200, 220 C, outlet air temperatures is controlled about 75 C, blower speed was adjust in 2500 rpm.. To control outlet temperature at 75 C, the pressure air and feed rate were increased or decreased. The pressure air and feed rate were affected by inlet air temperature, an increase inlet air temperature, the pessure air and feed rate increased. After spray drying process has done, characteristic of final product should be analyzed. The physical and chemical characteristics of final products were evaluated. 3. Method of Analysis 3.1. Bulk density (Bhandari et al., 1992) Bulk density was determined by the tapping method. Two grams of powder were loosely weighed into 10 ml graduate cylinder. The cylinder containing the powder was tapped on a flat surface to a constant volume. The final volume was recorded and bulk density was calculated by dividing the sample weight by the volume. 3.2. Color Analysis (Quek et al., 2007) Color values of dried samples (L, a, and b) were measured by using ColorQuestXE/Hunter Lab (USA). 3.3. Solubility (%) (Sanphakdee, 2007) Weigh powder sample 0,5 g and mixed with 50mL of distilled water (25 C) in an 100mL beaker glass. Then it was agitated using a magnetic stirer (size 2 mm X 7mm) at a speed of 600rpm. The residue was filtrated on a filter paper No 4 and using the vacuum pump. The filter paper with an insoluble solid was placed in an oven set at 102 ± 2 C until the weight was constant. The solubility(%) was calculated by using the following equation: ( ) Where m1 is weight of filter paper and insoluble solid after dried by oven, m2 is weight of dried filter paper, and m is weight of powder sample. 3.4. Hygroscopicity (%) (Jaya and Das, 2004, modification) A saturated solution of potassium nitrite salt (equilibrium relative humidity = 79.5±2% at 20ºC) was kept in glass wash bottle having two passage for air inlet and outlet. A diaphragm type vacuum pump was used to suck the air through the salt solution. Take filter paper in pump and weigh it until constant, then add powder sample 0,5 g and it was spread uniformly in the filter paper. The increase in weight of the sample at every 15 min was noted. This measurement was continued till the difference between two succesive weighings not exceed by 0.5%. The entire operation was carried out in a room maintained at 20ºC. 13

Figure 8. The Equipment of Hygroscopicity Analysis The hygroscopicity, HG (%) was calculated by using equation as following. Where b (g) is the increase in weight of powder, a (g) weight of powder sample, and Wi (%wb) is moisture content of powder. 3.5. Moisture content (AOAC, 2000) Moisture content of the sample was determined according to the oven method. The moisture can was cleaned and dried in hot air oven for 12 hr, then cooled in desiccators and the weight which was measured by digital balance was recorded. The sample was weighed and placed into the moisture can then dried in a hot air oven at 105ºC overnight until the weight was constant. The can containing sample was cooled in a dessicators. The weight of the can and sample was determined by using digital balance. The weight of dried sample was also calculated to determined its moisture content. 3.6. Total polyphenol content The total polyphenol content was determined by spectrophotometry, using gallic acid as standar, according to the method described by the International Organization for Standardization (ISO) 14502-1. Briefly, 1.0 ml of the diluted sample extract (50-100 fold dilution) was transferred in duplicate to separated tubes containing 5.0 ml of a 1/10 dilution of Folin-Ciocalteu s reagentin water. Then, 4.0 ml of a sodium carbonate solution (7.5% w/v) was added. The tubes were then allowed to stand at room temperatures for 60 min before absorbance at 765nm was measured against water. The total polyphenol was expressed as gallic acid equivalents (GAE) in g/100g material. The concentration of 14

polyphenols in samples was derived from a standard curve of gallic acid ranging from 10 to 100 µg/ml. C V DF %DM W = gallic acid concentration (µg/ml) obtained from calibration curve = Volume of tea extract solution (ml) = Dillution factor = % dry matter = Weight of tea sample (g) 3.7. Antioxidant Activity using DPPH Antioxidant activity (DPPH free radical scavenging activity) was determined with DPPH scavenging activity and slightly modification according method by Talcott et al 2003. Pipette extracted sample 50 µl through test tubes, then add with 1950 µl of 1,1-diphenil-2- picrylhydrazil (DPPH) methanolic solution. The mixture is thoroughly vortex-mixed and kept in dark for 30 min. The absorbance is measured later, at 517 nm. A calibration curve was prepared using a standard solution of Trolox (0, 200, 400, 600, 800, 1000µM) and the results were expressed on both fresh weight basis (fw) and dry weight basis (dw) as mmol Trolox Equivalent/100g. 3.8. Caffeine and Catechins Analysis Preparation of sample Add to the instant tea (0.500±0.001) g in the flask approximately 25 ml of hot water (max 50ºC). The sample was mixed in room temperature. After that, add 5.0 ml acetonitrile and it was mixed again. Preparation of Standards Use the % purity from the certificate to prepare the stock standard solution. The individual standard solution of GC, EGC, C, EC, EGCG, CF, GCG, ECG, CG were prepared by dissolving them in a small volume of metanol, to generate a stock concentration of 999.0, 313.6, 412.0, 880.0, 911.8, 1036, 1000, 469, 832.0 and 514.8 µg/ml respectively. The mixed stock standard solution was prepared by mixing an equal volume of each stock standard. Working standard solutions were prepared by dilution of the mixed stock solution and then filtered through a 0.45µm PTFE filter before HPLC analysis. HPLC analysis HPLC analysis of standards and samples was conducted on Water 966 high performance liquid chromatography comprising vacuum degasser, quaternary pump, autosampler, thermostatted column compartment, and photo diode array detector. The column used was a Platinum EPS C18 reversed phase, 3µm (L 53 x i.d 7mm). Mobile phase eventually adopted for this study was water/acetonitrille (87:13) containing 0.05% (v/v) trifluoroacetic acid (TFA) with the flow rate of 2 ml/min. Absorption wavelength was 15

selected at 210 nm. The column was operated at 30ºC. The sample injection was 20 µl. Peaks were identified by comparing their retention times and UV spectra in the 190-400 nm range with standards. The standard was injected before sample for made calibration curves. The caffeine and catechins content were calculated using their respective calibration curves. 3.9. Product Recovery (%) (Sanphakdee, 2007) Product recovery is mainly determined by powder collection efficiency. Material loss in a spray drying system is due mostly to the attachment of sprayed droplets and dry powder to the wall of the apparatus and the cyclone s poor efficiency in collecting fine particle. Product recovery was calculated from the total solid content that determined form moisture content that heat in the oven at 103±2ºC for 6 hours. Where a (g) is weight of powder product (dry basis), b (ml) is volume of feed, and c (total solid content of tea extract concentrated. 3.10. Energy Consumption (Kamaruddin et al, 1989) Energy analysis is used to calculate the amount of energy at each stage in the production system. Analysis of this energy can be used to understand and improve how, where and when energy that used efficiently and effectively. This analysis can be used for identify networks and processes to obtain the final product. Energy consumption especially at spray drying stage was calculated by using the equation as following Where P (kilowatt) is power of spray dryer and t (hour) 3.11. Statistical Analysis Data were analyzed by one way ANOVA using the application of SPSS software. Mean value were compared using The Duncan s Multiple Range Test. 16

IV. RESULTS AND DISCUSSIONS A. CHEMICAL COMPOSITION OF DRIED GREEN TEA (Camellia sinensis var. Oolong No 12) Dried green tea that used in this study is shown in Figure 9. The chemical composition of dried green tea had been studied, such as moisture content, total polyphenol compound, antioxidant activity, caffeine and catechin content as shown in Table 4. Type of polyphenols that measured are caffeine content, total catechins, and 7 single catechins (Gallocatetchin, Epigallocatechin, Catechin, Epicatechin, Epigallocatechin gallate, Gallocatechin gallate, Epigallocatechin, Catechin gallate). The sample contains moisture content 6.05 ± 0.06 % w/w db, total polyphenol compound 14.98 ± 0.42% db, antioxidant activity 141.50 ± 6.8 mmol Trolox/100 g db, the caffeine content 2.77 ± 0.23 g/100 g db, and total catechins content about 12.04 ± 1.20 g/100 g db. The highest of single catechins that contain in dried green tea is EGCG about 4.72± 0.31 g/100 g db, and was followed by EGC about 3.66± 0.39 g/100 g db, EC about 0.89± 0.07 g/100 g db, GC about 0.77± 0.11 g/100 g db, C about 0.76± 0.06 g/100 g db, ECG about 0.73± 0.02 g/100 g db, GCG about 0.51± 0.10 g/100 g db, and CG was not detected. In measuring chemical composition of sample, determining of dilution factor for each chemical analysis have been studied. For determining chemical compound in dried green tea, total polyhenol compound and antioxidant activity analysis used dilution factor 25, for caffeine and catechin content used dilution factor 10. The determining of dilution factor for each chemical analysis is important in order to get absorbance value in spectrometer or peak value in hplc well. It means that absorbance value or peak value must be in the middle of standard curve. The dried green tea contains of moisture content about 6.05 ± 0.06 % w/w db, this value is similar with moisture content of crude tea standard. The moisture content of the final product (crude tea) should be less than 6% (Wan et al 2009). In this research, the highest of single catechins is EGCG and was followed by EGC, EC, and GC. From literature, EGCG is the most abundant catechin and it is followed by EGC, ECG, and EC. The catechin composition depends on the location of cultivation of the tea plant, variety of plant, season of harvest, and process conditions (Shi et al, 2009). Figure 9. Dried green tea (Camellia sinensis var. Oolong No 12) 17

