Chapter 1. Introduction

Transcription

1 Chapter 1 Introduction 18

2 1.1 Black tea Tea is one of the most widely consumed beverages worldwide and is made from the processed leaves of evergreen shrub Camellia sinensis. The beverage got its significance owing to the presence of high amounts of polyphenolic compounds and their associated antioxidant properties. In 2009, world tea production reached over 3.87 million tonnes (Table 1.1). The largest producers of tea are the People's Republic of China (35.5%) and India (20.7%), followed by Kenya, Sri Lanka and Turkey (Fig. 1.1). Traditionally, tea produced is classified into black (fully fermented), oolong (partially fermented) and green (unfermented) tea based on the period of fermentation, the leaves and buds have undergone during processing. This process is not a true fermentation but an enzymatic oxidation, herein simple polyphenols undergo an enzymatic polymerization by tea polyphenol oxidase leading to formation of complex condensation compounds. The amount of polyphenols in fresh leaf, green and black teas are in the range 30-35%, 10-25% and 8-21%, respectively (Lunder, 1992). The polyphenolic composition of green, oolong and black tea leaves is mainly responsible for the taste, colour, astringency and delightful aroma of their infusion. Black tea production accounted for 75% of global tea production in 2009, with India as the major producer as well as the largest consumer ( The other three major black tea producing countries are Sri Lanka, Kenya and Indonesia (Table 1.2). Considerable interests have been developed in the past decade in unraveling the beneficial health effects of tea, particularly its polyphenolic 19

3 Table 1.1 World production of tea and major producing countries (2009) Country Production (tonnes) China 1,375,780 India 800,000 Kenya 314,100 Sri Lanka 290,000 Turkey 198,601 Vietnam 185,700 Indonesia 160,000 Japan 86,000 Argentina 73,425 Iran 40,000 Bangladesh 60,000 Malawi 52,559 Uganda 48,663 ther countries 185,409 Total 3,870,237 Source: FASTAT, 2009 Fig 1.1 Distribution of world tea production (2009) 20

4 Table 1.2 Black tea production by major producing countries (2009) Country India 815 Sri Lanka 305 Kenya 238 Indonesia 131 China 65 Bangladesh 54 Source: FASTAT, 2009 Production (tonnes) components and its antioxidant activity. Catechins, TFs and TRs are the three important groups of polyphenols present in tea. The formation and mechanism of these compounds during processing as well as their respective biological activities are of great importance and of scientific and commercial interest. Consumption of tea flavonoids has been linked to lower incidences of chronic diseases such as cardiovascular disease and cancer. The antioxidant activity of phenolic compounds is due to their redox properties, allowing them to scavenge reactive oxygen species, such as superoxide radical, singlet oxygen, hydroxyl radical, nitric oxide, nitrogen dioxide and peroxynitrite, which play important roles in carcinogenesis (Wan et al., 2008). Green tea extracts are powerful antioxidants, mainly owing to the presence of high catechins content and reported to have stronger antioxidant activity and lower toxicity than synthetic antioxidants like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and DL- tocopherol (Chen and Wan, 1994). In the case of black tea, the process used in the 21

5 manufacture, is known to decrease the levels of monomeric catechins to a much greater extent of polymerization that leads to the formation of TFs and TRs which are also known to possess antioxidant activities (Lin and Lin-Shiau, 2008). Satoh et al. (2005) compared the antioxidant activities of various types of teas and showed that 2,2-Di-phenyl-1-picrylhydrazyl (DPPH) scavenging activity decreased from steamed-green tea, roasted-green tea, oolong tea to black tea. Black tea possesses many biological effects besides being an effective antioxidant (Vasundhara and Jaganmohan Rao, 2009). The fermented teas, including oolong, black and pu-erh teas are more effective than unfermented green tea in suppressing the body weight and lipogenesis in rats (Lin and Lin- Shiau, 2008). 1.2 Traditional method of black tea manufacture The traditional process of black tea manufacture from fresh green tea leaves is described by Balentine et al. (1997; 2004). The composition of fresh tea leaves is given in Table 1.3. The process comprises four major steps: withering, rolling, fermentation and firing (Fig 1.2). Withering is a process whereby the freshly plucked tea leaves are stored until the moisture content is reduced to about 55-72%. Withering causes protein breakdown leading to an increase in free amino acids, soluble carbohydrate and caffeine, changes in organic acid also takes place. The withered leaves are crushed by rolling or maceration in order to break down the leaf cell structure and bring enzymes and the substrate polyphenols into contact. 22

