109

Chapter 5

Membrane clarification

of Black tea extracts

110

5.1 Significance and focus of the work

Decreaming is an important step in the process to meet the cold stability

requirements of RTD tea. Conventional decreaming employing any

separation technique would result in loss of flavour, color, and taste including

health enhancing polyphenols since tea cream composition is similar to tea.

Membrane technology has the potential to overcome some of the

disadvantages inherently associated with the conventional decreaming

methods. In the present study, various MF and UF membranes were

screened for the clarification of black tea extract. Besides establishing clarity,

storage stability, and assessing the tea quality parameters, efforts were made

to obtain maximum yield of tea solids as well as greater polyphenols content

in the clarified product in the membrane process. Identification and

quantification of various tea components responsible for haze and tea cream

formation, partitioned by the membranes during processing were also

attempted. Besides, evaluated the antioxidant potential of reject stream tea

solids, in order to find an appropriate utility while evolving a comprehensive

solution to clarification of tea extracts.

5.2 Clarity and storage stability of membrane processed tea extracts

Black tea extract obtained under optimized conditions was used as a feed

stock (0.61% solids content and 0.35% total polyphenols content) in the first

set of experiments for assessing the selectivity of various MF (pore size 450

and 200 nm) and UF (MWCO 25, 50, 100 and 500 kDa) membranes for the

clarification process. The black tea extracts clarified by different MF and UF

membranes were stored under refrigerated conditions (5°C) to assess the

111

product stability. Clarity was measured in terms of turbidity and stability was

measured in terms of tea cream content in extract samples immediately after

membrane processing and also after storage (Table 5.1). Immediately after

membrane clarification, extracts were bright and visibly clear with very low

turbidity values. Turbidity of black tea extract reduced from 11.72 NTU to less

than 0.60 NTU after membrane processing and the reduction was higher with

UF (0.12-0.25 NTU) compared to MF (0.52-0.60 NTU) in relation to their pore

size and MWCO. Turbidity of extracts increased during storage and 450 nm

membrane permeate reached the maximum turbidity of 2.85 NTU after 30

days of storage at RT. The increase in turbidity with storage under cold

conditions was greater compared to ambient conditions (Table 5.1).

However, turbidity values of membrane processed extracts measured at a

beverage strength of 0.25 g/200 ml, were well within an acceptable limit (~4

NTU) while the unprocessed extract reached a turbidity of 25.23 NTU.

Transmission/absorbance has been used as a measure of clarity by

various researchers (Kawakatsu et al., 1995; Ramarethinam et al., 2006;

Sugiyama & Ueoka, 2006; Evans & Bird, 2006). Transmission spectra of

centrifuged extract sample showed that transmission increased with increase

in wavelength in the visible range and transmission at 660 nm was

conveniently chosen for measurement (Fig. 5.1). Transmission values

generally showed a similar trend as that of turbidity values with respect to

membrane pore size and storage (Table 5.1). But the transmission values did

not correlate well with their corresponding turbidity values specifically in the

case of cold stored samples (r2 = 0.207) questioning their suitability as index

of absolute measurement of clarity. On the other hand, inverse transmission

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Table 5.1

Clarity, color and tea cream content of membrane processed black tea extracts

Membrane pore Turbidity (NTU) Transmission (%) Hunter colour Tea cream (µg/ml)

Size / MWCO RT Cold RT Cold L a b Cold

Storage days 0 14 30 0 30 0 30 30 0 0 0 0 30

Feed 11.72±0.51 15.81±0.42 23.11±1.53 13.72±1.21 25.23±1.14 84.2 68.3 62.3 48.2 5.3 24.6 86.81±0.12 254.33±0.12

UF Permeates

25 kDa 0.12±0.03a 0.29±0.03

a 0.83±0.05

a 0.24±0.04

a 4.86±0.07

a 92.3 87.4 82.4 69.7 -2.7 8.3 -* - *

50 kDa 0.16±0.04a 0.50±0.03

b 1.21±0.07

b 0.25±0.03

a 3.49±0.08

b 90.3 80.3 79.1 63.5 -1.4 20.6 - * - *

100 kDa 0.18±0.03a 0.32±0.04

c 1.52±0.07

c 0.26±0.03

a 2.75±0.04

c 89.9 75.1 72.3 62.3 -0.9 19.3 - * - *

500 kDa 0.25±0.03b 0.39±0.03

c 1.83±0.05

d 0.27±0.01

a 3.26±0.10

d 90.1 73.5 70.2 59.7 1.3 22.3 - * - *

MF Permeates

200 nm 0.52±0.04c 0.80±0.02

d 2.16±0.15

e 0.37±0.01

b 3.93±0.08

e 88.2 76.3 71.5 56.4 3.1 22.2 - * - *

450 nm 0.60±0.07c 0.82±0.06

d 2.85±0.13

f 0.40±0.02

b 4.04±0.01

e 86.6 74.5 73.2 56.8 2.7 22.6 - * - *

Feed 0.6% (soluble solids 6.07 mg/ml); VCR-5.

Turbidity, transmission @ 660 nm and color measured at a strength of 0.25 g/200 ml; RT; Room temperature, 26°C; Cold, 5-6°C; L, Lightness; +a, red; -a, green; +b, yellow; *Not

observed.

Values are expressed as mean ± standard deviation (n=2 runs) of triplicate analyses.

Mean values, denoted by a different letter along a column, are significantly different at P 0.05 with respect to an increase in membrane MWCO/pore size.

113

Fig. 5.1. Visual spectra of black tea extracts

values correlated better with the corresponding TSS values for RT (r2 = 0.796)

as well as cold stored (r2 = 0.729) extracts suggesting its greater dependency

on the solids present in the extract.

The Hunter colour values, ‘a’ and ‘b’ of the crude black tea extract were

positive, suggesting that red and yellow are the principal colours in black tea

extracts. There are two predominant groups of pigments in black tea infusion;

TFs are reddish-orange in colour and TRs are red-brown pigments (Owuor et

al., 2006). Hunter color values measured immediately after processing

showed that lightness ‘L’ increased while redness and yellowness decreased

with membrane processing. Lower MWCO UF membranes (25-100 kDa)

drastically affected redness value of the tea extract while yellowness was not

greatly affected except with UF-25 membrane (Table 5.1). Evans et al. (2008)

114

made a similar observation wherein UF significantly removed haze with a

corresponding reduction in lightness. According to these researchers, this

would enable using higher solids concentrations of ultrafiltered solutions in

iced tea production. Instead, it may be desirable to improve the clarity without

losing much of the original color of tea liquor. Accordingly, MF seemed to be

more advantageous than UF. The results also showed that it is appropriate to

measure clarity in terms of turbidity rather than absorbance or transmission or

lightness ‘L’ values.