Table 4. The chemical compositions of dried green tea Chemical Composition Moisture Content Total Polyphenol Antioxidant activity Caffeine Catechins GC (Gallocatechin) EGC (Epigallocatechin) C (Catechin) EC (Epicatechin) EGCG (Epigallocatechin gallate) GCG (Gallocatechin gallate) ECG (Epicatechin gallate) CG (Catechin gallate) Amount 6.05 ± 0.06% w/w wb 14.98 ± 0.42% db 141.50 ± 6.8 mmol Trolox/100 g db 2.77 ± 0.23 g/100 g db 12.04 ± 1.20 g/100 g db 0.77± 0.11 g/100 g db 3.66± 0.39 g/100 g db 0.76± 0.06 g/100 g db 0.89± 0.07 g/100 g db 4.72± 0.31 g/100 g db 0.51± 0.10 g/100 g db 0.73± 0.02 g/100 g db Not detected B. EXTRACTION In this research, extraction method used the best condition from previous study, like temperature of the hot water: 90 C and with regarding water: 1: 20 (w / v), the time of extraction 60 minutes, and ph value was 5,0 (Butsoongnern, 2006). The best temperature of extraction is about 80-90 C because it can defend the antioxidant of tea (Fulder, 2004). The solvent that used in this research was water because it has lower cost, available, and not contain of side-effect. Besides, polyphenols content in green tea is soluble in the water and it contains of antioxidant (Stahl, 1969). The chemical composition of green tea extract had been studied, such as total poliphenol compound, antioxidant activity, caffeine and catechin content as shown in Table 5. For determining chemical compound in green tea extract, total polyhenol compound and antioxidant activity analysis used dilution factor 25, and for caffeine and catechin content used dilution factor 50. The sample contains total polyphenol content 49.91 ± 0.33µg/mL, antioxidant activity 0.45 ± 0.02 mmol Trolox/L, the caffeine content 27.22 ± 0.05 µg/ml, and total catechins content about 116.81 ± 0.19 µg/ml. The highest of single catechins that contain in green tea extract is EGCG about 39.64 ± 0.25 µg/ml, and was followed by EGC about 33.62 ± 0.11 µg/ml, GC about 12.91± 0.11 µg/ml, GCG about 9.88 ± 0.32 µg/ml, EC about 8.47 ± 0.11 µg/ml, ECG about 6.16 ± 0.22 µg/ml, C about 6.15 ± 0.16 µg/ml. Chemical Composition Total Polyphenol Antioxidant activity Caffeine Catechin GC EGC C EC EGCG GCG ECG Table 5. The chemical compositions of green tea extract Amount 49.91 ± 0.33µg/mL 0.45 ± 0.02 mmol Trolox/L 27.22 ± 0.05 µg/ml 116.81 ± 0.19 µg/ml 12.91± 0.11 µg/ml 33.62 ± 0.11 µg/ml 6.15 ± 0.16 µg/ml 8.47 ± 0.11 µg/ml 39.64 ± 0.25 µg/ml 9.88 ± 0.32 µg/ml 6.16 ± 0.22 µg/ml 18

C. FREEZE CONCENTRATION In this study, the ice maker machine was used to make the concentrated green tea. Ice maker machine is one of machine that using freeze concentration method and it is shown in Figure 10. The principle of this machine, it will resulting in a slurry of ice crystals in a fluid concentrate. The ice crystals were then removed in some way, in this study it used centrifuge machine for separating the ice crystals and a concentrated product. The total solid in fluid concentrate will increase as longer of freeze concentration time. This step will be stopped, if the total solid reached 3, 6, and 9% of solid. For measure the total solid, it used refractometer and it will be confirmated with oven method. Figure 10. Ice cream maker machine Figure 11. Centrifuge machine 19

The measurement of Total Dissolved Solids (TDS) in this research used two kinds of measurement, such as hand recfractometer and oven method. Hand refractometer is a equipment to measure TDS that content in fruit, food product that contain of fruit, and sucrose solution (Nielsen, 1996). Nowadays, hand refractometer is used in process of made a solution in the industry, like milk industry and beverage industry. The principle of hand refractrometer is measure the index of refraction from the food that contain of carbohidrat. The unit of refratometer is Brix that equal with percentage of sucrose solution (g sucrose/ 100 g sample). Because of this research used green tea extract as the sample, which it is non-sucrose sample, the calibration of hand refractometer measurement with oven method must be done. Green tea concentrated were analyzed TDS (total dissolved solids) with Refractometer ( brix) and then compared the amount of brix with the amount of TDS from oven method to make sure that TDS in tea concentrated has reached 3, 6, 9% TDS. There is a relationship between of brix and Total Dissolved Solid as shown in Figure 12. The relationship is a linear regression with equal of regression y = 0.842x + 0.142 and R 2 = 0.997. The disadvantages of freeze concentration compared to evaporation and reverse osmosis have include higher capital cost, higher operating cost, and excessive loss of product during the ice separation (Helman et al, 1992). In this study, an increase in concentration of concentrated tea, will decrease percentage of recovery concentrated tea on freeze concentration step. Total solid 10 9 8 7 6 5 4 3 2 1 0 y = 0.842x + 0.142 R² = 0.997 0 2 4 6 8 10 12 Brix Figure 12. The Relationship between of Brix (refractometer) and Total Dissolved Solid (oven method) The recovery of concentrated tea from freeze concentration method (ice cream maker) is shown in figure 13. It was calculated from total solid in concentrated tea after freeze concentration dividing with total solid in extract tea before freeze concentration. The percentage of concentrated tea recovery from ice cream maker will decrease as increase the total solid concentration of green tea. This could be explained because the ice crystal is likely to contain sample solid at some extent. The solid present in ice crystal has the higher concentration with the longer freeze concentration process. Moreover, the solid recovery from the ice crystal by centrifuge cannot give the 100% yield. This lead to the higher loss of recovery in the green tea concentrate obtained from the longer freeze concentration process. An increase in concentration of tea concentrated will decrease percentage of recovery because ice separation with centrifuge more difficult and the loss of product during the ice separation increase. 20

Recovery (%) 120 100 80 60 40 20 0 96.91 71.15 68.36 3 6 9 Concentrated Tea (% Total solid) after freeze concentration Figure 13. Recovery of concentrated tea from freeze concentration step that used ice cream maker machine (%solid/solid) D. CONCENTRATED GREEN TEA The chemical composition of concentrated of green tea had been studied, such as total poliphenol compound, antioxidant activity, caffeine and catechin content as shown in Table 6. For determining chemical compound in concentrated green tea 3%, 6%, and 9 % have total polyhenol compound and antioxidant activity analysis used dilution factor 200, 400, and 600, respectively, and for caffeine and catechin content used dilution factor 50, 100, 175, respectively. Table 6. The chemical composition of concentrated green tea Chemical Composition Sample Conc. Tea 3% Conc. Tea 6% Conc.Tea 9% Total Polyphenol (µg/ml) 45.68 ± 0.15 b 52.99 ± 0.75 a 46.42 ± 2.89 b Antioxidant activity 0.51 ± 0.00 b 0.55 ± 0.00 a 0.39 ± 0.02 c (mmol Trolox/L) Caffeine (µg/ml) 33.43 ± 0.10 a 32.82 ± 0.43 a 25.31 ± 1.44 b Catechin (µg/ml) GC EGC C EC EGCG GCG ECG 139.46 ± 0.20 a 17.74 ± 0.07 b 46.17 ± 0.11 a 10.57 ± 0.02 a 11.56 ± 0.00 a 37.41 ± 0.10 a 10.67 ± 0.17 a 5.35 ± 0.07 a,b 138.16 ± 0.90 a 20.12 ± 0.16 a 44.63 ± 0.30 b 9.69 ± 0.01 b 11.81 ± 0.16 a 35.51 ± 0.16 b 11.30 ± 0.13 a 5.11 ± 0.01 b 111.40 ± 2.00 b 12.99 ± 0.32 c 34.58 ± 0.28 c 6.01 ± 0.17 c 8.35 ± 0.31 a 35.43 ± 0.59 b 8.58 ± 0.50 b 5.48 ± 0.16 a 1 Values are mean ± SD (n=2) 2 Value in a column followed by different letters are significantly (p<0.05) different. Concentrated green tea 3%, 6%, and 9% contain total polyphenol content 49.91 ± 45.68 ± 0.15, 52.99 ± 0.75, 46.42 ± 2.89 µg/ml, respectively. Concentrated green tea 3%, 6%, and 9% contain antioxidant activity 0.51 ± 0.00, 0.55 ± 0.00, 0.39 ± 0.02 mmol Trolox/L, respectively. Total polyphenol (µg/ml) and antioxidant activity (mmol Trolox/L) in concentrated tea 6% solid is more higher than concentrated 3% because it were concentrated. But total polyphenol and antioxidant activity decrease when concentration in feed increase until 9%, it because there are 21

some chemical compound that looses in freeze concentration method. The highest of single catechins that contain in concentrated green tea is EGC, and it was followed by EGCG, GC, EC, GCG, C, and ECG. E. PRODUCTION TEA POWDER BY SPRAY DRYER There are many things that affect on spray drying process, in spray drying condition such as inlet air temperature, outlet air temperature, blower speed, inlet and outlet humidity, and in feed condition, such as feed concentration, feed temperature, and feed flow. In this research, parameter which used feed concentration and inlet air temperature. Then, the blower speed was adjusted at 2500 rpm and the outlet air temperature was controlled at 75 C. To control outlet temperature at 75 C, the pressure air and feed rate were increased or decreased. The pressure air and feed rate were affected by inlet air temperature, an increase inlet air temperature, the pessure air and feed rate increased. To control outlet temperature at 75 C with inlet temperature 180 C, the pressure air that used about 2-5 psi and feed rate about 3-5 L/hr, to control outlet temperature at 75 C with inlet temperature 200 C, the pressure air that used about 15-20 psi and feed rate about 15-20 L/hr, and to control outlet temperature at 75 C with inlet temperature 220 C, the pressure air that used about 17.5-25 psi and feed rate about 25-30 L/hr. This condition will affect the time of drying. An increase inlet air temperature, will reduce the time of drying because the drying process will occur faster. In this research, it used JMC spray dryer as shown in Figure 14.Green tea powder was produced by JMC-minilab spray dryer as shown in Figure 15. The results of physical and chemical analyses shown in Tables 7 and 8. Figure 14. JMC-minilab Spray Dryer 22

Figure 15. Green Tea Powder 1. Physical Analysis Results Conc (%) 3 6 9 The physical analysis results of green tea powder shown in Table 7. The physical results were found variated and the significant differences were found among the samples of each physical analysis. Inlet Temp. ( C) Bulk Density (g/ml) Table 7. The physical analysis results of green tea powder L a b Solubility (%db) Hygroscopicity (%) 180 0.4511 ± 0.0050 b 71.95 ± 0.03 d 3.50 ± 0.07 f 30.08 ± 0.24 f 79.67 ± 0.85 c 17.84 ± 1.16 a 200 0.4278 ± 0.0011 c 70.89 ± 0.22 e 4.15 ± 0.03 d 33.56 ± 0.45 b 78.64 ± 0.04 c 15.75 ± 0.45 b 220 0.4262 ± 0.0011 c 71.14 ± 0.29 e 3.86 ± 0.09 e 31.97 ± 0.62 c 80.03 ± 0.04 c 8.84 ± 0.33 e 180 0.4069 ± 0.0017 d 68.07 ± 0.23 f 4.91 ± 0.04 c 34.05 ± 0.47 b 77.65 ± 0.17 c.d 15.94 ± 0.22 d 200 0.4000 ± 0.0003 e 72.42 ± 0.68 d 4.14 ± 0.12 d 31.20 ± 0.69 d 75.56 ± 1.02 d 12.69 ± 1.02 c 220 0.3972 ± 0.0004 e 73.05 ± 0.31 c 4.13 ± 0.01 d 30.65 ± 0.06 d 75.56 ± 0.34 d 10.87 ± 0.52 b 180 0.5014 ± 0.0008 a 70.38 ± 0.15 e 5.79 ± 0.08 a 37.37 ± 0.08 a 89.54 ± 0.22 a.b 16.56 ± 0.56 a.b 200 0.4522 ± 0.0010 b 74.41 ± 0.08 a 5.42 ± 0.15 b 37.31 ± 0.51 a 87.96 ± 0.54 b 12.37 ± 1.29 c 220 0.3933 ± 0.0007 f 73.81 ± 0.1 b 4.87 ± 0.02 c 34.31 ± 0.30 b 91.17 ± 1.12 a 12.52 ± 0.32 c 1 Values are mean ± SD (n=3) 2 Value in a column followed by different letters are significantly (p<0.05) different. 23