6 Table 1.3 Composition of fresh green tea leaves Components Fresh tea leaves (% dry weight) Flavanols 25.0 Flavonols and flavonol glycosides 3.0 Polyphenolic acids and depsides 5.0 ther polyphenols 3.0 Caffeine 3.0 Theobromine 0.2 Amino acids 4.0 rganic acids 0.5 Monosaccharide 4.0 Polysaccharides 13.0 Cellulose 7.0 Protein 15.0 Lignin 6.0 Lipids 3.0 Chlorophyll and other pigments 0.5 Ash 5.0 Volatiles 0.1 Source: Balentine et al., 1998 During fermentation the simple flavanoids in green tea leaves are oxidized by endogenous tea enzymes, polyphenol oxidase (PP) and peroxidase (PD) to produce, the more complex polyphenols that impart a bright red color and the astringent flavour to black tea. Tea fermentation is truly an enzymatic polymerization and is not a typical fermentation process as against the terminology practiced in the tea industry. The role of enzymes in the fermentation process for conversion of green tea leaves to black tea is well summarized (Sanderson and Coggon, 1977; Roberts, 1962). Fermented tea is 23

7 Fig. 1.2 Process for the manufacture of black tea leaf 24

8 fired (dried) with hot, dry air reducing the moisture content of the leaves to less than 5%. Firing of tea arrests fermentation by inactivating enzymes and results in improvement of color and creates the final balance of tea aroma. Following drying, the tea is sorted and graded to yield a commercial black tea product. 1.3 Ready-to-drink tea Tea processing has undergone many changes over the last 100 years, from loose to blended tea, tea packets, tea bags, instant tea, and finally ready-to-drink (RTD) tea. As consumers look for healthier alternatives to soft drinks, RTD tea has become a dynamic category in the world market. USA, China, Japan and Europe are the important markets for RTD tea, which is catching up with other countries as well. The tea beverage is generally prepared by brewing tea leaves in freshly boiled water for a few minutes and perhaps adding milk and sugar. In many countries, tea is more commonly enjoyed as an iced beverage (iced tea). However, such a beverage cannot be prepared by infusing traditionally manufactured tea leaves in cold water since many of the tea compounds responsible for its organoleptic properties are only sparingly soluble in cold water. Traditionally, cold tea is prepared by infusion of tea leaves and then taste enhancers like sugar or lemon juice are added, which is then cooled (>30 min) before consumption. Many methods have been proposed for manufacturing cold water infusing tea leaf that offered the convenience of not having to boil water and wait for it cool down, and the benefit of the fresh brewed tea taste. A more 25

9 convenient option is to use cold water soluble tea powders for the preparation of iced tea. However, for many consumers the quality of the final beverage from instant tea powder is not equal to that prepared from hot infused tea. Besides, use of powders is perceived to be artificial and therefore unnatural (Goodsall et al., 2004). The other alternative, instant tea beverages are typically available to consumers as packaged products in cans, bottles and other sterile containers, single strength beverage ready for consumption (RTD) or as a concentrate which is diluted with water to form a drinkable tea beverage (Agbo and Spradlin, 1995). Since RTD tea offers greater convenience, it is gaining more popularity. The reference for consumer acceptance is a product that would resemble in its color, flavor and taste as iced tea made from a hot infused tea. Industrially, RTD tea is generally prepared by using tea extracts or reconstituted tea powder with addition of sugar, lemon/peach juice, citric acid and colorants to modify its flavour, taste and colour. Besides, various additives are used as stabilising agents. Such type of cold teas, available in the market, does not ideally meet the consumers' demands who are looking for additive-free foods of high nutritional value (Todisco et al., 2002) Problems associated with RTD teas ne of the most relevant problems encountered in the production of natural and additive-free RTD cold tea is its instability due to development of haze and formation of tea cream. It gives discoloration and precipitation of complexed substances, affecting the visual appeal, flavour and colour, besides reduces the 26

10 shelf stability (Todisco et al., 2002). Since the expected shelf life of RTD tea is commonly 6 to 12 months under refrigeration, the stability of infusion is of great importance Tea cream Strong aqueous black tea infusion becomes turbid, changing from clear, deep red to light brown or orange suspension as it cools down. The coloured precipitate, whose formation causes turbidity, is known as tea cream. In simple words, cold-water insolubles are known as tea cream in the art. It is a very finely divided colloidal precipitate which imparts a distinct opacity to the clear liquor and comprises tannin complexes that comprise 15-35% of the total tea solids present in the infusion (Roberts, 1963). Tea catechins and their oxidation products when interact with caffeine, protein, pectins and metal ions in the extract form larger complexes that eventually precipitate out (Ekanayake et al., 2001). Tea cream contains many of the compounds that provide taste and colour in black tea and its formations cause both loss of taste and colour (Jobstl et al., 2005). Bradfield and Penney (1944) were the first to demonstrate a relationship between tea cream formation and tea quality (strength, pungency and briskness) in the infusion. The color of the tea cream is determined by the ratio of TRs to TFs. A high content of TRs results in the production of dull cream and bright cream would be associated with high TFs content. Triacetidin is a pink colored compound and may also play a part in determining the color of the cream 27