Tea cream formation leads to precipitation affecting the stability of tea

products and it is expected that a well clarified product is less prone to tea

cream formation. The tea cream particles make the light scatter when the

light passes through the infusion, giving the infusion a hazy appearance

(Liang and Xu, 2003). Tea cream formation was not observed with any of the

membrane processed samples for 30 days even under cold conditions of

storage (Table 5.1). Although these measurements were not made at a

uniform concentration, the original concentrations (TSS 1.83-4.19%) were well

above the normal beverage strength (0.25 g/200 ml; 0.125%). These results

showed that both UF and MF meet the primary requirements of processing, in

terms of improving clarity and retarding tea cream formation. However, MF

retained the original tea colour components to a greater extent and as such

may be preferred over UF.

5.3. Permeation characteristics of tea solids

The economic feasibility of a process very much depends on the yield which

is measured in terms of permeated solids in a membrane clarification process.

115

Rejection of tea components including cream components such as proteins

and pectins increased with decrease in MWCO/pore-size of the membranes

(Table 5.2). Influence of membrane pore-size on permeation of various tea

components is presented in Figure 5.2. Tea solids including polyphenols

showed a significant increase in their permeation with pore-size. Permeation

of catechins was much greater than total polyphenols owing to their relative

size and composition. Proteins and pectins are generally considered to be

predominantly associated with cream formation. Permeation of pectins did

not increase to the extent of total tea solids while proteins showed a similar

trend as that of total tea solids in the UF range. The results advocated the

necessity to look at higher pore-size membranes in place of low MWCO UF

membranes conventionally employed for clarification purposes.

Permeation of solids increased with increase in pore-size and the

recovery ranged between 18% and 63% at VCR 5. The highest solids

recovery was obtained with MF-450 membrane. In a similar manner,

permeation of polyphenols varied between 13% and 51% with a bearing on

the pore size of the membrane and the lowest recovery was obtained with UF-

25 membrane. Ramarethinam et al. (2006) observed a 43% reduction in

polyphenols during UF of green tea extract. PES has been earlier proposed

as one of the quality parameters while optimizing the extraction conditions in

terms of tea quality (Chapter 3). In a similar manner, polyphenol content-in-

permeated solids (PPS) could be used as a quality index for membrane

clarified products. Membrane processing reduced the PPS as the molecular

size of majority of non-polyphenolic components such as caffeine and sugars

are smaller facilitating their permeation over high molecular weight

116

Table 5.2

Rejection of tea components by UF and MF membranes

Membrane pore Rejection (%) PPS TF/TR Polyphenols/

size / MWCO Soluble solids Polyphenols Caffeine Protein Pectin (g/g) Pectin

Feed 0.573 0.036 62.4

UF permeates

25 kDa 77.41±0.03a 83.40±0.14

a 67.65±0.07

a 80.50±0.28

a 86.00±0.42

a 0.421 0.048 73.9

50 kDa 73.37±0.09b 80.55±0.07

b 58.75±0.07

b 75.05±0.27

b 83.90±0.00

a 0.419 0.040 75.5

100 kDa 62.65±0.16c 74.00±0.14

c 34.75±0.06

c 60.35±0.14

c 80.25±0.21

b 0.400 0.038 82.1

500 kDa 42.79±0.07d 56.65±0.07

d 6.40±0.21

d 42.98±0.28

d 69.35±0.35

c 0.434 0.033 88.3

MF permeates

200 nm 25.89±0.03e 39.95±0.07

e 2.85±0.05

e 40.80±0.25

e 65.85±0.35

d 0.464 0.034 109.7

450 nm 21.43±0.21f 35.90±0.00

f 1.05±0.07

f 39.70±0.21

f 62.10±0.57

e 0.467 0.035 105.5

Feed 0.6%: Soluble solids 6.07±0.15; Polyphenols 3.48±0.12; Protein 0.58±0.14; Pectin 0.055±0.011 mg/ml; Caffeine 310±10 µg/ml; VCR-5.

PPS-Polyphenols in permeated solids

Values are expressed as mean ± standard deviation (n=2 runs) of triplicate analyses.

Mean values, denoted by a different letter along a column, are significantly different at P 0.05 with respect to an increase in membrane MWCO/pore size.

117

Fig. 5.2 Influence of membrane pore size on permeation of various

tea components

polyphenols, specifically TRs. PPS was higher in MF (0.464-0.467 g/g) than

UF (0.400-0.434 g/g) permeates, however, both were much lower than the

feed (0.573 g/g). TRs are responsible for colour, body and taste while TFs

determine the briskness, brightness and quality of the liquor (Venkateswaran

et al., 2002). Higher TF/TR ratio is positively correlated to tea quality and

market price (Cloughley, 1980). The molecular size of TFs is in the range of

500-3000 Da while that of TRs is 700-40,000 Da (Todisco et al., 2002).

Consequently, low MWCO UF membranes showed higher TF/TR value in

their processed stream compared to feed owing to the rejection of high

molecular weight TRs (Table 5.2). On the other hand, TF/TR values of MF

permeate was closer to feed indicating that these membranes did not exhibit

118

any significant selectivity between TFs and TRs.

Caffeine binds only weakly to many tea cream components supporting

that it does not play an essential part in, and is not required for the initiation of

tea cream process (Jobstl et al., 2005). Caffeine stimulates tea cream

formation only in the presence of sufficient substances containing galloyl

group (Liang and Xu, 2003). There was practically no elimination of caffeine

during MF as indicated by their very low rejection values (Table 5.2). Evans

et al. (2008) showed that the caffeine transmitted through UF membranes

easily and was thus found in higher relative concentrations in the permeated

solids. It is a low molecular weight compound (194 Da), nevertheless its

concentration varied in the UF permeates; rejection increased with decrease

in MWCO of the membrane (Table 5.2) which suggested that

permeation/rejection is not only controlled by the actual pore size of the

membrane but also by the dynamic layer formation. It is a natural

phenomenon in feeds containing substances such as proteins and pectins to

have the tendency to form a secondary layer on the actual membrane surface

which would act as a dynamic membrane layer. Similar observation has been

reported during membrane processing of honey containing naturally occurring

enzymes (Barhate et al., 2003) and soy protein extracts (Vishwanathan et al.,

2011).