1.1 Bulk Density of Green Tea Powder Bulk density is defined as the mass of particles occupied by a unit volume of bed. Bulk density of the powder variated between 0.3933-0.5014 g/ml, the powder with treatment 9% solid concentration in feed and inlet air temperature 180 C is sample that has the highest bulk density and the powder with treatment 9% solid concentration in feed and inlet air temperature 220 C has the lowest one. Significant differences were found among the samples. Bulk density values were found in this research to be in range of bulk density values of instant tea produced by using similar technology about 0.298-0,450 g/ml (Nadeem et al, 2011). Table 7 shows powder bulk density decreases as inlet air temperature increases. This is caused by evaporation rates are faster when inlet temperature increase and the products dry to more porous of fragmented structure. Walton (2000) reported that increasing the drying air temperature generally produces a decrease in bulk and particle to be hollow. Besides, bulk density value also depend on moisture content of the powder, as a product of the higher moisture would tend to have a higher bulking weight caused by the presence of water (Chegini and Ghobadian, 2005). Increasing the solid concentration of the feed from 3 to 6% however which be related to increased total solid content, can reduce the moisture content and it causes bulk density value decrease. The highest bulk density value was grained by concentration 9% and inlet air temperature 180 C because of the sample are sticky on the chamber wall, the more stick nature of a powder is associated with a high bulk density, as the particles that tend sticky together leave less interspaces between them and consequently result in a smaller bulk volume (Goula & Adamopoulos, 2008). 1.2 Color of Green Tea Powder Color is one of important sensory attributes of food and a major quality parameter in dehydrated food. During drying, color may change because of chemical or biochemical reaction. Enzymatic oxidation, Maillard reactions, caramelization, and ascorbic acid browning are some of the chemical reaction that can occur during drying and storage. The changes in color during airdried sample was significantly higher compared to freeze-drying of strawberries. Discoloration and browning during air drying may be result of various chemical reactions including pigment destruction (Farias and Ratti, 2009). The attributes as indicator in determining color are L, a, b, and hue values. L value indicates the brightness of sample with range 0 (black) to 100 (white). The a value indicates a micture colors of red and green. The +a value indicates red color with range 0-100, while a value indicates green color with range 0-(-80). The b value indicates a combination of yellow and blue. +b range for 0-70 indicates yellowness while b range for 0-(- 70) for blueness (Francis, 1996). The results of green tea powder color were shown at Table 7. Color of of the powders are variated, for L value between 68.07-74.41, powder with treatment 9% solid concentration in feed and inlet air temperature 200 C has the highest L value and powder with treatment 6% solid concentration and inlet air temperature 180 C has the lowest one. For a value variated between 3.50-5.79, powder with treatment 9% solid concentration and inlet air temperature 180 C has the highest a value and powder with treatment 3% solid concentration and inlet air temperature 180 C has lowest a value. For b value variated between 30.08-37.37, with treatment 9% solid concentration and inlet air temperature 180 C has highest b value, while powder with treatment 24

3% solid concentration in feed and inlet air temperature 180 C has the lowest one. Based on ANOVA, significant differences were found among the samples. According to Nadeem et al (2011) when the inlet air temperature increased, the L values decreased while the b values increased. This implied that the color of the powders became little darker at higher drying temperatures. But, the result in this study is not same with literature, the L value of powder increased significantly as concentration of feed and inlet temperature increase, except on concentration 3%, the higher temperature will decrease the L value. The a and b value of powder increased significantly as concentration of feed increase and as inlet temperature decrease. This implied that the different concentrations of feed and inlet temperatures on product resulted the varieted and different color significantly (p<0.05). The higher of inlet temperature and feed concentration affect the time of drying. Contact of powder with inlet and outlet air temperature made browning reaction occurs faster. Besides, The Maillard reaction may occur in this research because green tea contains carbohydrate about 7% dry weight of tea leaves ( Chako et al, 2010). However, freeze concentration also give the effect of reducing color in the feed preparation and it resulted different color of origin. 1.3. Solubility of Green Tea Powder Many factor affect the solubility, including processing conditions, storage condition, composition, ph, density, and particle size. It has been found that increasing product temperatures is accompanied by increasing protein denaturation, which decreases solubility (Okos, 1992). But, Goula and Adamopoulos (2005) reported that the more soluble powder at the high drying temperatures. The solubility results on Table 7 show that inlet air temperature gave the various result of powder solubility but an increases of feed concentration until 9% can increase percentage of solubility. The solubility of green tea powder was found vary from 75.56-91.17%. The best condition which has the highest percentage of solubility is 9% solid concentration in feed and inlet temperature 220 C and the lowest solubility was found in tea powder with treatment on 6% solid concentration in feed and inlet air temperature 200 and 220 C. The powder with treatment 9% solid concentration in feed and inlet air temperature has the highest percentage of solubility because that condition has the lowest bulk density and time of drying. A low bulk density (< 0.4 g/ml)is required for good dispersibility of nonfat drymilk. It was found that particle agglomeration, which increases particle size, increased sinkability. The larger particles were less soluble and the longer drying time was required to made the dry large particles (Okos, 1992). 1.4. Hygroscopicity of Green Tea Powder The result of percentage of hygroscopicity ia variated about between 8.84-17.84%. The highest percentage of hygroscopicity was found in the tea powder with treatment 3% solid concentration in feed and inlet air temperature 180 C and the lowest percentage of hygroscopicity was found in the tea powder with treatment 3% solid concentration in feed and inlet air temperature 220 C. According GEA Niro Research Laboratory, green tea powders in this study have nonhygroscopic (<10%) to hygroscopic (15.1-20.0%) characteristic. In this study, inlet temperature give affect on hygroscopicity but feed concentration not give the affect. This observation is similar to that repored by other researchers, increases in inlet air temperature lead to hygroscopicity and as results to lower caking-degree (Goula & Adamopoulos, 2010). 25

2. Chemical Analysis Results Conc (%) 3 6 9 The chemical analysis results of green tea powder shown in Table 8. The chemical results were found variated and the significant differences were found among the samples of each chemical analysis, except caffeine. Inlet Temp. ( C) Table 8. The chemical analysis results of green tea powder Moisture content (%db) Total Polyphenols (%w/w db) Antioxidant (mmol Trolox/100 g db) Catechin (g/100 g db) Caffeine (g/100 g db) 180 3.33 ± 0.13 b 30.55 ± 0.85 a 226.38 ± 7.22 b.c 21.77 ± 0.45 a.b.c.d 5.37 ± 0.04 a 200 2.76 ± 0.25 c 27.44 ± 0.04 b 221.15 ± 6.33 c 21.14 ± 0.42 a.b.c.d 5.09 ± 0.25 a 220 3.77 ± 0.13 a 27.27 ± 0.04 b 218.80 ± 0.98 c 23.23 ± 2.28 a 5.64 ± 0.47 a 180 2.76 ± 0.02 c 30.69 ± 0.17 a 247.77 ± 5.65 a 20.69 ± 0.81 b.c.d 4.96 ± 0.52 a 200 2.67 ± 0.01 c 29.98 ± 1.02 a 235.62 ± 0.27 a.b.c 19.28 ± 1.49 d 5.02 ± 0.37 a 220 2.77 ± 0.06 c 29.89 ± 0.34 a 232.54 ± 4.20 a.b.c 20.43 ± 0.21 c.d 5.37 ± 0.16 a 180 3.11 ± 0.12 b 29.69 ± 0.22 a 241.67 ± 5.38 a.b 22.24 ± 0.09 a.b.c 5.16 ± 0.61 a 200 2.33 ± 0.15 d 30.34 ± 0.54 a 247.63 ± 5.10 a 22.97 ± 0.88 a.b 5.63 ± 0.24 a 220 3.22 ± 0.08 b 30.16 ± 1.12 a 243.63 ± 16.51 a.b 20.60 ± 0.05 b.c.d 5.37 ± 0.18 a 1Values are mean ± SD (n=2) 2Value in a column except caffeine followed by different letters are significantly (p<0.05) different. 2.1 Moisture content of Green Tea Powder Moisture content expresses the amount of water present in a moist sample. Two bases are widely used to express moisture content, namely moisture content wet basis and moisture content dry basis. Base on Table 8, powders moisture content varied from 2.33-3.77% wet basis. The highest moisture content was found in tea powder with treatment feed concentration 3% and inlet air temperature 220 C and the lowest polyphenol content was found in tea powder with treatment feed concentration 9% and inlet air temperature 200 C. The dried product that produced by spray dryer usually has moisture content below 5% (Singh and Heldman, 2009). Powders moisture contents were found to be in the targeted range of <5 g/100g as it is suggested to be in the range of 3-5 g/100g for instant tea powder to provide better stability during packaging and storage (Sinija & Mishra, 2008). Base on the results, moisture content was significantly influenced by inlet air temperature and feed concentration. An increase in inlet air temperature until 200 C gave a decrease in moisture content significanly. The increase inlet temp. until 220 C gave an increase moisture content. It cause by drying temperature difference between air inlet and air outlet. the higher the temperature difference. the lower ability to produce a unit weight of product of constant residual moisture content from a constant solid feedstock (Master, 1991). Besides. the moisture content of the spray-dried powders decreases with the increase in inlet and outlet air temperature (Quek et al, 2007; Tonon et al, 2008). An increase feed concentration will require more higher inlet temperature of spray drying. The sample contain 9% concentration and it is dried with inlet air 26