11 (Wickremasinghe and Perera, 1966). Creaming is valued by professional tea tasters as a contributory indication of tea quality and a visual assessment of creaming down by a tea taster, is a means whereby it is judged whether the tea contains sufficiently high amounts of TFs, TRs and caffeine, and whether they are present in relative proportions for the tea to be considered satisfactory quality. But it is considered as a negative factor in instant tea processing industry as it affects the appearance and dispersibility of iced tea beverages (Roberts, 1963). Tea cream formation in black tea depends on many factors like fermentation time, temperature, duration of extraction and water-to-tea ratio (Liang and Xu, 2003). Concentration, composition, ph and temperature-time history of the infusion also affect cream formation (Tolstoguzov, 2002). Tea cream formation is governed by various types of interactions, including polyphenol caffeine (with or without lipid) and polyphenol protein interactions (Jobstl et al., 2005). The principal constituents of black tea cream are TRs, TFs and caffeine (Roberts, 1962) and 97% of the tea cream consists of normal constituents of black tea (Smith, 1968). The approximate composition of tea cream produced in Assam tea infusion (1:40) was reported to be 15% TFs, 65% TRs, 14% caffeine, 3% ash, which included potassium (1%) and calcium (0.2%). The remaining 3% included non-caffeine nitrogenous compounds and other minor constituents (Smith, 1968). Nagalaksmi et al. (1983) brought out the differences between the tea creams isolated at ambient as well as at 4 C. Theanine is the major amino 28

12 acid constituent of the tea infusion but does not take part in cream formation, proteins to a larger extent and soluble caffeine to a lesser extent seem to play a significant role in the composition of tea cream isolated at ambient temperature in comparison to that isolated at 4 C. The polymeric polyphenols do not contribute significantly to cream formation at ambient temperature, but are the major constituents of cream isolated at lower temperatures. The nitrogenous compounds especially caffeine complexes with highly acidic phenolic groups of tea polyphenols, namely, TFs and TRs via the formation of hydrogen bonds. These hydrogen bonds are stable at lower temperatures and increasingly become unstable at higher temperatures. Liang et al. (2002) reported that caffeine, gallocatechin (GC) and epigallocatechin gallate (EGCG) are the predominant compounds in green tea cream, while TRs, GC and TFs in black tea cream. ther tea components like protein, pectin and calcium also take part in tea cream formation by coprecipitating with insoluble complexes (Liang et al., 2002). Gallated compounds offer more hydroxyl groups for hydrogen bonding for tea cream formation. Also oxidation products of catechins have a stronger creaming capacity than the unoxidized catechins. Hence, the cream formation is lesser in green tea compared to oolong and black tea. However, average size of cream particles is bigger in green than black tea Decreaming methods RTD tea is generally produced from instant tea powder. Decreaming is an 29

13 important step in the process to meet the cold stability requirements of the product. Conventional decreaming methods (removal of the precipitating complexes) include clarification by centrifugation/filtration after adjustments in temperature, enhancing the solubility by employing chemicals and enzymes, and other equivalent techniques or combination of these methods. Enzymatic approaches have overcome some of the disadvantages associated with other methods, namely, cold water extraction, chill decreaming, chemical stabilization and chemical solubilization (Rutter and Stainsby, 1975; Clark et al., 1984; Mishkin, 1962; Tsai, 1987). 1.4 Enzymatic treatments during black tea processing The role of enzymes in tea processing has been recognized for nearly four decades and its application to improve the quality of tea. Enzyme treatment have been given at three stages of black tea processing, i.e. prior to fermentation of tea, prior to extraction of black tea and to the extract; to improve soluble solids yield, cold water extractability/solubility and decrease in tea cream formation as well as to improve the clarity. Tannase is the most commonly employed enzyme for tea processing. This enzyme hydrolyses esters of phenolic acids, including the gallated polyphenols found in tea. Therefore, tea compounds are generally used as substrate for assessing the activity of tannase. Although tannase can be obtained from plant, animal and microbial sources, it is mainly produced by the latter. Aguilar et al. (2007) has summarized the various types of bacteria, yeast 30

14 and fungi employed in tannase production. Filamentous fungi of the Aspergillus genus and bacteria of the Bacillus genus have been widely used for producing tannase. Phenolic compounds such as gallic acid, pyrogallol, methyl gallate and tannic acid induces tannase synthesis (Bajpai and Patil, 1997). The major commercial food applications of tannase are in the production processes of instant tea, acorn liquor and gallic acid. The tannase of some Aspergillus strains has a molecular weight around kda. Their activity and stability ph are and , respectively, whilst optima temperatures ranged from 35ºC to 40ºC. Tannase is stable for several months at 30ºC (Belmares et al., 2004). The enzyme is commercialized by many companies with different catalytic units depending on the product presentation. Various cell-wall-digesting enzymes have also been employed which react and modify plant cell wall biopolymers. Cell-wall digesting enzyme is an enzyme which breaks down one or more tea cell-wall constituents to simpler materials and thus reduces the structural integrity or increases the permeability of the cell wall. Plant cell walls are composed primarily of cellulose, but contain lesser amounts of proteins, hemicellulose, pectins, and lipids. Accordingly, cell-wall digestive enzymes include cellulase and hemicellulase, proteases such as papain, pectinase, dextranase, lysozyme and lipases (Tsai, 1987) Enzymatic preconversion treatment to green tea leaves The four major catechins (Fig. 1.3) in green tea leaf are epicatechin (EC) and epigallocatechin (EGC) and the gallated forms of these catechins (bearing a 31