Earlier studies revealed that tea cream components are also co-

extracted in direct proportion along with other tea components (Chapter 3).

Apart from major tea cream components, TFs and TRs, other tea components

like caffeine, protein, pectin and calcium also co-precipitate along with

insoluble complexes (Liang et al., 2002). A widely supported hypothesis is

119

that complexation is exhibited by both polyphenols-protein and caffeine-

polyphenols in tea cream formation (McManus et al., 1981). Protein is a

major compound that participates in tea cream formation by forming

complexes with polyphenols and smaller compounds (McManus et al., 1985).

Todisco et al. (2002) attempted to eliminate proteins in tea infusion, by

employing ultrafiltration (MWCO 40 kDa). The ultrafiltered product thus

obtained remained stable up to 2 months but the extent of elimination of

protein was not established. In the present study, low MWCO UF membranes

displayed greater rejection of protein compared to MF membranes (Table

5.2). Accordingly, proteins were eliminated to an extent of ~81% by UF-25

while the elimination was only ~40% by MF membranes. TCA solubility test

revealed that the majority of these protein components extracted into the tea

extract were NPN compounds, such as theanine, amino acids and peptides

besides caffeine. Their molecular sizes are much smaller compared to true

proteins, which explained the lower rejection experienced by MF membranes.

The endogenous polysaccharide, especially polygalacturonic acid,

takes part in tea cream formation in black tea by forming loosely bound

complexes with polyphenolic and proteinaceous materials (Millin et al., 1969).

There is little information about the complexation of pectin with other

constituents of tea. Owing to its larger molecular size, pectins were

eliminated to a larger extent by UF (71-88%) and MF (66-68%) membranes.

Even partial removal of pectins could retard generation of secondary

cloudiness and sedimentation in products (Nosaka, 1993). Kawakatsu et al.

(1995) reported pectin elimination to the extent 30-50% in green tea

employing UF (MWCO 30-100 kDa). Although PP/pectin ratio improved with

120

all the membranes used in the study, the ratio was much higher with MF

membranes (Table 5.2).

Following fermentation of black tea, only approximately 10% of the tea

catechins are oxidized to TFs and 75% are converted to TRs while 15%

remained as simple catechins (Wu et al., 1998). The molecular size of four

major catechins, EC, EGC, ECG and EGCG are 290.27, 306.27, 442.37 and

458.37 Da, respectively. Permeation of catechins was relatively higher owing

to their smaller molecular size (290-458 Da) compared to oxidized

polyphenols, TFs and TRs (Tables 5.2 and 5.3). Among the individual

catechins, EC displayed the highest permeation followed by EGC, ECG and

EGCG in accordance with their molecular sizes. Although catechins were

much smaller in their molecular size compared to the MWCO of the UF

membranes used in the study, low MWCO UF membranes rejected these

compounds to a substantial extent that could be attributed to the dynamic

layer formation. Permeation of EC and EGCG in UF-500 were 96.2% and

86.4%, respectively while the permeation of individual catechins was above

95.7% in MF-450.

Although both UF and MF improved clarity and retarded tea cream

formation in tea extracts, high MWCO UF (500 kDa) and MF membranes

offered additional advantages in terms of greater retention of original tea colour

and recovery of tea solids including polyphenols in the clarified extract.

Besides, these membranes resulted in higher polyphenols-pectin ratios

indicating that permeation of desirable tea components like polyphenols were

higher than the undesirable components like pectin. This is likely to lead to

reduced tea cream formation in the processed extract. Accordingly,

121

Table 5.3

Individual catechin contents and recovery in membrane processed black tea extracts

Membrane pore

size / MWCO

Catechins (µg/ml) Total

catechins

EC EGC ECG EGCG Total Recovery (%)

Feed 18.35±0.10 96.61±0.13 67.83±0.12 185.03±0.12 367.82

UF Permeates

25 kDa 5.42±0.03 30.93±0.12 20.82±0.16 54.01±0.02 111.18 24.18

50 kDa 11.01±0.10 55.30±0.11 35.61±0.02 76.43±0.12 178.35 38.79

100 kDa 14.20±0.26 65.42±0.13 44.86±0.01 95.91±0.11 220.39 47.93

500 kDa 17.60±0.15 88.64±0.09 60.12±0.10 159.93±0.10 326.29 71.97

MF Permeates

200 nm 17.81±0.15 91.53±0.13 62.95±0.10 162.83±0.10 335.12 72.89

450 nm 18.15±0.19 94.42±0.10 65.57±0.12 177.10±0.13 355.34 77.29

Feed 0.6% (soluble solids 6.07 mg/ml); VCR-5.

Values are mean ± standard deviation of duplicate analyses.

122

subsequent studies were planned with UF-500 and MF membranes at various

higher feed concentrations to assess the recovery and productivity under

diafiltration mode of operation.

5.4 Diafiltration of black tea extracts

5.4.1 Storage stability of processed extracts

Membrane processing was carried out at four different feed concentrations

and the primary and the composite permeates obtained after diafiltration were

stored under RT and cold conditions. Turbidity and tea cream content were

measured in permeates immediately after processing and also after storage

(Table 5.4 and 5.5). Clarity of membrane processed tea extracts decreased

with diafiltration and increase in feed concentration at all conditions,

immediately after processing as well as under various storage conditions.

The reduction in clarity with increase in concentration was higher with

increase in pore size. However, diafiltration did not significantly affect clarity

at higher feed concentrations (5% and 10%). RTD tea beverage is generally

considered clear if the turbidity is below 50 NTU (Tsai, 1987). However, for

assessing the membrane clarification performance it was considered

appropriate to set a notional limit of at least 50% reduction in normal tea brew

turbidity (11.8 NTU). Accordingly, UF produced membrane permeates at all

feed concentrations meeting this criteria (<5.9 NTU) even after 30 days of

cold storage, while MF gave acceptable clarity in the processed extract only at

lower concentrations (0.6% and 2%). Lightness increased with diafiltration

while it decreased with increase in feed concentration up to 2% beyond which

it remained nearly unaffected (Table 5.5).