temperature 180 C. the sample cannot dry properly and it stuck in the wall and result the higher of moisture content. 2.2 Total Polyphenols Content The Folin-Ciocalteu assay is one ofthe oldest methods developed to determine the content of total poliphenols. The total polyphenols of green tea powder variated from 27.27-30.69% w/w db. The highest total polyphenols content was found in tea powder with treatment 6% solid concentration in feed and inlet air temperature 180 C about 30.69 ± 0.17 % w/w db and the lowest polyphenol content was found in tea powder with treatment 3% solid concentration in feed and inlet air temperature 220 C about 27.27 ± 0.04 % w/w db. An increase inlet temperature 180-220 C decrease polyphenols content and increase feed concentration not affects and results variated amount of total polyphenols. Another research reported a slight decrease in total polyphenols content of the spray dried soybean extract by increasing the inlet air temperature (Georgetti et al., 2008). 2.3 Antioxidant Activity Antioxidant activity (DPPH free radical scavenging activity) was determined with DPPH scavenging activity. The DPPH (2,2-diphenyl-1picrylhydrazyl) system offers a stable radicalgenerating procedure. It is sensitive enough to detect active principles at low concentrations. Antioxidant activity of green tea powder variated from 218.80-247.77 mmol Trolox/100 g db. The highest antioxidant activity was found in tea powder with treatment 6% solid concentration in feed and inlet air temperature 180 C about 247.77 ± 5.65 mmol Trolox/100 g db and the lowest polyphenol content was found in tea powder with treatment 3% solid concentration in feed and inlet air temperature 220 C about 218.80 ± 0.98 mmol Trolox/100 g db. An increase inlet temperature 180-220 C decrease antioxidant activity slightly, but an increase feed concentration, it will increase antioxidant activity. Based on the results, the highest antioxidant activities of green tea powder (247.77 ± 5.65 mmol Trolox/100 g db) due to the high content of total polyphenols compounds (30.69 ± 0.17 % w/w db) as well as. It similar with another research that reported the total antioxidant activity of the samples can be substantially associated with the phenolic substances present in the mountain tea spray dried. There was a significant positive correlation (P < 0.0001 and r 2 0.98239) between the results of the Total polyphenols and antioxidant activity of the samples (Nadeem et al, 2011). 2.4 Catechins and Caffeine (g/100g db) Content of Green Tea Powder The Catechins of green tea powder variated significantly from 19.28-23.23 g/100 g db and the caffeine is not different significantly 4.96-5.64 g/100 g db. The highest total catechins was found in tea powder with treatment 3% solid concentration in feed and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 6% solid concentration in feed and inlet air temperature 200 C. The highest caffeine was found in tea powder with treatment 3% solid concentration in feed and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 6% solid concentration in feed and inlet air temperature 180 C. An increase inlet temperature and feed concentration results variated amount of cathechins and not influences significantly to caffeine content. The catechins compound such as GC, EGC, C, EGCG, GCG, and ECG shown in Table 9. The results shown EGC and EGCG are the higher catechin compound in the powder. The EGC 27

content of green tea powder variated significantly from 5.86-7.37 g/100g db with treatment 3% solid concentration in feed and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 9% solid concentration in feed and inlet air temperature 220 C. The EGCG content of green tea powder variated significantly from 4.62-7.02g/100g db with treatment 3% solid concentration in feed and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 6% solid concentration in feed and inlet air temperature 200 C. Table 9. Catechins and caffeine content (g/100g db) of green tea powder Catechins and Caffeine compound Tea Powder with difference Concentration(%) and Inlet Temp. ( C) 3% 6% 9% 180 C 200 C 220 C 180 C 200 C 220 C 180 C 200 C 220 C Catechins GC 2.70 ± 0.11 c.d 0.06 d 0.23 a.b 0.08 a.b.c.d 0.10 a.b.c.d 0.02 a 0.05 a.b.c 0.18 b.c.d 0.05 a EGC 6.89 ± 0.22 a.b 0.06 a.b 0.76 a 0.04 a.b 0.25 a.b 0.18 a.b 1.41 a 0.22 a.b 0.13 b C 1.32 ± 0.01 c.d 0.05 d 0.13 c.d 0.15 c.d 0.04 b.c.d 0.07 a.b.c 0.03 a.b 0.07 a.b 0.00 a EC 1.77 ± 0.01 b 0.08 b 0.18 a.b 0.04 b 0.04 b 0.06 b 0.14 a.b 0.03 a.b 0.67 a EGCG 6.49 ± 0.11 a.b 0.06 a.b 0.73 a 0.32 b.c 0.74 c 0.18 b.c 0.96 a.b 0.25 a 0.08 b.c 0.99 ± 1.06 ± GCG 1.61 ± 0.02 ECG 1.02 ± 0.01 a.b 0.05 a.b 1.64 ± 1.60 ± 1.45 ± 1.61 ± 1.46 ± 1.58 ± 1.41 ± 1.53 ± 0.07 a 0.18 a 0.14 a 0.17 a 0.01 a 0.18 a 0.11 a 0.06 a 0.08 a 0.05 a.b 0.15 b 0.02 a.b 0.30 a.b 0.03 a 0.03 a 0.86 ± 0.74 ± 0.93 ± 0.91 ± 1.10 ± 1.05 ± 5.37 ± 5.09 ± 5.64 ± 4.96 ± 5.02 ± 5.37 ± 5.16 ± 5.63 ± 5.37 ± Caffeine 0.04 a 0.25 a 0.47 a 0.52 a 0.37 a 0.16 a 0.61 a 0.24 a 0.18 a Total of 21.77 ± 21.14 ± 23.23 ± 20.69 ± 19.28 ± 20.43 ± 22.24 ± 22.97 ± 20.60 ± Catechins 0.45 a.b.c.d 0.42 a.b.c.d 2.28 a 0.81 b.c.d 1.49 d 0.21 c.d 0.09 a.b.c 0.88 a.b 0.05 b.c.d 1 Values are mean ± SD (n=2) 2 Value within a row with different letters are not significant different(p<0.05) The GC content of green tea powder variated significantly from 2.54-3.09 g/100g db with treatment 6% solid and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 3% solid and inlet air temperature 200 C. The EC content of green tea powder variated significantly from 1.73-2.38g/100g db with treatment 9% solid and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 6% solid and inlet air temperature 220 C. The C content of green tea powder variated significantly from 1.25-1.64g/100g db with treatment 9% solid and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 3% solid and inlet air temperature 200 C. The GCG content of green tea powder variated significantly from 1.41-1.64g/100g db with treatment 3% solid and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 9% solid and inlet air temperature 200 C. The ECG content of green tea powder variated significantly from 0.74-1.10g/100g db with treatment 9% solid and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 3% solid and inlet air temperature 200 C. 28

2.5. Correlation coefficients (r) and determination coeffisien (r 2 ), for association between total polypenols and antioxidant activity of green tea powder. In this study, the correlation coefficient (r) between the total polyphenols and DPPH scavenging activity (antioxidant activity) of green tea powder is 0.73. The total polyphenols was positively correlation with antioxidant activity. It has determination coeffisien (r 2 ) about 0.533 and it is shown in Figure 16. In another research, the determination correlation (r 2 = 0.309) between the total polyphenol and the antioxidant activity of grape seed extract was not significant (p<0.05). There are compounds with a strong reducing power but a weak scavenging power; these compounds may have interfered with the correlation between total polyphenol and antioxidant activity. But there another research that reported there was a significant positive correlation (P < 0.0001 and r 2 0.98239) between the results of the total polyphenols and antioxidant activity of the mountain tea spray dried (Nadeem et al, 2011). Trolox(mmol/100 g db) 260 255 250 245 240 235 230 225 220 215 210 y = 6.55x + 41.42 R² = 0,533 27 28 29 30 31 32 Total polyphenols(%w/w db) Trolox(mmol/100 g db) Linear (Trolox(mmol/100 g db)) Figure 16. The correlation between total polyphenols and antioxidant activity in green tea powder (correlation coefficient (r) is 0.73) In this study, determination coefficient (r 2 ) = 0.533, approximately 60% of the antioxidant activity of the spray dried green tea extract may be attributed partly to the contribution of the phenolic compounds. This unclear relationship between the antioxidant activity and the total polyphenols may be explained in numerous ways. In fact, the total polyphenols content does not incorporated all antioxidants present in the extract (Djeridane et al., 2006). This is the reason why samples with similar concentration of the total polyphenol may vary in their scavenging activity.(tabart et al., 2009). In addition, the occurrence of synergism between the chemical compounds in the tea powder makes the antioxidant activity dependent of the chemical structure of the antioxidant substance and interaction between them, besides its concentration. This is the reason why tea powder with similar concentrations of total phenolics, may vary significantly in their antioxidant activities. But another research said that there is a high correlation was demonstrated between the total polyphenol content and antioxidant in green tea from Argentina (DPPH assays) capacities with correlation coefficients (r) was 0.9561 for the total polyphenols and DPPH (Anesini et al., 2008). 29

Correlation coefficients (r), for association between single catechin of green tea powder and antioxidant activity. The analysis of catechins, caffeine content, and antioxidant activity in this research were duplicated for each sample and mean values of the duplicated tests are presented. The contents of single catechin and caffeine were calculated and based on the dry tea weight and correlated with antioxidant activity in green tea powder and it is shown in Table 10. Linear regressive analysis and principal component analysis were carried out on software of SAS institute for Windows (version 9.0, Inc., 2000). Based on the results, correlation analyses between single catechin and antioxidant activity show that GC, C, and EC in green tea powder were positively correlation with antioxidant activity. As show in Table 11, the content of GC (r=0.11001, P=0.6639), EGC (r=-0.17041, P=0.4990), C (r=0.55958, P=0.0157), EC (r=0.46615, P=0.0512), EGCG (r=-0.23031, p=0.3579), GCG (r=-0.29023, p=0.2427), ECG (r=-0.06356, p=0.8022), and CF (r=-0.20416, p=0.4159 in green tea powder. The Catechin (C) and Epicatechin (EC) was highly significant (p 0.05) with antioxidant activity. According Pardo et al (2011), the catechins and antioxidant activity in grape seed extract generally showed decreases after the thermal. Based on literature, the decrease in ECG and EGCG may have affected the antioxidant activity of the extracts because these phenolic compounds are known to have more scavenging power than the flavan-3-ols that clearly increased: gallic acid, gallocatequin, and catechin), due to their stearic conformation and the presence of the gallate group joined to the C ring treatments,but they were not always significant (p<0.05). It different with result of this study that found ECG and EGCG have negatively correlation with antioxidant activity. Table 10. Correlation coefficients (r) between single catechin and antioxidant activity in green tea powder Single of catechin and caffeine (g/100g) Caffeine Antioxidant Activity (µmol Trolox/100 g db) GC 0.1100 0.66 EGC - 0.1704 0.50 C 0.5596* 0.02 EC 0.4662* 0.05 EGCG - 0.2303 0.36 GCG - 0.2902 0.24 ECG - 0.0636 0.80 R Caffeine - 0.2042 0.42 *Represents significant difference at 95% probability level (P 0.05) (See at Appendix 21) 3 Recovery of Spray Drying, Water Removal, and Energy Consumption p The recovery of spray dryer, amount of water removal, and energy consumption have been studied. The percentage spray drying recovery variated from 12.26-63.1%, the highest percentage of spray drying was found in tea powder with treatment 6% solid concentration in feed and inlet air temperature 220 C and the lowest total catechins was found in tea powder with treatment 9% solid 30