15 gallic acid (GA) moiety), epicatechin-3-gallate (ECG) and epigallocatechin-3- gallate (EGCG). During oxidative fermentation of green tea, all these catechins undergo oxidative biotransformation, through their quinones, into dimeric compounds known as TFs and higher molecular weight compounds known as TRs, which are components of black tea (Goodsall et al., 2000). TFs comprise several well-defined catechin condensation products that are characterized by their benzotropolone ring (Fig. 1.4). TRs are a group of undefined molecules with a large variance in molecular weight. TFs and TRs are responsible for the orange and brown colors of black tea infusions and products as well as making significant contributions to the astringency and body of the made tea. TRs are larger in size and darker in color than TFs. The oxidative polymerizations are a combination of biochemical oxidations mediated by PP and/or PD enzymes present in the leaf and chemical reactions of reactive species. The general reaction catalyzed by tannase (flavanol gallate esterase) is the cleavage of gallate ester linkages, both on gallated catechins and also from other gallated compounds within the leaf (Goodsall et al., 2000). Tannase action is also expected to hydrolyze gallated ester linkages of TFs and TRs releasing gallic acid in black tea. Galloyl groups are important in cream formation and tannase has been used extensively for the degallation and solubilization of black tea cream. EGCG and ECG are the most abundant catechins in fresh tea leaves and tannase treatment hydrolyzes EGCG to yield. EGC and gallic acid, and ECG to yield EC and gallic acid by cleaving their ester bonds. Several studies have been made with tannase treatment on green tea leaf to simplify the mixture 32

16 H H (-)-Epicatechin (EC) (-)-Epigallocatechin (EGC) H H C C H (-)-Epicatechin-3-gallate (ECG) H (-)-Epigallocatechin-3-gallate (EGCG) Fig 1.3 Chemical structures of major catechins Source: Goodsall et al., 2000 of catechins before the start of the fermentation (Sanderson and Coggon, 1974; Sanderson et al., 1977; Goodsall et al., 2000; 2004; Balentine et al., 2004). 33

17 H H H H C Theaflavin (TF1) (EC+EGC) H Theaflavin-3-gallate (TF2a) (EC+EGCG) H H H C C H H H C Theaflavin-3 -gallate (TF2b) (ECG+EGC) H Theaflavin-3,3 -digallate (TF3) (ECG+EGCG) Fig 1.4 Chemical structures of theaflavin and major theaflavin gallates Source: Goodsall et al.,

18 1.4.2 Enzymatic treatment of black tea before/during extraction Enzymatic treatments have been attempted with black tea by treating withtannase and cell wall digesting enzymes either before (Tsai, 1987) or during extraction (Lehmberg et al., 1999a;b;c) to improve its quality in terms of stability, and cold water solubility as well as extraction yield. Cell wall-digesting enzyme breaks down one or more cell wall constituents to simpler materials and thus reduces the structural integrity or increase the permeability of the cell wall Enzymatic treatment to extract Tannase has been recommended for hydrolysis of cream to lower molecular weight compounds, reducing turbidity and increasing cold water solubility (Sanderson and Coggon, 1974). This treatment to tea extract (Takino, 1976; Agbo and Spradlin, 1995) converts at least a portion of the insoluble solids of tea cream to a cold water-soluble form. The enzyme based method eliminates or reduces the need for inorganic and organic materials normally employed in the chemical solubilization methods. This approach is rightly referred as Enzymatic solubilization of cream and also as Enzymatic clarification of extract Transformation of tea polyphenols with the action of endo and exogenous enzymes PP and PD are the two natural endogenous enzymes present in fresh tea leaves responsible for fermentation (oxidation process) in the conversion of green tea leaves to black tea. During this fermentation process, all catechins 35

19 undergo enzymatic oxidation and condensation to form dimeric and polymeric compounds, TFs and TRs (Fig. 1.5A). The role of PD is limited as endogenous H 2 2 produced by PP is largely consumed by catalase active in tea. Tannase hydrolyses esters of phenolic acids including the gallated polyphenols. Tannase treatment to green tea hydrolyses gallated catechins ECG and EGCG to EC and EGC, respectively, with cleavage of gallic acid from their ester bonds. This results in producing enhanced levels of TF1, which is the oxidation and condensation product of EGC and EC. During the subsequent fermentation process TFs and TRs are formed with a higher proportion of their ungallated forms (Fig. 1.5 B) compared to untreated sample (Fig 1.5A). An ideal ratio of EGC(G):EC(G)::3:1 in green tea, facilitates only TF1 production but in practice a small amount of etf acid may also be formed if endogenous H 2 2 becomes available to activate PD. Although tannase treatment is aimed at complete degallation, it may not happen. In tannase treated samples, gallic acid produced could be a measure of extent of degallation. Addition of H 2 2 during oxidative fermentation is beneficial as it can activate endo-pd that could oxidize gallic acid and EC into etf acid (Fig 1.5C). However, if the addition is made during the beginning of fermentation it could be detrimental to the formation of TF1. It is preferable to add H 2 2 after allowing sufficient time for maximum TF1 formation and subsequent addition could lead to etf acid formation utilizing free gallic acid which would otherwise affect the taste of tea. Such an approach will not interfere either with the formation of beneficial TF1. Treatment to black tea leaves and extracts with tannase, degallates 36