123

Table 5.4

Influence of feed concentration and diafiltration on clarity of membrane processed black tea extracts

Membrane pore Turbidity (NTU)

size / MWCO RT Cold

Storage days 0 30 0 30

P1 PC P1 PC P1 PC P1 PC

Feed 0.6% 11.75±0.05 23.10±1.53 13.72±1.21 25.22±1.14

500 kDa 0.25±0.03a 0.59±0.01

a 1.83±0.05

a 2.03±0.03

a 0.27±0.01

a 0.86±0.01

a 3.26±0.10

a 3.49±0.04

a

200 nm 0.52±0.04b 0.86±0.01

b 2.16±0.15

b 2.25±0.02

b 0.37±0.01

b 1.25±0.02

b 3.93±0.08

b 4.30±0.03

b

450 nm 0.60±0.07b 1.24±0.01

c 2.85±0.13

c 2.95±0.02

c 0.40±0.01

b 1.45±0.04

c 4.04±0.21

b 4.58±0.06

b

Feed 2% 13.81±0.02 22.83±0.02 14.92±0.01 27.41±0.01

500 kDa 0.47±0.01a 0.75±0.01

a 1.76±0.03

a 1.96±0.03

a 0.56±0.03

a 0.96±0.01

a 3.48±0.02

a 4.75±0.04

a

200 nm 0.59±0.01b 0.99±0.01

b 1.88±0.02

b 2.19±0.02

b 1.26±0.03

b 1.55±0.04

b 4.08±0.04

b 5.09±0.04

b

450 nm 0.90±0.01c 1.90±0.01

c 3.24±0.04

c 3.85±0.01

c 1.36±0.04

c 1.98±0.01

c 4.89±0.04

c 5.18±0.03

b

Feed 5% 33.82±0.02 42.91±0.01 34.91±0.01 37.34±0.02

500 kDa 0.79±0.01a 0.86±0.01

a 2.07±0.04

a 2.49±0.01

a 0.55±0.04

a 0.96±0.01

a 2.49±0.04

a 2.77±0.00

a

200 nm 0.99±0.01b 1.25±0.01

b 2.36±0.03

b 2.59±0.01

b 1.25±0.01

b 1.55±0.04

b 5.17±0.06

b 7.06±0.00

b

450 nm 1.89±0.00c 1.96±0.01

c 3.85±0.04

c 4.07±0.04

c 1.86±0.04

c 1.98±0.01

c 6.78±0.06

c 8.75±0.05

c

Feed 10% 45.52±0.02 52.92±0.02 46.82±0.02 55.23±0.02

500 kDa 1.24±0.01a 1.46±0.01

a 2.46±0.03

a 2.65±0.05

a 0.85±0.04

a 1.07±0.04

a 3.81±0.08

a 4.73±0.01

a

200 nm 1.99±0.01b 2.17±0.01

b 3.06±0.03

b 3.48±0.02

b 1.38±0.06

b 1.88±0.06

b 5.83±0.01

b 7.20±0.08

b

450 nm 3.20±0.01c 3.17±0.02

c 5.45±0.04

c 5.76±0.03

c 2.79±0.09

c 3.18±0.01

c 9.19±0.06

c 10.77±0.09

c

P1 - First permeate (80% permeation); PC - Composite permeate (Feed 0.6%, 6 step DF with 180 ml; Feed 2-10%, 9 step DF with 270 ml).

Turbidity measured at a strength of 0.25 g/200 ml; RT- Room temperature, 26°C; Cold 5-6°C. Values are expressed as mean ± standard deviation of duplicate analyses.

Mean values, denoted by a different letter along a column within the same group of feed strength, are significantly different at P 0.05 with respect to an increase in membrane MWCO/pore size.

124

Table 5.5

Influence of feed concentration and diafiltration on stability and colour of membrane processed black tea extracts

Membrane pore Tea cream (µg/ml)

Hunter colour size / MWCO L a b

Storage days 0 30 0 0 0

P1 PC P1 PC P1 PC P1 PC P1 PC

Feed 0.6% 17.51±0.42 52.42±0.07 48.2 5.3 24.6

500 kDa - * 0.15±0.01

a -

* 0.43±0.01

a 59.7 60.2 1.3 1.8 22.3 20.5

200 nm - * 0.41±0.01

b -

* 0.96±0.01

b 56.4 58.9 3.1 3.5 22.2 21.4

450 nm - * 0.64±0.02

c -

* 1.04±0.03

b 56.8 58.2 2.7 3.8 22.6 21.6

Feed 2% 69.32±0.07 111.21±0.07 45.1 6.7 25.8

500 kDa 0.33±0.04a 0.41±0.01

a 0.58±0.07

a 0.67±0.01

a 50.1 58.0 1.1 1.0 19.8 18.4

200 nm 0.64±0.02b 0.75±0.04

b 1.54±0.02

b 2.05±0.01

b 49.4 50.1 3.5 3.1 20.1 19.0

450 nm 1.06±0.01c 1.14±0.02

c 1.92±0.01

c 2.48±0.01

c 48.6 53.1 3.8 4.2 23.8 20.3

Feed 5% 380.03±0.07 398.04±1.13 43.2 8.1 27.1

500 kDa 1.07±0.01a 1.75±0.02

a 1.63±0.01

a 2.64±0.02

a 50.0 58.0 1.0 0.8 19.1 18.1

200 nm 1.53±0.01b 1.79±0.01

a 3.91±0.01

b 4.79±0.01

b 49.5 53.8 3.6 3.0 21.2 19.0

450 nm 2.52±0.01c 2.83±0.02

b 4.84±0.02

c 5.25±0.01

c 50.6 55.2 4.1 4.2 24.2 20.3

Feed 10% 502.51±0.19 584.51±0.71 40.1 9.0 28.3

500 kDa 1.83±0.03a 2.34±0.20

a 2.93±0.04

a 4.12±0.13

a 50.2 57.2 1.4 0.9 20.3 19.2

200 nm 4.03±0.03b 4.82±0.01

b 5.78±0.02

b 6.58±0.02

b 48.1 52.8 3.8 3.2 20.1 20.3

450 nm 5.31±0.02c 6.06±0.03

c 6.61±0.01

c 7.91±0.02

c 50.1 52.1 5.0 4.1 22.1 20.3

P1 - First permeate (80%); PC - Composite permeate (Feed 0.6%, 6 step DF with 180 ml; Feed 2-10%, 9 step DF with 270 ml). Color measured in RT stored samples at a strength of 0.25

g/200 ml; L, Lightness; +a, red; +b, yellow; Tea cream values measured in cold stored samples and normalized to a uniform solids concentration of 0.125%; *Not observed.