concentration in feed and inlet air temperature 180 C. The amount of water removal variated from 0.62-1.98 kg water removal/h, the highest water removal was found in tea powder with treatment 3% solid and inlet air temperature 220 C and the lowest water removal was found in tea powder with treatment 6% solid and inlet air temperature 180 C. The energy consumption variated from 12.88-91.28 KwH, the highest energy consumption was found in tea powder with treatment 3% solid and inlet air temperature 180 C and the lowest energy consumption was found in tea powder with treatment 9% solid and inlet air temperature 220 C. The results as shown in Table 11 were affected by the inlet air temperature. An increase inlet temperature gave an increase percentage of recovery, amount of water removal, and decrease energy consumption/batch production. Tea powder with condition 3% solid concentration in feed and inlet air temperature 220 C has a high recovery, the highest amount of water removal, and a low energy consumption. Feed concentration showed no influence to percentage of recovery and amount of water removal, but it influence to energy consumption/batch, because in this study used the difference volume of feed for each concentration in order to got the same total solid in the feed. So, the higher feed concentration, the lower volume that used, result the lower of energy consumption. Table 11. The percentage of recovery, amount of water removal, and energy consumption of spray drying process Total Solid (%) 3 6 9 Inlet Temp. ( C) Recovery (%) kg of Water removal/hr Energy Consumption/batch (KwH) 180 35.52 0.71 91.28 200 55.84 1.74 37.33 220 59.09 1.98 32.85 180 26.4 0.62 50.77 200 53.46 1.13 28.00 220 63.10 1.26 25.01 180 12.26 0.74 27.44 200 38.75 1.23 16.61 220 50.03 1.58 12.88 F. EFFECT OF EXTRACTION, FREEZE CONCENTRATION, AND SPRAY DRYING ON CHEMICAL PROPERTIES The quality of product especially chemical quality is greatly influenced by production method. The effect of green tea powder production method such as extraction, freeze concentration, and spray drying on its chemical properties such as total polyphenols content, catechins, caffeine, and antioxidant capasity have been studied. Total polyphenols, catechins, and caffeine content in dried tea, extract tea, concentrated tea, and tea powder are presented in Table 12. Their antioxidant activies are shown in Figure 17. Amount of total polyphenols, catechins, caffeine, and antioxidant activity of sample decreases significantly (p<0.05) after it were treated such as extraction, concentration, and spray drying. It means there is going to lose on the chemical qualities of products in production method. Besides, decreased percentage of 31

recovery concentrated tea, freeze concentration step also decreased the chemical qualities of concentrated tea as its concentation increasing. Sample Table 12. Total polyphenols, catechin, and caffeine contents in dried tea, extract tea, concentrated tea, and tea powder Total Polyphenols (g/3000g dried tea) Catechin (g/3000g dried tea) Caffeine (g/3000g dried tea) Dried Tea 444.45 ± 2.76 a 339.21 ± 34.08 a 82.58 ± 6.38 a Extract Tea 347.48 ± 0.43 b 268.65 ± 0.44 b 62.59 ± 0.11 b Concentrated Tea Tea Powder 3% solid 295.06 ± 0.96 c 225.23 ± 0.32 c 53.99 ± 0.00 c 6% solid 264.95 ± 3.75 d 172.69 ± 1.12 d 41.02 ± 0.54 d 9% solid 208.87 ± 13.01 e 146.21 ± 2.63 e 33.22 ± 1.89 e 3% 180 C 103.61 ± 2.87 i 73.74 ± 1.56 g,h 18.18 ± 0.14 h,i 3% 200 C 146.96 ± 0.19 f 113.19 ± 2.27 f,g 27.25 ± 1.36 f,g 3% 220 C 153.07 ± 0.20 f 130.26 ± 12.85 e,f 31.60 ± 2.66 e,f 6% 180 C 59.06 ± 0.33 l 39.76 ± 1.58 i 9.53 ± 1.01 j 6% 200 C 116.94 ± 3.96 h 75.00 ± 5.81 g,h 19.54 ± 1.43 h 6% 220 C 137.26 ± 1.56 g 93.74 ± 0.97 g 24.63 ± 0.75 g 9% 180 C 23.72 ± 0.17 m 17.74 ± 0.07 j 4.12 ± 0.49 k 9% 200 C 77.34 ± 1.37 j 58.41 ± 2.23 h,i 14.32 ± 0.61 i 9% 220 C 98.3 ± 3.63 i 68.64 ± 2.62 h 17.45 ± 0.58 h,i 1 Values are mean ± SD (n=2) 2 Value in a column followed by different letters are significantly (p<0.05) different. Figure 17. Antioxidant activity in dried tea, extract tea, concentrated tea, and tea powder (mol Trolox/3000g dried tea) 32

The effect of extraction method on the sample were found. The results are total polyphenol will decrease until 21.82% (444.45 ± 2.76 g/3000g dried tea in dried sample will decrease to be 347.48 ± 0.43 g/3000g dried tea in extract tea), antioxidant activity will decrease until 10.54%, catechin content will decrease until 20.80%, and caffeine content will decrease until 24.20%. The effect of freeze concentration method on the sample were found. An increase in concentration of concentrated tea will decrease chemical properties. To make concentrated tea 3% total solid, freeze concentration method will decrease total polyphenol until 15.08% (347.48± 0.43 g/3000g dried tea in extract tea will decrease to be 295.06 ± 0.96 g/3000g dried tea in concentrated tea 3% total solid), antioxidant activity will decrease until 10.14%, catechin content will decrease until 16.16%, and caffeine will decrease until 13.74%. To made concentrated tea 6% total solid, freeze concentration method will decrease total polyphenol until 23.75%, antioxidant activity will decrease until 24.93%, catechin content will decrease until 35.72%, and caffeine will decrease until 34.46%. To made concentrated tea 9% total solid, freeze concentration method will decrease total polyphenol until 39.89%, antioxidant activity will decrease until 52.33%, catechin content will decrease until 45.58%, and caffeine will decrease until 46.92%. The chemical qualities of the tea powders is very diverse because it depends on the production process from the freeze concentration until drying process. The extraction method does not affect on the chemical quality of the powder because used one condition of extraction method and it was controlled during extraction. Freeze concentration affects the quality of tea powder because initial of the chemical quality in concentrated 3, 6, and 9% are different because of loss quality during this step. Spray drying also affects the quality and yield of tea powder because inlet temperature that is used different and it affects the time of drying and % recovery of the powder. The drying time and temperatures influence on the final product because low temperatures generally have a positive influence on the quality but require longer processing times (Martin et al., 1992). The effect of spray drying on the sample were found and the results were decrease variated. The decrease of the total polyphenol content in powder was found vary from 48.12-88.64%, with concentration 3% and inlet temperature 220 C as the lowest decrease of total polyphenol and concentration 9% and inlet temperature 180 C as the highest decrease of total polyphenol after spray drying. The decrease of total catechins in tea powder after spray drying was found vary from 42.16-87.87%. The lowest decrease of catechin content was found in tea powder with concentration 3% and inlet temperature 220 C as the lowest decrease of total polyphenol and the lowest polyphenol content was found in concentration 9% and inlet temperature 180 C. The decrease of the caffeine in powder was found vary from 39.96-87.60%, with concentration 6% and inlet temperature 220 C as the lowest decrease of caffeine and concentration 9% and inlet temperature 180 C as the highest decrease of caffeine after spray drying. The decrease of the antioxidant in powder was found vary from 77.22-94.46%, with concentration 9% and inlet temperature 220 C as the lowest decrease of caffeine and concentration 9% and inlet temperature 180 C as the highest decrease of caffeine after spray drying. Based on the resuts, antioxidant activity is drop after spray drying process. The decrease of the antioxidant activity in powder was found vary from 77-99.42 with the lowest decrease was found in treatment with 9% solid concentration in feed and inlet temp. 220 C, while the highest decrease was found at 9% solid concentration in feed and inlet temp. 180 C. An increase 33

inlet temp. will increase antioxidant activity because spray drying time in the higher inlet temp is more shorter, then the time contact between tea powder and air inlet more shorter. Therefore, antioxidant activity in the powder with inlet temp. 220 C is found more higher than the powder with inlet temp. 180 C. The similar experiment from other researchers using polyphenols solution at concentration 0.05% in etanol 20% didnot show any significant decrease in antioxidant activiy after heat treatments at temperatures 65-180 C. Perhaps, thus different fact is because of the much of lower concentration of polyphenols than those applied in this study (Pardo et al., 2011) In this research, the single of catechin compounds have been studied and it were affected by process, such as extraction and freeze concentration as shown in Table 13. The highest of single catechins that contain in dried green tea is EGCG, and it was followed by EGC, EC, GC, C, ECG, and GCG. The highest of single catechins that contain in green tea extract is EGCG, and was followed by EGC, GC, GCG, EC, ECG, and C. While, the highest of single catechins that contain in concentrated green tea is EGC, and it was followed by EGCG, GC, EC, GCG, C, and ECG. Table 13. The single of catechin compound in dried, extract, and concentrated/feed tea Catechins Sample (g/3000g dried tea) Dried tea Extract tea Feed Conc 3% Feed Conc 6% Feed Conc 9% GC 21.70 ± 3.99 b 29.68 ± 0.24 a 28.65 ± 0.11 a 25.15 ± 0.19 a,b 17.04 ± 0.42 c EGC 103.02 ± 11.76 a 77.31 ± 0.24 b 74.56 ± 0.17 b 55.79 ± 0.37 c 45.38 ± 0.36 c C 21.56 ± 2.19 a 14.15 ± 0.36 c 17.06 ± 0.03 b 12.11 ± 0.02 c 7.89 ± 0.22 d EC 25.08 ± 2.79 a 19.48 ± 0.26 b 18.67 ± 0.00 b 14.76 ± 0.19 c 10.96 ± 0.41 d EGCG 132.89 ± 8.57 a 91.16 ± 0.57 b 60.42 ± 0.16 c 44.38 ± 0.20 d 33.22 ± 0.77 d GCG 14.37 ± 3.99 b,c 22.71 ± 0.73 a 17.23 ± 0.27 b 14.12 ± 0.17 b,c 11.25 ± 0.66 c ECG 20.58 ± 0.80 a 14.16 ± 0.50 b 8.64 ± 0.11 c 6.38 ± 0.01 d 7.19 ± 0.21 d 1 Values are mean ± SD (n=2) 2 Value in a column followed by different letters are significantly (p<0.05) different. The single of catechins compound in tea powder have been studied and it was affected by spray drying condition, and the results shows in Tables 14, 15, and 16. In powders which have treatment feed concentration 3%, inlet air temperature 180 C, 200 C, 220 C will decrease the single catechins compound, such as GC will decrease 68%, 53%, 42% ; EGC 69%, 52%, 45%; C 74%, 61%, 56%; EC 68%, 52%, 45%; EGCG 64%; 42%, 35%; GCG 68%, 52%, 47%; ECG 60%, 39%, 31%, respectively. In powders which have treatment feed concentration 6%, inlet air temperature 180 C, 200 C, 220 C will decrease the single catechins compound, such as GC will decrease 78%, 56%, 44%, respectively; EGC 77%, 55%, 49%; C 78%, 54%, 44%; EC 77%, 54%, 46%; EGCG 76%; 85%, 44%; GCG 78%, 69%, 48%; ECG 74%, 55%, 33%, respectively. While, In powders which have treatment feed concentration 9%, inlet air temperature 180 C, 200 C, 220 C will decrease the single catechins compound, such as GC will decrease 87%, 59%, 42%, respectively; EGC 87%, 61%, 58%; C 84%, 49%, 32%; EC 85%, 57%, 29%; EGCG 86%, 46%, 44%; GCG 90%, 64%, 59%; ECG 90%, 61%, 52%, respectively. 34