20 A Traditional method of conversion of green tea leaves to black tea a Green tea catechins Ungallated- EC, EGC Gallated- ECG, EGCG PP/PD* Aerobic Catechins Gallocatechin quinones TFs Ungallated -TF1 Gallated - TF3, gallate TF3 gallate TF3,3 digallate TRs Ungallated Gallated a Typical catechin composition in green tea - EC:1-3%; EGC:3-6%; ECG:3-6%; EGCG:8-12% (Harbowy and Balentine,1997). * Role of PD may be limited since catalase active in tea removes peroxides as they form Usually the oxidation is not complete and some quantity of simple catechins and TFs are present along with TRs. Typical polyp henols composition in black tea: simple catechins- 15%; TFs-15%; TRs-70% (Collier et al., 1973). B Tannase preconversion treatment to green tea leaves b Green tea catechins Tannase Ungallated - EC, EGC Gallated - ECG, EGCG Anaerobic Degallation EGCG ECG b Usually the degallation and oxidation reactions may not be complete and some quantity of simple catechins and TFs may be including TF1 are present along with TRs and GA. EGC + GA EC + GA PP/PD Aerobic EGC+EC TF1+GA TFs+TF1+GA TRs Ungallated Gallated + GA Fig.1.5 Expected tannase oxidation products of green/black tea catechins during enzymatic conversion Contd., 37

21 C Tannase - Peroxide treatment to green tea leaves c Green tea catechins Ungallated - EC, EGC Gallated - ECG, EGCG Tannase Anerobic Degallation EGCG ECG EGC + GA EC + GA PP/PD Aerobic EGC+EC TF1+GA TFs+TF1+GA EC+GA PD H 2 2 TRs Ungallated Gallated etf acid + etf acid c Some amount of simple catechins, TFs and may be traces of TF1 and GA are present along with TRs and etf acid D Tannase treatment to black tea leaves/extracts d Black tea leaves Simple catechins TFs, TRs Tannase Degallation EGCG ECG Gallated TF Gallated TRs EGC + GA EC + GA TF1 Ungallated TRs TF1+ TFs + TRs Ungallated Ungallated Gallated + GA d Some amount of simple catechins may be present along with TRs, TFs (including traces of TF1) and GA Fig.1.5 Expected tannase oxidation products of green/black tea catechins during enzymatic conversion 38

22 gallated TFs and TRs as well as catechins releasing gallic acid (Fig 1.5D). The production of gallic acid is a direct measure of hydrolytic activity of tannase and free gallic acid at elevated levels result in a metallic note affecting the taste of tea to a greater extent Relative merits of various approaches The tannase treatment is useful at any process stage of its application although the benefits are more when applied in the pre-fermentation stage as it results in the formation of polyphenolic compounds that are less prone to become permanently insoluble, thereby increasing cold water solubility, higher yield, preventing tea cream formation and higher clarity. However, it may be desirable to apply enzyme treatment either during extraction or to the extract from the viewpoint of ease of adoption in the manufacturing process. In the latter approaches, black tea leaf can be used as produced in the usual manner. Enzymatic treatment of tea leaves in the solid state is preferable over enzymatic clarification after the extract is prepared, since a separate enzyme inactivation step can be avoided. While each of these processes is successful to varying degree towards improving the quality of RTD beverages either directly or indirectly, each has inherent disadvantages too. With due considerations to application and adoption in the manufacturing process, enzymatic treatment to black tea before extraction may be preferred. The feasibility and economy of instant tea products are very much dependent on the extract yield. Most of the work on enzymatic processing of black tea is focused on solids extractability, tea 39

23 cream solubilization, and clarity but there are no reports on polyphenols recovery. The future attempts should also aim at improving the extractability of polyphenols without affecting the quality (polyphenol-to-soluble solids and TF/TR ratios) of black tea extract in addition to above objectives. 1.5 Extraction of black tea RTD tea is often made using reconstituted spray dried tea powder. The extraction efficiency is a critical factor in determining the economics of an instant tea production process. Besides the extract yield, the quality of the soluble powder obtained is an equally important factor, which decides the quality of final converted products. Tea is valued for its colour, strength, briskness and flavour of the liquor and first three of these could be correlated to the TFs and TRs content of black tea liquor (Clougley, 1980). Earlier attempts revealed that TFs content is an important factor in determining black tea quality (Hilton and Ellis, 1972). Liang et al. (2003) analysed the chemical composition, colour differences of black tea infusions and their relationships with sensory quality assessed by professional tea tasters as an attempt to develop an objective method of quality evaluation. ne of the earliest experiments by Natarajan et al. (1962) examined the brewing behaviour of four different grades of black tea, extraction rates of different constituents, and effect of water-to-tea ratio, water temperature and infusion time. Extensive studies were conducted by Spiro and co-workers on extractability of black tea (Price and Spiro, 1985). All the above studies on 40