Values are expressed as mean ± standard deviation (n=2 runs) of duplicate analyses. Mean values, denoted by a different letter along a column within the same group of feed strength,

are significantly different at P 0.05 with respect to an increase in membrane MWCO/pore size.

125

Tea cream formation in processed extracts increased with feed

concentration as well as with storage which was higher with MF compared to

UF. Normalized teas cream values of crude and membrane processed

extracts are presented in Table 5.5. DF facilitates greater recovery of solids

as well as other tea compounds responsible for cream formation. As a result

DF generally increased the cream formation at all feed concentrations.

However, the reduction in tea cream formation achieved by UF and MF

membranes at lower feed concentrations was of one order of magnitude in

membrane processed extracts even after 30 days of cold storage (<2.48

µg/ml) compared to fresh crude extract (17.51µg/ml), indicating their

acceptability.

5.4.2. Recovery of tea solids and polyphenols

Increase in feed concentration affected the recovery of solids and

polyphenols. When the feed concentration was increased from 0.6% to 10%,

solids recovery decreased from 45.6% to 37.9% with UF-500 membrane in a

single step process. The drop in polyphenols recovery was more drastic from

34.6% to 8.6%. DF improved the recovery of solids, polyphenols and TFs in

the processed stream under all conditions (Table 5.6). Solids and

polyphenols recovery improved with each step up to three steps of diafiltration

beyond which the increase was insignificant (Figs. 5.3 and 5.4) suggesting a

limit for diafiltration. Although DF displayed greater influence with increasing

feed concentration, recoveries at higher feed concentrations could not match

0.6% feed concentration. Expectedly recoveries were greater with larger pore

size MF membrane. Highest recoveries of solids (76.1%) and polyphenols

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Table 5.6

Solids and polyphenols recovery and productivity during diafiltration of black tea extracts

Membrane pore Recovery (%) Solids Solids

size / MWCO Total soluble solids Total polyphenols PPS (g/g) TF/TR Processing processed permeated

P1 Pc P1 Pc P1 Pc P1 Pc time (h) (kg/m2/h) (kg/m

2/h)

Feed 0.6 %

500 kDa 45.60±0.14a 52.60±0.28

a 34.60±0.14

a 54.52±0.21

a 0.434 0.591 0.035 0.037 3.2 0.075 0.039

200 nm 59.40±0.13b 71.25±0.07

b 48.15±0.21

b 72.15±0.49

b 0.463 0.584 0.035 0.037 2.1 0.112 0.080

450 nm 62.50±0.14c 76.10±0.01

c 51.35±0.07

c 76.30±0.28

c 0.470 0.574 0.035 0.038 1.9 0.127 0.096

Feed 2%

500 kDa 41.10±0.14a 57.30±0.14

a 23.35±0.07

a 45.23±0.14

a 0.403 0.500 0.034 0.037 8.7 0.102 0.061

200 nm 50.75±0.07b 67.05±0.07

b 29.50±0.42

b 59.40±0.14

b 0.431 0.565 0.033 0.037 6.0 0.148 0.090

450 nm 51.40±0.14b 69.15±0.06

c 30.65±0.21

b 61.50±0.15

b 0.424 0.572 0.034 0.038 5.5 0.161 0.112

Feed 5%

500 kDa 34.40±0.14a 55.55±0.07

a 21.55±0.07

a 43.70±0.28

a 0.379 0.485 0.035 0.036 41.5 0.051 0.028

200 nm 43.45±0.07b 66.30±0.14

b 25.45±0.07

b 54.55±0.07

b 0.355 0.501 0.035 0.037 28.5 0.075 0.049

450 nm 47.85±0.06c 68.35±0.07

c 25.70±0.28

b 59.80±0.14

c 0.325 0.532 0.035 0.039 24.0 0.089 0.061

Feed 10%

500 kDa 37.85±0.07a 51.65±0.21

a 8.60±0.14

a 21.70±0.28

a 0.152 0.280 0.033 0.036 69.5 0.058 0.030

200 nm 39.75±0.05b 61.45±0.21

b 21.70±0.28

b 47.70±0.14

b 0.364 0.511 0.035 0.036 55.5 0.073 0.045

450 nm 43.75±0.07c 66.40±0.28

c 23.65±0.21

c 52.45±0.21

c 0.359 0.523 0.035 0.037 48.0 0.077 0.051

P1- First permeate (80% permeation); PC- Composite permeates after diafiltration (3 step DF with 90 ml).

Feed 0.6%: Soluble solids 6.07±0.15; Polyphenols 3.48±0.12 mg/ml: PES 0.573 g/g; TF/TR 0.036

Values are expressed as mean ± standard deviation of duplicate analyses.

Mean values, denoted by a different letter along a column within the same group of feed strength, are significantly different at P 0.05 with respect to an increase in membrane

MWCO/pore size.

127

Fig. 5.3 Improvement in tea solids recovery during diafiltration

128

Fig. 5.4 Improvement in polyphenols recovery during diafiltration

129

(76.3%) were obtained with MF-450 membrane at 0.6% feed concentration

under DF mode. As discussed earlier, PPS should be a primary parameter

which should be looked at along with TF/TR ratio for optimizing the membrane

process conditions in terms of tea quality. Improvement in polyphenols

recovery was much higher compared to solids recovery with all the three

membranes under all conditions of feed concentrations employed in DF mode

of operation. Although PPS value improved with DF, improvement beyond

the feed value was achieved only at lower feed concentrations.