Table 14. The single catechins content in powder green tea with treatment 3% solid concentration in feed Catechins Sample (g/3000g dried tea) Feed Conc 3% Powder 3% 180 C Powder 3% 200 C Powder 3% 220 C GC 28.65 ± 0.11 a 9.13 ± 0.36 d 13.57 ± 0.34 c 16.74 ± 1.31 b EGC 74.56 ± 0.17 a 23.31 ± 0.74 c 35.71 ± 0.30 b 41.33 ± 4.28 b C 17.06 ± 0.03 a 4.47 ± 0.05 c 6.67 ± 0.27 b 7.51 ± 0.71 b EC 18.67 ± 0.00 a 5.98 ± 0.02 c 8.86 ± 0.42 b 10.23 ± 0.99 b EGCG 60.42 ± 0.16 a 21.98 ± 0.38 c 34.91 ± 0.30 b 39.33 ± 4.08 b GCG 17.23 ± 0.27 a 5.43 ± 0.07 c 8.19 ± 0.38 b 9.17 ± 0.99 b ECG 8.64 ± 0.11 a 3.44 ± 0.02 c 5.27 ± 0.27 b 5.94 ± 0.48 b 1 Values are mean ± SD (n=2) 2 Value in a column followed by different letters are significantly (p<0.05) different. Table 15. The single catechins content in powder green tea with treatment 6% solid concentration in feed Catechins Sample (g/3000g dried tea) Feed Conc 6% Powder 6% 180 C Powder 6% 200 C Powder 6% 220 C GC 25.15 ± 0.19 a 5.42 ± 0.16 d 10.98 ± 0.39 c 14.16 ± 0.10 b EGC 55.79 ± 0.37 a 12.82 ± 0.07 d 25.19 ± 0.99 c 28.30 ± 0.81 b C 12.11 ± 0.02 a 2.68 ± 0.29 d 5.55 ± 0.14 c 6.79 ± 0.32 b EC 14.76 ± 0.19 a 3.34 ± 0.08 c 6.81 ± 0.17 b 7.94 ± 0.26 b EGCG 44.38 ± 0.20 a 10.79 ± 0.61 d 6.81 ± 2.89 c 24.90 ± 0.81 b GCG 14.12 ± 0.17 a 3.07 ± 0.27 d 5.65 ± 0.66 c 7.39 ± 0.06 b ECG 6.38 ± 0.01 a 1.64 ± 0.10 d 2.86 ± 0.58 c 4.25 ± 0.10 b 1 Values are mean ± SD (n=2) 2 Value in a column followed by different letters are significantly (p<0.05) different. Table 16. The single catechins content in powder green tea with treatment 9% solid concentration in feed Catechins Sample (g/3000g dried tea) Feed Conc 9% Powder 9% 180 C Powder 9% 200 C Powder 9% 220 C GC 17.04 ± 0.42 a 2.29 ± 0.04 d 6.98 ± 0.45 c 9.94 ± 0.16 b EGC 45.38 ± 0.36 a 6.01 ± 1.13 c 17.89 ± 0.56 b 19.06 ± 0.41 b C 7.89 ± 0.22 a 1.27 ± 0.02 d 4.04 ± 0.18 c 5.34 ± 0.00 b EC 10.96 ± 0.41 a 1.59 ± 0.11 c 4.76 ± 0.07 b,c 7.73 ± 2.19 b EGCG 33.22 ± 0.77 a 4.70 ± 0.77 c 17.94 ± 0.63 b 18.58 ± 0.28 b GCG 11.25 ± 0.66 a 1.16 ± 0.15 b 4.01 ± 0.27 b 4.59 ± 0.18 b ECG 7.19 ± 0.21 a 0.73 ± 0.24 c 2.80 ± 0.07 b 3.42 ± 0.09 b 1 Values are mean ± SD (n=2) 2 Value in a column followed by different letters are significantly (p<0.05) different. 35

The lowest of the single catechins decreasing, GC (41.57%), EGC (44.57%), EGCG (34.91%), GCG (46.78%), ECG (31.25%) in powder with treatment 3% solid concentration in feed and inlet air temperature 220 C. While, for the lowest C and EC decreasing in powder with treatment 9% solid concentration in feed and inlet air temperature 220 C. An increase inlet air temperature gave an increase the single catechins content in powder tea. Whereas, an increase feed concentration gave a decrease the single catechins content in powder tea. Base on the results, powder with treatment 3% solid concentration and inlet temperature 220 C has the highest chemical yield for total polyphenol, total catechins, and the single catechins compound. Powder with treatment 6% solid concentration and inlet temperature 220 C has the the highest of caffeine content. Powder with treatment 9% solid concentration and inlet temperature 220 C has the highest of antioxidant activity, but the lowest for total polyphenol, total catechins, caffeine, and antioxidant activity was had by powder with treatment 9% solid concentration and 180 C. This could be explained because that condition results the highest weight of powder from spray dryer so its contain more amount of chemical quality. This condition results the highest weight of powder because the higher inlet temperature of spray drying might be descrease the chemical quality of the powder, but it requires shorter processing time, the sample can be dried and not sticky in the wall chamber, so it can produce more recovery of powder. For condition 9% solid concentration in feed and inlet temperature 180 C results the lowest amount of chemical quality because it has the lowest weight of powder. An increases concentration of the sample, it requires more higher inlet temperature and temperature 180 C is not enough for drying sample which contain concentration 9%. This treatment cause stickness of sample in the wall of spray dryer chamber. 36

V. CONCLUSIONS AND RECOMMENDATION A. CONCLUSIONS The effect of spray drying condition on physical and chemical properties of dried green tea extract were observed. It were found that different solid concentration of 3, 6, 9% in feed and inlet air temperatures of 180, 200, and 220 C affected the variation of physical and chemical properties of green tea powder. An increase inlet air temperature, resulted in a significant decrease (p<0.05) in bulk density, hygroscopicity, total polyphenols and antioxidant activity. An increase of solid concentration in feed gave an increase in tea powder solubility, L, a, b value, and antioxidant activity. However, the total polyphenols contents were not affected by the increase of solid concentration. For spray drying process, an increase in inlet air temperature gave an increase in recovery and amount of water removal. On the other hand, it gave a shorter time of drying and a higher reduction of energy consumption. An increase in solid concentration of feed yielded a decrease in recovery, and resulted the variation in the amount of water removal. It also allowed a short time of drying and reduce of the energy. The powder which has the best physical and chemical properties is that produced by the application of 3% solid concentration in feed and inlet air temperature 220 C. It has a low of bulk density 0.4262 ± 0.0011 g/ml, a high L value 71.14 ± 0.29, a low a value 3.86 ± 0.09 and b value 31.97 ± 0.62, a high solubility 80.03 ± 0.04%, a low hygroscopicity 8.84 ± 0.33%, the highest total catechins 23.23 ± 2.28 g/100g db with a high amount of EGC, EGCG, and GC, the highest of caffeine content 5.64 ± 0.47, a high recovery of spray drying 59.09%, the highest of water removal 1.98 kg/hr, and a low energy consumption. The powder has an antioxidant activity 218.80 ± 0.1 mmol Trolox/100 g db. B. RECOMMENDATIONS The recomendations for this study are using another technique for increasing the concentration of feed, example vacuum evaporator, in order to decrease the loss of product during concentrate the sample. Besides, the addition of drying aids, like maltodextrins, modified starches, and arabic gum are needed in order to obtain good product recovery and stability. 37

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APPENDICES 41

Appendix 1. Condition of Spray Drying Process Conc. %Total Solid (oven) Condition of Spray Drying Process Inlet Outlet blower Inlet humidity Temp. ( C) Temp. ( C) speed Outlet humidity 3% 3.0529 180 75 2500 69-73% / 32.8 C 71% / 30.5 C 3.0529 200 75 2500 62% / 32 C 59% / 32.8 C 3.0529 220 75 2500 72-73% / 29.3 C 67-69% / 30.1 C 6% 5.9862 180 75 2500 68% / 29.8 C 70% / 30.1 C 5.9862 200 75 2500 71% / 29.5 C 70% / 28.9 C 5.9862 220 75 2500 75% / 28.9 C 72% / 29.3 C 9% 8.9595 180 75 2500 65%/ 31.6 C 66% / 30.9 C 8.9595 200 75 2500 70% / 28.9 C 73% / 27.7 C 8.9595 220 75 2500 60% / 32 C 65% / 29.8 C 42

Appendix 2. Clasification of Hygrospocity Type of Powder Product (GEA Niro Research Laboratory) Hygroscopicity Non hygroscopic: ;<10% Slightly hygroscopic: 10.1-15% Hygroscopic: 15.1-20% Very hygroscopic: 20.1-25% Extremely hygroscopic: >25% 43