24 extractability of tea were conducted under brewing/infusing conditions close to inhome infusion preparations. The extraction conditions employed in the instant tea manufacture are aimed towards the maximum recovery of solids and are usually harsher compared to the brewing conditions used for tea preparations. Extraction conditions namely, solvent-solute ratio, extraction time and temperature greatly influence the extraction process. Long (1977) conducted a series of bench scale experiments on extraction of black tea and followed it up with pilot-scale studies on batch-simulated continuous counter-current extraction with an aim to develop a commercial extraction process (Blogg and Long, 1980). All these studies were focused on the yield of black tea considering its importance in the manufacture of instant tea. However, not much research effort has gone in to the direction of extractability of polyphenols, which contribute to the organoleptic properties Enzymatic extraction of black tea There are several reports on enzymatic treatment of tea leaves with common cell-wall digesting enzymes such as pectinases, cellulases, amylases and proteases prior to extraction to improve the extract yield and also with tannase to improve the cold water extractability. Tsai (1987) employed an enzyme solution containing tannase in conjunction with enzymes such as cellulase, pectinase and hemicellulase with an objective to improve the yield of cold-water soluble solids from black tea. The likely mechanism by which this process works is that upon imbibition of enzyme solution by black tea leaves and swelling of leaf tissues, the 41

25 enzymes are absorbed into or onto the tissues, causing the release of immobilized tea solids from leaf material and hydrolysis of released tannins to provide a higher yield of cold-water soluble tea solids. Extraction yield increased by combined enzymatic treatment compared to tannase treatment alone. Tannase-pectinase treatment gave greater extraction yield and cold-water solubility compared to tannase-cellulase treatment. Due to the action of tannase upon released tea solids, gallic acid and other organic acids are released, causing a decrease in ph and it may be necessary to adjust the ph, subsequently to a desired level with any food compatible base. Many researchers followed this combined enzyme treatment approach with some variations. Lehmberg et al. (1999b) extracted black tea using a solution containing a cocktail of enzymes. However, there are no reports to improve the extractability of polyphenols along with improving the overall extract yield. 1.6 Membrane clarification of tea extracts There is an increasing demand for foods that are more closely resemble the original raw materials and have a healthy or natural image, and have fewer synthetic additives, or have undergone fewer changes during processing (Fellows, 2009). It is necessary to respond to these pressures from the consumers. Although, enzymatic approaches have overcome some of the disadvantages associated with conventional decreaming methods, even enzymes are not preferred in the production of additive-free natural products. In 42

26 this perspective, membrane technology is an alternate approach and a mild physical process, which could overcome most of these disadvantages. Membrane processing that involves the principles of separation by size and shape of molecules or particles is a simple procedure. It offers several advantages over conventional processing methods as they are convenient and easy to scale-up. Pressure driven membrane processes are often identified by the range of size of solutes they separate, namely, reverse osmosis (R) or hyperfiltration, nanofiltration (NF), ultrafiltration (UF), and microfiltration (MF). Commercial membrane devices are available in four major types, namely plate and frame, tubular, spiral-wound and hollow fiber. Membrane technology for the processing of fruit juices and beverages has been applied mainly for clarification using UF and MF, and for concentration using R. Even though pressure-driven, these processes are attractive and cost effective, since the absence of phase change and inter-phase mass transfer necessitates less energy (Raman et al., 1994). Commercially, the major impact of membranes has been for the clarification of apple juice (Girard and Fukumoto, 2000). MF and UF have been replacing conventional fining and filtration methods for clarifying apple juices. Advantages of UF and MF over conventional methods include reduction in enzyme consumption, elimination of fining agents and their associated problems, and production with a continuous simplified process (Keefe, 1984; Rösch, 1985). During enzymatic polymerization, ~10% of the catechins are converted to TFs, bisflavanols and other oligomers with molecular weights of Da 43