Membrane processing did not drastically alter the TF/TR ratio (Table

5.6). During primary processing TF/TR ratios reduced marginally, however,

improved marginally above the feed value during the subsequent DF step.

Caffeine was rejected to a small degree with increase in concentration, more

so with UF-500. Expectedly DF facilitated their permeation (Table 5.7) but did

not reach the extent of dilution (97.5%) employed owing to the secondary

layer formation. Any reduction in caffeine content is likely to positively

influence the briskness index of tea beverage (Venkateswaran et al., 2002).

Permeation of four major catechins was according to the reverse order

of their molecular size in the primary step; EC recovery was higher followed

by EGC, ECG and EGCG. However, level of recovery of individual catechins

was nearly equal to one another after diafiltration. Total recovery of catechins

in primary and composite permeates increased with pore size of the

membrane and decreased with increase in feed concentration (Table 5.7).

DF improved the total catechins recovery by 6.5% to 14.5% and the maximum

recovery of catechins obtained was 89.9% as against 76.3% of polyphenols

(450 nm; 0.6% feed).

130

Table 5.7

Recovery of caffeine and catechins during diafiltration of black tea extracts

Recovery (%)

Membrane pore size/

Caffeine EC EGC ECG EGCG Total catechins

MWCO P1 Pc P1 Pc P1 Pc P1 Pc P1 Pc P1 Pc

Feed 0.6 %

500 kDa 75.9±0.10 84.8±0.12 76.7±0.17 86.3±0.17 73.4±0.13 81.9±0.13 71.1±0.12 83.3±0.15 69.1±0.14 81.9±0.12 71.4 83.4

200 nm 77.7±0.12 87.5±0.13 78.2±0.14 89.6±0.13 75.8±0.16 88.6±0.14 74.2±0.17 88.4±0.13 73.5±0.12 88.7±0.11 74.3 88.8

450 nm 79.2±0.11 93.0±0.16 80.1±0.15 90.5±0.12 78.2±0.17 90.2±0.15 77.4±0.14 89.6±0.17 76.5±0.10 89.1±0.10 77.3 89.9

Feed 2%

500 kDa 72.5±0.10 77.2±0.10 74.7±0.13 81.0±0.14 70.2±0.16 76.7±0.10 70.9±0.11 78.1±0.13 69.3±0.11 78.5±0.10 69.9 78.6

200 nm 74.0±0.12 83.7±0.12 75.4±0.12 82.3±0.12 74.8±0.13 81.3±0.12 75.0±0.10 83.6±0.10 73.0±0.11 84.8±0.12 73.8 83.0

450 nm 77.0±0.10 87.1±0.13 78.6±0.11 84.4±0.11 76.0±0.12 83.2±0.13 76.8±0.10 82.5±0.10 75.2±0.12 81.8±0.10 75.7 83.0

Feed 5%

500 kDa 70.6±0.10 74.7±0.11 72.4±0.10 77.9±0.10 67.7±0.10 75.7±0.13 66.3±0.13 75.8±0.10 64.3±0.12 72.4±0.13 65.7 75.5

200 nm 72.2±0.11 80.4±0.17 73.4±0.13 80.1±0.12 73.6±0.11 79.8±0.11 70.3±0.10 80.1±0.11 68.5±0.12 76.9±0.15 70.1 79.2

450 nm 74.0±0.10 84.1±0.15 75.7±0.13 82.3±0.13 74.8±0.13 82.9±0.14 72.0±0.10 82.8±0.10 70.6±0.10 78.0±0.10 71.9 81.5

Feed 10%

500 kDa 64.8±0.13 71.7±0.16 68.6±0.14 73.4±0.15 66.6±0.15 72.5±0.10 64.3±0.11 70.9±0.12 61.5±0.14 69.5±0.10 63.7 71.6

200 nm 69.5±0.12 76.9±0.13 70.0±0.10 76.2±0.13 70.2±0.16 76.1±0.13 69.4±0.10 75.6±0.10 67.3±0.13 72.8±0.13 68.7 75.2

450 nm 72.1±0.15 79.6±0.15 71.3±0.10 80.6±0.15 72.3±0.14 80.0±0.14 71.1±0.10 79.1±0.10 70.5±0.10 78.2±0.10 71.4 79.5

P1 - First permeate (80% permeation); PC - Composite permeate (Feed 0.6%, 6 step DF with 180 ml; Feed 2-10%, 9 step DF with 270 ml).

Feed 0.6%: Soluble solids 6.07 mg/ml; Caffeine 313.0; EC 18.3; EGC 96.5; ECG 67.2; EGCG 187.4 µg/ml.

Values are mean ± standard deviation of duplicate analyses.

131

Pectins (64.5-74.7%) were eliminated to a larger extent and did not

vary much with either feed concentration or membrane pore size in the

primary step (Table 5.8), as their rejection was controlled by the dynamic

membrane layer. Despite their relatively larger molecular size, DF facilitated

their permeation, leading to reduction in the overall elimination of pectins

(45.7-52.5%). Nevertheless, the reduction of pectins achieved is substantial

and expected to offer a significant benefit considering their role in tea cream

formation.

Minerals are rapidly extracted in to black tea infusion (Pintauro, 1970).

Magnesium content was higher followed by calcium, iron and zinc contents in

the raw and membrane processed extracts (Table 5.8). Chao and Chiang

(1999b) investigated the participation of various minerals in semi-fermented

(Paochung) tea and reported that calcium is the only mineral significantly

involved, with ~66% of it present in the original infusion participating in the

cream formation. Also in green and oolong tea infusions, calcium ions were

more easily precipitated with polyphenols than other cations (Guo and Chen,

1991). All the minerals including calcium were eliminated only to a lesser

extent which reduced further when followed by DF (Table 5.8). The storage

stability established with membrane clarified extracts suggested that minerals

such as calcium are not playing a vital role in the clarified environment of tea

extracts.