Appendix 3. Data and Calibration Curve of Gallic Acid Standard by Spectrophotometer Con. Abs.1 Abs.2 Abs.3 Average SD 0 0.0758 0.0756 0.0759 0.0758 0.0002 10 0.2040 0.2166 0.2157 0.2121 0.0070 20 0.3402 0.3243 0.3339 0.3328 0.0080 30 0.4762 0.4515 0.4592 0.4623 0.0126 40 0.5703 0.5941 0.5609 0.5751 0.0171 50 0.7070 0.7258 0.6792 0.7164 0.0133 60 0.8341 0.8265 0.8351 0.8319 0.0047 70 0.9340 0.8864 0.9263 0.9156 0.0256 80 1.0452 1.0402 1.0579 1.0478 0.0091 90 1.1513 1.1698 1.1567 1.1593 0.0095 100 1.2461 1.2966 1.2720 1.2716 0.0253 Gallic acid calibration curve Absorbance 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 y = 0.0119x + 0.0966 R² = 0.9987 0 20 40 60 80 100 120 Conc. Gallic acid (ug/ml) 44

Appendix 4. Data of Trolox and DPPH preparation; Data of Standard Trolox; Standard Trolox Calibration Curve by Spectrophotometer Trolox + DPPH preparation Name MW Weight Vol. MeOH Con. (g) (ml) (mm) Trolox 250.32 0.0250 10 9987.22 DPPH 394 0.0024 100 60.91 Reaction Std or sample 50 ml + 2000 ml DPPH incubate at RT for 30 min in a dark place. Measure A 517 nm using MeOH as blank Standard trolox Level Con. A 517 %Inhibition (mm) 1 2 3 mean 1 0 1.0364 0.9335 0.9589 0.9763 0.00 2 200 0.8568 0.8486 0.7278 0.8111 16.92 3 399 0.6424 0.7035 0.7014 0.6824 30.10 4 599 0.5327 0.5294 0.3724 0.4782 51.02 5 799 0.3003 0.2862 0.3013 0.2959 69.69 6 999 0.1476 0.1707 0.1763 0.1649 83.11 % Inhibition 100.00 80.00 60.00 40.00 20.00 0.00-20.00 Trolox y = 0.0851x - 0.6762 R² = 0.9963 0 200 400 600 800 1000 1200 Concentract (um) 45

Appendix 5. Data of Concentration Caffeine and Catechin in Mix Standard Concentration Con.Mix G GC EGC C EC EGCG CF GCG ECG CG (µg/ml) 99.9 39.2 40.18 100 97 80 100 40.18 98 49 1 0.20 0.08 0.08 0.20 0.19 0.16 0.20 0.08 0.20 0.10 2 1.00 0.39 0.40 1.00 0.97 0.80 1.00 0.40 0.98 0.49 3 5.00 1.96 2.01 5.00 4.85 4.00 5.00 2.01 4.90 2.45 4 9.99 3.92 4.02 10.00 9.70 8.00 10.00 4.02 9.80 4.90 5 19.98 7.84 8.04 20.00 19.40 16.00 20.00 8.04 19.60 9.80 6 39.96 15.68 16.07 40.00 38.80 32.00 40.00 16.07 39.20 19.60 7 59.94 23.52 24.11 60.00 58.20 48.00 60.00 24.11 58.80 29.40 8 79.92 31.36 32.14 80.00 77.60 64.00 80.00 32.14 78.40 39.20 9 99.90 39.20 40.18 100.00 97.00 80.00 100.00 40.18 98.00 49.00 46

Appendix 6. Data and Curve of Gallocatechin standard GC Level Conc Peak area (mv*sec) SD %RSD Peak area RF-GC (µg/ml) 1 2 3 Mean (mv*sec) 1 0.08 2295 393 896 1344.9 150.1024 0.90 0.087500 2 0.39 ND ND ND ND ND ND ND ND 3 1.96 13472 9022 14117 12204 2774.2 22.73262 12.20 0.160607 4 3.92 117960 98942 99996 105633 10688.8 10.11882 105.63 0.037110 5 7.84 433241 428290 425265 428932 4026.6 0.93874 428.93 0.018278 6 15.68 889802 891797 886389 889329 2734.8 0.30751 889.33 0.017631 7 23.52 1361222 1361419 1354988 1359210 3657.4 0.26908 1359.21 0.017304 8 31.36 1811274 1870638 1809024 1830312 34941.5 1.90904 1830.31 0.017134 9 39.20 2131986 2127202 2292481 2183890 94073.3 4.30760 2183.89 0.017950 3000.00 GC Peak area (mv*sec) 2000.00 1000.00 y = 57.622x - 43.399 R² = 0.9955 0.00 0.00 10.00 20.00 30.00 40.00 50.00-1000.00 Con. (ug/ml) 47

Appendix 7. Data and Curve of Epigallocatechin Standard EGC Level Conc Peak area (mv*sec) SD %RSD Peak area RFE- EGC (µg/ml) 1 2 3 Mean (mv*sec) 1 0.08 12348 9901 580 7610 6209.6 81.60145 7.61 0.010560 2 0.40 ND ND ND ND ND ND ND ND 3 2.01 46516 45031 39105 43551 3921.0 9.00332 43.55 0.046130 4 4.02 60674 54821 51140 55545 4808.1 8.65615 55.55 0.072338 5 8.04 277513 272498 267033 272348 5241.6 1.92460 272.35 0.029506 6 16.07 846070 838482 838159 840904 4477.1 0.53241 840.90 0.019113 7 24.11 1363869 1366317 1361497 1363894 2410.1 0.17671 1363.89 0.017676 8 32.14 1818552 1878280 1818431 1838421 34519.0 1.87764 1838.42 0.017485 9 40.18 2150500 2153638 2333291 2212476 104640.3 4.72956 2212.48 0.018161 Peak area (mv*sec) 3000.00 2000.00 EGC 1000.00 y = 58.038x - 82.110 R² = 0.994 0.00 0.00 10.00 20.00 30.00 40.00 50.00-1000.00 Con. (ug/ml) 48

Appendix 8. Data and Curve of Catechin Standard C Level Conc Peak area (mv*sec) SD %RSD Peak area RF-C (µg/ml) 1 2 3 Mean (mv*sec) 1 0.20 46661 45593 7427 33227 22349.8 67.26408 33.23 0.006019 2 1.00 4381 4718 3579 4226 585.1 13.84539 4.23 0.236630 3 5.00 258152 249477 231391 246340 13653.5 5.542547 246.34 0.020297 4 10.00 496033 462270 474490 477598 17094.7 3.579307 477.60 0.020938 5 20.00 871562 880836 873651 875350 4864.8 0.55575 875.35 0.022848 6 40.00 1613818 1616021 1613928 1614589 1241.4 0.076884 1614.59 0.024774 7 60.00 2425475 2430889 2429159 2428508 2765.1 0.113862 2428.51 0.024707 8 80.00 3225592 3355028 3222453 3267691 75652.3 2.315162 3267.69 0.024482 9 100.00 3805808 3815782 4067067 3896219 148042.7 3.799651 3896.22 0.025666 Peak area (mv*sec) C y = 39.359x + 44.271 R² = 0.999 5000.00 4000.00 3000.00 2000.00 1000.00 0.00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 Con. (ug/ml) 49

Appendix 9. Data and Curve of Epicatechin Standard EC Level Conc Peak area (mv*sec) SD %RSD Peak area RF-EC (µg/ml) 1 2 3 Mean (mv*sec) 1 0.19 44738 43550 40904 43064 1962.7 4.557543 43.06 0.004505 2 0.97 ND ND ND ND ND ND ND ND 3 4.85 229877 219701 205325 218301 12335.7 5.650788 218.30 0.022217 4 9.70 483862 449854 458421 464046 17688.0 3.811684 464.05 0.020903 5 19.40 913945 916884 906668 912499 5259.3 0.576358 912.50 0.021260 6 38.80 1762054 1760126 1758253 1760144 1900.6 0.107978 1760.14 0.022044 7 58.20 2672613 2678775 2676645 2676011 3129.5 0.116948 2676.01 0.021749 8 77.60 3555755 3690704 3549852 3598770 79671.6 2.213856 3598.77 0.021563 9 97.00 4200623 4204674 4453436 4286244 144806.4 3.378398 4286.24 0.022631 Peak area (mv*sec) 5000.00 4000.00 3000.00 2000.00 1000.00 EC y = 45.016x + 16.906 R² = 0.999 0.00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 Con. (ug/ml) 50

Appendix 10. Data and Curve of Epigallocatechin gallate Standard EGCG Level Conc Peak area (mv*sec) SD %RSD Peak area RF- (µg/ml) 1 2 3 Mean (mv*sec) EGCG 1 0.16 35914 32970 22961.3 2081.7 9.066209 22.96 0.006968 2 0.80 ND ND ND ND ND ND ND ND 3 4.00 ND ND ND ND ND ND ND ND 4 8.00 36723 24152 20291.7 8889.0 43.80635 20.29 0.394251 5 16.00 602610 596321 573670 590867 15221.4 2.57611 590.87 0.027079 6 32.00 1795609 1791354 1785725 1790896 4957.9 0.276839 1790.90 0.017868 7 48.00 2896843 2893573 2895324 2895247 1636.4 0.056519 2895.25 0.016579 8 64.00 3884732 4016829 3894688 3932083 73560.8 1.870785 3932.08 0.016276 9 80.00 4627886 4646375 4833889 4702717 113974.1 2.42358 4702.72 0.017011 Peak area (mv*sec) EGCG y = 62.623x - 209.555 R² = 0.992 6000.00 5000.00 4000.00 3000.00 2000.00 1000.00 0.00-1000.000.00 20.00 40.00 60.00 80.00 100.00 Con. (ug/ml) 51

Appendix 11. Data and Curve of Gallocatechine gallate Standard GCG Level Conc Peak area (mv*sec) SD %RSD Peak area RF-GCG (µg/ml) 1 2 3 Mean (mv*sec) 1 0.08 ND ND ND ND ND ND ND ND 2 0.40 ND ND ND ND ND ND ND ND 3 2.01 163453 54484.3 #DIV/0! #DIV/0! 54.48 0.036873 4 4.02 56960 49114 49144 51739.3 4521.3 8.738526 51.74 0.077659 5 8.04 372415 375248 360921 369528 7587.3 2.053235 369.53 0.021747 6 16.07 823357 823338 819065 821920 2472.5 0.300823 821.92 0.019554 7 24.11 1284788 1285181 1296132 1288700 6439.0 0.499652 1288.70 0.018707 8 32.14 1729687 1789176 1728673 1749179 34642.4 1.980496 1749.18 0.018377 9 40.18 2073892 2078048 2270978 2140973 112607.1 5.259623 2140.97 0.018767 Peak area (mv*sec) 2500.00 2000.00 1500.00 1000.00 500.00 GCG y = 54.638x - 66.134 R² = 0.9952 0.00-500.000.00 10.00 20.00 30.00 40.00 50.00 con. (ug/ml) 52