27 and 75% of them are converted to TRs in black tea. The size of TRs is reported to be in the range of Da ( m) (Todisco et al., 2002). Green tea extracts initially contain high levels of unoxidized flavanols, especially monomeric catechins such as EC, ECG, EGC and EGCG that impart a desired taste (astringency) to the beverage. Unfortunately these catechins remaining in the extract will still be oxidized over time to the less desirable oxidized polyphenols (Ekanayake et al., 2001). Evans and Bird (2006) suggested that potentially a physical barrier could be used to separate polyphenols including TRs from the larger cream aggregates since the majority of black tea cream particles formed (84.8%) is in the size range of m (Liang and Xu, 2001). However, it may be desirable to employ techniques to prevent/reduce tea cream formation while retaining natural characteristics. In the last few decades, some attempts have been made employing membrane technology (pressure driven) for the clarification of extracts from black and green tea demonstrating its capability. Attempts made with black tea extracts are described below. Wickremasinghe (1977) developed a patented method for preparing cold water soluble tea concentrates and powders by selectively removing high molecular weight compounds such as chlorophyll, protein, polypeptides and polysaccharides while retaining the polyphenolic compounds by subjecting the black tea extract to filtration through a gel (polymerized dextran or polyacrylamide), porous glass granules or a UF membrane. In the UF process, extract is prefiltered through glass wool and 35 ml of ethanol (for 25 g black tea) is added to 200 ml of hot extract (60 C) to prevent membrane clogging before UF 44

28 (20 kda; 0.1 MPa). The membrane selectively removed high molecular weight compounds while allowing permeation of caffeine, polyphenols and amino acids. The ph of the resulting extract (4.8) is adjusted to 5.1 and conventionally processed to obtain a water soluble tea concentrate or powder. Todisco et al. (2002) studied the clarification of infusions from commercial black tea leaves using a 40 kda ceramic tubular membrane with a focus to eliminate proteins that interact with soluble tannins and precipitate in the infusion during storage. The purpose of the work was to integrate between the optimum infusion until a limiting polyphenol concentration is achieved and UF process to produce a stable tea with high polyphenols content and reproducible color quality. Flux and polyphenols rejection were studied over wide range of operating conditions ( kpa; m/s; 50 C). Low rejection of polyphenols (~12%) and high flux 150 LMH was observed at the highest flow velocity and 120 kpa. Polyphenols concentration and color parameters (CIE L, a, b) remained stable, and no visible haze was observed in the ultrafiltered product for up to 2 months stored in dark bottles at -4 C. Corresponding untreated infusions showed a strong reduction of lightness and yellowness whereas redness increased probably as a consequence of oxidation. There was a slight decrease in the polyphenols concentration in the direct infusion owing to the precipitation. However, protein content was not estimated in permeates which would have established whether proteins were eliminated during UF and their role in the cream formation. Evans and Bird (2006) examined UF as a clarifying procedure with two flat 45

29 sheet polymeric membranes of equal MWC (30 kda) made of fluoropolymer (FP) and regenerated cellulose (RC) in a cross-flow system using reconstituted spray dried black tea. The permeate quality was analysed in terms of haze and color (CIE tristimulus values) at 35 C. Haze was characterized by the absorbance at 900 nm, corresponding to an absorbance minimum of a centrifuged sample. Color and haze were compared before and after UF (0.1 MPa TMP, 0.44 m/s and 50 C) at similar solids concentrations. Both the membranes were effective in reducing the haze by at least an order of magnitude. Lightness and yellowness increased considerably after UF. Permeate of FP membrane showed greater haze and redness indicating its potential for transmission of larger molecular weight compounds compared to RC membrane. At 0.1 MPa TMP and after 30 min of operation, FP and RC membranes showed a steady flux of 23.0 and 32.1 LMH, rejecting 21% and 27% of solids, respectively. The membrane was effective in rejecting haze and cream aggregates, but transmitted lower molecular weight compounds that led to a relatively low overall rejection of solids. As the TMP increased, both the membranes rejected more solids. These researchers also evaluated the solute-membrane fouling interactions during UF. Being more hydrophobic, FP membrane showed more fouling tendency than RC membrane with hydrophilic tea components that led to surface modification of FP membrane resulting in a more hydrophilic surface than the original membrane. This demonstrated the advantage of using a moderately hydrophobic membrane for tea liquor filtration in terms of greater flux following 46

30 multiple fouling and cleaning cycles closer to fluxes similar to those obtained with hydrophilic materials. Hydrophobic materials generally offer greater chemical and thermal stability compared to hydrophilic membrane materials and therefore, preferred for industrial applications. Evans et al. (2008) in a subsequent study investigated the efficiency of separation and final product quality using different MWC RC and FP membranes (10, 30 and 100 kda). The FP membranes generally showed lower fluxes than the RC membranes. FP-10 had the lowest steady state flux of 14 LMH and RC-100 displayed the highest with 32 LMH. All RC membranes showed similar solids (69-73%) and polyphenols (~90%) transmission. However, the FP membranes displayed greater variations in their transmission. FP-30 provided the highest solids (73%) as well as polyphenols (~90%) transmission while FP-10 (65%) and FP-100 (62.5%) gave lower solids transmissions. Caffeine transmitted through both the types of membranes easily and was thus found in higher relative concentrations in the permeated solids. The haze 900 nm) had been significantly removed by membrane filtration (<0.002) compared to unfiltered ( ) and commercial ice tea (0.025) samples. Correspondingly, lightness had also increased significantly. According to these researchers, this would enable using higher solids concentrations of ultrafiltered solutions in iced tea production. Instead, it would be a good preposition to improve the clarity without losing much of the original color of tea liquor. RC-100 gave the reddest and yellowest solution among all the membranes. The FP membranes were significantly rougher than the RC 47