132

Table 5.8

Influence of diafiltration on elimination of pectin and minerals in membrane processed black tea extracts

Elimination (%)

Membrane pore Pectin Calcium Magnesium Iron Zinc

size / MWCO P1 Pc P1 Pc P1 Pc P1 Pc P1 Pc

0.6% extract

500 kDa 71.30±0.14a 51.25±0.07

a 21.51±0.01 12.61±0.01 24.31±0.11 18.41±0.08 24.81±0.03 13.41±0.01 25.23±0.01 17.83±0.02

200 nm 67.85±0.07b 49.50±0.42

a 21.42±0.02 11.64±0.02 22.21±0.01 17.32±0.03 24.44±0.02 12.21±0.02 21.54±0.02 15.42±0.01

450 nm 66.65±0.21b 47.35±0.07

a 21.43±0.04 10.41±0.01 23.74±0.03 10.31±0.01 24.22±0.01 10.82±0.01 20.22±0.03 10.54±0.01

2% extract

500 kDa 72.20±0.14a 52.50±0.28

a 29.54±0.02 24.21±0.03 22.53±0.04 18.43±0.02 23.23±0.04 15.32±0.02 23.42±0.05 19.32±0.03

200 nm 68.40±0.14b 48.30±0.00

b 28.53±0.01 20.44±0.01 22.01±0.01 16.44±0.05 24.64±0.05 14.43±0.03 24.44±0.04 18.23±0.02

450 nm 64.45±0.35c 45.70±0.14

c 28.42±0.01 19.42±0.02 20.60±0.01 15.35±0.07 25.46±0.02 13.63±0.01 22.71±0.01 17.11±0.01

5% extract

500 kDa 73.30±0.14a 52.30±0.00

a 24.61±0.02 18.51±0.02 20.82±0.02 19.21±0.02 25.27±0.05 18.54±0.01 25.62±0.03 21.61±0.04

200 nm 68.40±0.14b 48.45±0.21

b 24.32±0.01 18.22±0.03 21.42±0.03 17.52±0.03 23.61±0.04 16.67±0.06 23.34±0.02 19.70±0.02

450 nm 68.70±0.28b 47.40±0.28

b 22.54±0.02 20.72±0.02 21.63±0.06 14.23±0.05 20.82±0.07 14.66±0.01 25.76±0.01 18.66±0.07

10% extract

500 kDa 74.70±0.14a 52.15±0.21

a 25.23±0.02 19.33±0.02 22.55±0.02 19.55±0.03 22.53±0.08 19.11±0.02 27.42±0.03 22.63±0.03

200 nm 73.10±0.14a 49.45±0.49

b 25.31±0.04 21.22±0.01 21.86±0.03 18.63±0.04 23.61±0.09 17.62±0.03 21.54±0.04 19.61±0.06

450 nm 70.60±0.00b 47.40±0.14

b 24.52±0.01 22.41±0.01 21.64±0.07 15.42±0.01 22.60±0.05 15.34±0.01 21.93±0.01 18.52±0.04

P1- First permeate (80% permeation); PC - Composite permeate (Feed 0.6%; 6 step DF with 180 ml, Feed 2-10%, 9 step DF with 270 ml).

Feed 0.6%: Soluble solids: 6.07±0.15; Pectin: 0.056±0.011 mg/ml; Minerals: Calcium 1.58±0.12; Magnesium 3.69±0.14; Iron 0.87±0.12; Zinc 0.29±0.11 mg/l.

Pectin and mineral values are mean ± standard deviation of duplicate analyses.

Mean values, denoted by a different letter along a column within the same group of feed strength, are significantly different at P 0.05 with respect to an increase in membrane MWCO/pore

133

5.4.3. Membrane selection

Membrane processing took a longer processing time at higher feed

concentrations. Processing time reduced with increase in membrane pore-size,

accordingly higher the pore-size greater was the throughput. When the

productivity was analysed in terms of throughput/processed solids, process

duration and membrane area, it revealed that 2% feed concentration gave higher

productivity followed by 0.6% concentration (Table 5.6). The trend remained

more or less same when the productivity was analysed in terms of permeated

solids (Fig 5.5). Assessment of primary quality criteria, turbidity and tea cream

contents suggested that both 0.6% and 2% feed concentrations were acceptable.

Besides, another quality parameter, PPS also favoured only the above two feed

conditions. DF helped to improve the TF/TR ratio of composite permeates

marginally higher than the original level of feed under all conditions employed.

However, solids and polyphenols recoveries obtained with 0.6% feed

concentration and MF-450 combination were excellent with any other

combination employed (Fig 5.6 and 5.7). Diafiltration was effective in improving

solids and polyphenols recovery but suggested a limit (100-150%) so that

elimination of pectins is not dropped below 50% (Fig 5.8). Accordingly, it is

desirable to employ MF as a means of clarification of tea extracts, preferably at

low feed concentration under diafiltration mode of operation.

5.5 Reject stream of membrane clarification process

Membrane clarification process resulted in a yield of ~76% of the total feed solids

134

Fig. 5.5 Productivity during membrane processing at various feed

concentrations (DF- 3 steps)

Fig. 5.6 Recovery of tea solids during membrane processing at various feed

concentrations (DF- 3 steps)

135

Fig. 5.7 Recovery of polyphenols during membrane processing at

various feed concentrations (DF- 3 steps)

Fig. 5.8 Effect of diafiltration on tea components (DF-3 steps)

136

with a nearly matching recovery of polyphenols (MF-450 nm at 0.6% feed

concentration after 150% DF). This analysis revealed that the retentate stream

of the membrane process is not to be treated as a reject waste stream as it

contains a substantial amount of polyphenols. Therefore, a comparative

assessment of anti-oxidant potential of tea solids present in various membrane

process streams was carried out employing DPPH method to evolve a

comprehensive solution for the clarification process.

5.5.1 Antioxidant activity and phenolic content of black tea solids

A two-step 100% DF improved the polyphenols recovery from 51% to 65% in the

clarified extract. The radical scavenging activities (RSA) of tea solids of crude

and clarified extracts were not significantly different from each other while

retentate solids showed much greater RSA at all dosages (Fig. 5.9). After DF,

RSA marginally reduced in the cumulative permeate while drastically affected the

final retentate. Although higher phenolic content-in-solids (PS) could be probably

attributed to greater RSA in the primary retentate tea solids, RSA of other

process stream samples did not exhibit a high correlation corresponding to their

PS (Table 5.9). The composition of tea components in the membrane process

streams and their antioxidant contributions were analysed further to explain the

observed phenomenon.