Appendix 12. Data and Curve of Epicatechin gallate Standard ECG Level Conc Peak area (mv*sec) SD %RSD Peak area RF-ECG (µg/ml) 1 2 3 Mean (mv*sec) 1 0.20 33351 34269 22540 649.1 2.879876 22.54 0.008696 2 0.98 ND ND ND ND ND ND ND ND 3 4.90 170443 163453 160687 164861 5028.1 3.049899 164.86 0.029722 4 9.80 386359 372909 377111 378793 6880.9 1.816546 378.79 0.025872 5 19.60 886511 887579 877589 883893 5485.5 0.620604 883.89 0.022175 6 39.20 1832457 1819321 1826645 1826141 6582.5 0.360459 1826.14 0.021466 7 58.80 2834437 2835000 2832846 2834094 1117.1 0.039418 2834.09 0.020747 8 78.40 3791078 3929398 3783514 3834663 82129.8 2.141772 3834.66 0.020445 9 98.00 4506252 4511335 4565129 4527572 32624.5 0.720573 4527.57 0.021645 Peak area (mv*sec) 5000.00 4000.00 3000.00 2000.00 1000.00 ECG y = 47.834x - 38.911 R² = 0.998 0.00-1000.000.00 20.00 40.00 60.00 80.00 100.00 120.00 Con. (ug/ml) 53

Appendix 13. Data and Curve of Catechin Gallate Standard CG Level Conc Peak area (mv*sec) SD %RSD Peak area RF-CG (µg/ml) 1 2 3 Mean (mv*sec) 1 0.10 8168 7645 9716 8510 1076.9 12.65556 8.51 0.011516 2 0.49 ND ND ND ND ND ND ND ND 3 2.45 65801 62792 58652 62415 3589.4 5.750829 62.42 0.039253 4 4.90 158481 151682 153876 154680 3470.0 2.243356 154.68 0.031678 5 9.80 390771 398559 379321 389550 9676.9 2.484124 389.55 0.025157 6 19.60 821299 812873 814387 816186 4491.9 0.550358 816.19 0.024014 7 29.40 1274066 1278920 1276134 1276373 2435.8 0.19084 1276.37 0.023034 8 39.20 1720236 1808215 1729822 1752758 48266.0 2.75372 1752.76 0.022365 9 49.00 2052642 2072100 2065152 2063298 9860.6 0.477905 2063.30 0.023748 Peak area (mv*sec) 2500.00 2000.00 1500.00 1000.00 500.00 CG y = 43.760x - 28.485 R² = 0.998 0.00-500.000.00 10.00 20.00 30.00 40.00 50.00 60.00 Con. (ug/ml) 54

Appendix 14. Data and Curve of Caffeine Standard CF Level Conc Peak area (mv*sec) SD %RSD Peak area RF-CF (µg/ml) 1 2 3 Mean (mv*sec) 1 0.20 35046 32729 22592 1638.4 7.252083 22.59 0.008853 2 1.00 8610 8833 8264 8569 286.7 3.345865 8.57 0.116700 3 5.00 182649 177241 167583 175824 7632.3 4.34084 175.82 0.028437 4 10.00 357352 332481 339993 343275 12756.3 3.716041 343.28 0.029131 5 20.00 662590 668568 653289 661482 7699.5 1.163975 661.48 0.030235 6 40.00 1284839 1284653 1283294 1284262 843.5 0.065676 1284.26 0.031146 7 60.00 1946767 1948628 1949767 1948387 1514.4 0.077726 1948.39 0.030795 8 80.00 2605219 2693547 2599419 2632728 52750.3 2.003636 2632.73 0.030387 9 100.00 3089850 3094222 3272598 3152223 104270.4 3.307838 3152.22 0.031724 Peak area (mv*sec) 4000.00 3000.00 2000.00 1000.00 CF y = 31.976x + 13.163 R² = 0.999 0.00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 Con. (ug/ml) 55

Appendix 15. Calculation of Total Polyphenols in Tea Powder Tea ref Wt.sample %DM Extract DF Absorbance Linear eq. C (µg/ml) Total polyphenols (%w/w dry basis) Powder (g) Volume(mL) 1 2 m b 1 2 1 2 Mean SD 3% 180C 3% 200C 3% 220C 6% 180C 6% 200C 6% 220C 9% 180C 9% 200C 9% 220C 1 2.0031 96.67 250 50 0.5846 0.5442 0.0114 0.0143 50.03 46.48 32.29 30.01 2 2.0032 96.67 250 50 0.5458 0.5408 0.0114 0.0143 46.62 46.18 30.09 29.81 1 2.0012 97.24 250 50 0.5045 0.4988 0.0114 0.0143 43.00 42.50 27.62 27.30 2 2.0008 97.24 250 50 0.5019 0.4994 0.0114 0.0143 42.77 42.55 27.48 27.34 1 2.0035 96.23 250 50 0.4978 0.4889 0.0114 0.0143 42.41 41.63 27.50 26.99 2 2.0008 96.23 250 50 0.4987 0.4883 0.0114 0.0143 42.49 41.58 27.59 26.99 1 2.0042 97.24 250 50 0.5627 0.5527 0.0114 0.0143 48.11 47.23 30.85 30.29 2 2.0026 97.24 250 50 0.5667 0.5563 0.0114 0.0143 48.46 47.54 31.10 30.52 1 2.0043 97.33 250 50 0.5556 0.5141 0.0114 0.0143 47.48 43.84 30.43 28.09 2 2.0038 97.33 250 50 0.5696 0.551 0.0114 0.0143 48.71 47.08 31.22 30.17 1 2.001 97.23 250 50 0.5434 0.5373 0.0114 0.0143 46.41 45.88 29.82 29.48 2 2.0006 97.23 250 50 0.5478 0.5499 0.0114 0.0143 46.80 46.98 30.07 30.19 1 2.0023 96.89 250 50 0.5616 0.5118 0.0114 0.0143 48.01 43.64 30.93 28.12 2 2.0045 96.89 250 50 0.549 0.5366 0.0114 0.0143 46.90 45.82 30.19 29.49 1 2.0057 97.67 250 50 0.5688 0.5576 0.0114 0.0143 48.64 47.66 31.04 30.41 2 2.0036 97.67 250 50 0.5491 0.549 0.0114 0.0143 46.91 46.90 29.97 29.96 1 2.0033 96.78 250 50 0.5314 0.5358 0.0114 0.0143 45.36 45.75 29.24 29.49 2 2.0044 96.78 250 50 0.5594 0.5643 0.0114 0.0143 47.82 48.25 30.81 31.09 30.55 1.167 27.44 0.146 27.27 0.319 30.69 0.359 29.98 1.334 29.89 0.317 29.68 1.198 30.34 0.508 30.16 0.925 56

Appendix 16. Calculation of Antioxidant Activity in Tea Powder Tea Powder ref Wt. sample %DM Extract Vol DF A517 A 517 sample %Inhibition b m Trolox (mm) Trolox(µmol/100 g db) (g) (ml) control 1 2 1 2 1 2 1 2 Mean SD %RSD 3% 180C 3% 200C 3% 220C 6% 180C 6% 200C 6% 220C 9% 180C 9% 200C 9% 220C 1 2.0031 96.67 250 50 0.6175 0.4026 0.3836 34.80 37.88-3.8964 0.1122 344.90 372.33 222644 240347 2 2.0032 96.67 250 50 0.6175 0.4382 0.3699 29.04 40.10-3.8964 0.1122 293.52 392.10 189466 253099 1 2.0012 97.24 250 50 0.6175 0.4105 0.3859 33.52 37.51-3.8964 0.1122 333.50 369.01 214225 237033 2 2.0008 97.24 250 50 0.6175 0.4138 0.402 32.99 34.90-3.8964 0.1122 328.74 345.77 211208 222150 1 2.0035 96.23 250 50 0.6175 0.4072 0.4068 34.06 34.12-3.8964 0.1122 338.26 338.84 219313 219687 2 2.0008 96.23 250 50 0.6175 0.4098 0.4078 33.64 33.96-3.8964 0.1122 334.51 337.40 217173 219047 1 2.0042 97.24 250 50 0.6175 0.376 0.3632 39.11 41.18-3.8964 0.1122 383.30 401.77 245843 257692 2 2.0026 97.24 250 50 0.6175 0.3744 0.3825 39.37 38.06-3.8964 0.1122 385.60 373.91 247521 240017 1 2.0043 97.33 250 50 0.6175 0.3823 0.3917 38.09 36.57-3.8964 0.1122 374.20 360.63 239777 231083 2 2.0038 97.33 250 50 0.6175 0.3795 0.3938 38.54 36.23-3.8964 0.1122 378.24 357.60 242427 229198 1 2.001 97.23 250 50 0.6175 0.3885 0.3995 37.09 35.30-3.8964 0.1122 365.25 349.38 234670 224469 2 2.0006 97.23 250 50 0.6175 0.3917 0.3836 36.57 37.88-3.8964 0.1122 360.63 372.33 231748 239261 1 2.0023 96.89 250 50 0.6175 0.3877 0.3675 37.21 40.49-3.8964 0.1122 366.41 395.56 236084 254870 2 2.0045 96.89 250 50 0.6175 0.3829 0.3881 37.99 37.15-3.8964 0.1122 373.34 365.83 240284 235453 1 2.0057 97.67 250 50 0.6175 0.3898 0.3634 36.87 41.15-3.8964 0.1122 363.38 401.48 231868 256182 2 2.0036 97.67 250 50 0.6175 0.3637 0.3744 41.10 39.37-3.8964 0.1122 401.05 385.60 256174 246309 1 2.0033 96.78 250 50 0.6175 0.4149 0.3697 32.81 40.13-3.8964 0.1122 327.15 392.39 210923 252985 2 2.0044 96.78 250 50 0.6175 0.3569 0.3772 42.20 38.91-3.8964 0.1122 410.86 381.56 264751 245870 226.389 221.154 218.805 247.768 235.621 232.537 241.673 247.633 243.632 27602 12.1923 11548 5.2217 1119 0.5116 7356 2.9690 6466 2.7443 6204 2.6681 9055 3.7470 11494 4.6415 23154 9.5038 57

Appendix 17. Chromatogram of polyphenols in dried tea analyzed by HPLC-UV 58