31 membranes and increased fouling was present on rougher, more hydrophobic FP surfaces. The results demonstrated that flux and defined MWC are not adequate criteria in themselves to determine membrane selection. Surface science parameters are important both to the filtration properties of real liquors, and the resulting fouling and cleaning mechanisms. Pierre (2008) proposed a process for making a cold water soluble tea extract with good colour, low or no haze and acceptable yield, without addition of any chemicals or enzymes. According to the process, the cream fraction obtained after chill decreaming is solubilized in boiling water (1-15% concentration) and ultrafiltered ( kda) at ~45 C. The permeate fraction upon cooling to room temperature was found to be free from haze, which may be combined with the decreamed fraction obtained earlier from chill-decreaming step before or after concentration/drying. In the accompanying example, the above UF process (100 kda) greatly reduced the turbidity (0.65 NTU) compared to mere centrifugation (42.2 NTU) while processing solubilized primary cream fraction. Considering the increasing market demand for RTD tea, there is a great potential for adopting membrane technology in the production process to improve their stability and decrease the haze developed during refrigerated storage while retaining most of its natural characteristics. Although the above research works advanced the application of membrane technology for clarification of tea extracts, its efficacy has not been completely tested. For instance, none of the above researchers have studied polyphenols and solids recoveries in the process. 48

32 Besides, retentate stream is a very rich source of polyphenols and there are no attempts towards its recovery. It may be desirable to introduce this clarification technique to the primary extract considering the fact that RTD tea beverages are generally produced from reconstituted spray dried tea powder. However, majority of the earlier researchers have used reconstituted tea, which would have gone through a primary clarification process and may not be a representative sample for carrying out studies. Besides, most of the researchers relied upon absorbance/transmittance as a measure of clarity which could be misleading instead it may be desirable to assess in terms of direct turbidity units. It is also necessary to measure these quality parameters at uniform strength of samples for meaningful comparison. The clarification process needs are to be benchmarked in terms of low turbidity, high retention of polyphenols, high recovery of solids and storage stability. 1.7 Scope of the present investigation An exhaustive review of research carried out towards application of enzymes and membrane technology in the production of RTD black tea beverages forms a prelude to the present investigation. ne of the most relevant problems encountered in the industrial production of additive-free RTD cold tea is its instability due to development of haze and formation of tea cream. Conventional decreaming methods are associated with inherent disadvantages while membrane technology could overcome some of these disadvantages. Hence, 49

33 membrane technology has been investigated as a physical method for clarifying black tea extracts. RTD tea is often made using reconstituted spray dried tea powder. The extraction conditions employed are aimed at maximum recovery of tea solids with due consideration to the economics of the production process. Besides the extract yield, quality of soluble powder obtained is an equally important factor which decides the quality of final converted products. However, not much research effort has gone in to the direction of extractability of polyphenols and other tea solids. To begin with, the influence of extraction conditions on polyphenols content and tea cream constituents in black tea extracts was investigated. The role of enzymes in tea processing and its application to improve the quality of tea has been recognized for nearly four decades. There are several attempts on enzymatic treatment of tea leaves with common cell-wall digesting enzymes such as pectinases, cellulases, amylases and proteases prior to extraction to improve the extract yield and also with tannase to improve the cold water extractability. However, there are no attempts to improve the extractability of polyphenols along with improving the overall extract yield without affecting the tea quality. In this study, attempts were made employing enzyme assisted extraction to enhance the recovery of polyphenols besides ESY, maintaining a good balance of tea quality, using a cell-wall digesting enzyme (pectinase) and a tannin hydrolyzing enzyme (tannase). 50

34 Membrane technology has been explored for the clarification of extracts from black tea. Although the research carried out in the last three decades advanced the application of membrane technology for clarification of tea extracts, the efficacy has not been completely tested. For instance, there are no reports on polyphenols and solids recoveries in the process. RTD tea beverages are generally produced from reconstituted spray dried tea powder. Therefore, it is desirable to introduce this clarification technique to the primary extract. However, majority of the earlier researchers have used reconstituted tea for carrying out their studies, which may not be a representative sample as it would have gone through a primary clarification step in the production process. In the present investigation, membrane technology was assessed as a clarification method for black tea extract, obtained under optimized conditions, employing various MF and UF membranes with a focus on higher yield and greater retention of polyphenols. The retentate of the membrane clarification process contained a substantial amount of polyphenols and hence not to be treated as a reject waste stream. Attempts were made to evolve a comprehensive membrane process solution to clarification of tea extracts. A comparative assessment of anti-oxidant potential of tea solids present in various membrane process streams was carried out to establish a better utility for the retentate stream as a tea solids conserve. 51