Membrane processing in DF mode of operation (MF-450 at 0.6% feed

concentration) was described in the earlier sections. DF improved polyphenols

137

Fig. 5.9 Scavenging activity of black tea solids of membrane process

streams on DPPH radicals

recovery enabling PS value of processed/permeated solids equal to that of the

feed value.TF/TR ratio also improved marginally above the feed value. The

recovery of catechins (89.8%) was higher than polyphenols (76.3%). Mineral

contents including magnesium, calcium, iron and zinc were least affected by

membrane processing. The protein and pectin contents in the tea extract solids

were about 10% and 1%, respectively. From the permeation characteristics, it

could be construed that elimination of pectins was largely responsible for the

improved cold storage stability of membrane processed extracts.

138

Table 5.9

Phenolic content and ED50 value (DPPH) of black tea solids of various

membrane process streams

Sample ED50 (µg)

PS (g/g-tea solids) Aqueous extract Ethanol extract

Feed 0.584 48.16 ± 0.08 41.30 ± 0.14

MF fractions

P1 0.490 49.55 ± 0.07 47.85 ± 0.07 R1 0.899 27.65 ± 0.21 25.65 ± 0.07

DF fractions

PC 0.457 50.15 ± 0.07 47.10 ± 0.14 RF 0.399 43.45 ± 0.07 39.85 ± 0.07

P1 and R1 are primary permeate (80% permeation) and retentate; PC and RF are composite permeate and final retentate after 100% diafiltration, respectively. PS, polyphenols in tea solids.

ED50 of Trolox was 21.9 µg

Values are expressed as mean ± standard deviation of duplicate analyses.

The above analysis suggested that tea solids of feed, permeate and

retentate were significantly different only in terms of their protein and pectin

contents. On the other hand, the health benefits are associated not only with

polyphenols but also with other tea components such as polysaccharides,

proteins and pigments, but to varying extents. Yu et al. (2007) reported that

antioxidant activity by DPPH radical scavenging method decreased in the

following order for green tea solids: crude tea polyphenols > crude tea proteins >

crude tea polysaccharides with corresponding effective concentration values

(EC50) of 4.89, 41.04 and 376.39 µg/ml. Although roasted green tea contains

less total phenolics, it showed higher scavenging activity compared to oolong

and black tea. Satoh et al. (2005) reasoned out that this could be due to the

139

presence of non-phenolic antioxidant substances. Accordingly, proteins and

pectins would also contribute for the overall antioxidant activity of tea solids.

Hence, significant differences in RSA of tea solids were not observed among

crude extract, cumulative permeate and final retentate (Fig 5.9) despite

differences in their PS (Table 5.9), owing to contributions from other tea

components. The study revealed that although final retentate (Fig. 5.9) stream

may not be suitable for beverage applications, it is as good as original tea extract

in terms of its antioxidant activity (Fig. 5.9) indicating its suitability as a tea

conserve in functional foods.

5.5.2 Antioxidant scavenging efficacy of tea solids

The ED50 value is the dose of an antioxidant material required to scavenge 50%

of DPPH radicals. Lower ED50 value would reflect greater antioxidant activity of

the sample and vice versa. The ED50 values of various membrane fractions

(Table 5.9) were obtained from dose response curves of tea solids (Fig. 5.9).

The first retentate obtained in the primary MF step showed higher antioxidant

potential compared to its corresponding feed and permeate. After DF, there was

a significant drop in the PS of the final retentate. The process duration of primary

run is about 3 h while the final retentate remained in the process for nearly 16 h

which could have had a bearing on the polyphenolic activity of the retained

140

solids. Nevertheless, large difference in ED50 values between the final retentate

and permeates was not noticed which could be probably explained only by the

antioxidant activity contribution from other tea components. It emphasizes the

necessity to consider the combined effects of the complex bioactive compounds

when considering the overall benefits from the conserve.

5.5.3 Trolox equivalent antioxidant capacity (TEAC)

TEAC is a comparative antioxidant activity measure of bioactive compounds in

the sample with respect to a water soluble form of α-tocopherol (Trolox). TEAC

was calculated as the ratio between the slopes of the dose response curves of

tea solids sample and Trolox (Fig. 5.9). TEAC values of various membrane

fractions (Fig. 5.10) fell in a narrow range (0.438 – 0.504 µg/µg-tea solids) except

for the primary retentate (0.796 µg/µg-tea solids) in agreement with their

corresponding RSA and ED50 values. These TEAC values (~0.5 µg/µg-tea

solids) are quite significant indicating that the black tea solids possess

substantial antioxidant potential.

5.5.4 Ethanolic extraction of black tea solids

As an attempt to enrich the antioxidant compounds of tea solids, ethanol

extraction was employed to black tea solids obtained from different membrane

141

process streams. The TEAC of final retentate was 0.504 µg/µg-tea solids which

improved to 0.550 µg/µg-tea solids after enrichment by ethanolic extraction (Fig.

5.10). Ethanolic extracts of retentate samples showed 8-9% higher antioxidant

potential compared to the corresponding aqueous extract samples (Table 5.9).

Fig. 5.10 TEAC and polyphenols in tea solids (PS) of various

membrane process streams

(P1 and R1 are primary permeate and retentate; PC and RF are composite permeate and final

retentate)

The approach needs a detailed investigation to establish and exploit its potential

as a worthwhile enrichment method.

142

5.6 Conclusions

Membrane clarification of tea extracts offers the possibility of reducing tea cream

formation and haze in RTD tea and improve its stability during refrigerated

storage while retaining most of the natural quality characteristics of tea. The

study revealed that MF is preferred over UF membranes hitherto generally

employed for clarification of black tea extracts, meeting the requirements of RTD

tea beverages without any compromise on tea quality parameters. The results

also showed that processing crude extracts at low concentrations (<2.0%) offer

greater recovery of solids and polyphenols besides greater productivity per unit

area of membrane surface. Furthermore, the antioxidant potential (DPPH) of

reject stream tea solids of membrane process was comparable to the original tea

solids of the crude extract, suggesting that the final retentate stream containing a

substantial amount of tea solids could be converted in to a tea conserve. It is

emphasized that the clarification process needs are to be benchmarked in terms

of low turbidity, high retention of polyphenols, high recovery of tea solids and

storage stability, besides utilization of reject stream.