Recycling of Tea Waste: Simple and Effective Separation of Caffeine

Int. J. Sec. Metabolite, Vol. 4, Issue 2 (2017) pp. 78-89

Research Article

ISSN: 2148-6905 online Journal homepage: http://www.ijate.net/index.php/ijsm

Recycling of Tea Waste: Simple and Effective Separation of Caffeine and

Catechins by Microwave Assisted Extraction (MAE)

Gönül SERDAR

, Ezgi DEMİR, Münevver SÖKMEN

Karadeniz Technical University, Faculty of Science, Department of Chemistry, 61080, Trabzon, Turkey

Received: 24 August 2016 - Revised: 22 December 2016 - Accepted: 01 January 2017

Abstract: This study was designed to develop a cost effective green extraction method to separate valuable

chemicals from black tea waste (BTW) and tea dust called “caffeine dust” (CD). These materials are totally

unwanted residue and treated as rubbish. Two extraction methods were used for extraction of waste materials

obtained in three collection periods. In conventional extraction method, material was subjected to hot water

extraction at 80

C for 40 minutes. In microwave assisted extraction (MAE) method, different eco-friendly aqueous

solvents (water, citric acid-water or ethanol-water) were used under a controlled 600 W microwave power at 80

C for only 4 minutes treatment in a close microwave system. MAE and conventional method gave similar extract

yields for both waste materials but MAE was more successful and economical completing the extraction in shorter

time. Using citric acid-water solvent in MAE significantly increased the extraction of individual catechins such as

epicatechin (EC) and epigallocatechin gallate (EGCG). This study showed that tea waste could be utilized for

production of valuable chemicals using nontoxic solvents at extremely short extraction periods.

Keywords: Tea waste, caffeine, catechins, microwave assisted extraction,

1. Introduction

Black tea is one of most favored drink in all societies. Different methods are used for

black tea production to obtain its red bright infusion. Basic steps for production line are

withering, rolling, fermentation and drying. Among them, fermentation step is the most

significant process of making black tea since it defines the quality of the tea.

In Turkey, tea is planted in Eastern Black Sea region and harvested in three collection

periods; first collection is in May, second is in June, and the third is in August-September.

Almost half of black tea is processed by the government based tea processing plants in seven

grades [1]. All tea factories use there main techniques namely orthodox, rotorvan and CTC

(curling, tearing, crushing) or combination of these techniques. Çaykur is a government based

company and use either orthodox (nonpressing orthodox+sieving+pressing orthodox) or

modified Çaykur method (nonpressing orthodox+rotorvan+sieving+conical orthodox). During

these processes tea leaves are broken down to small particles and this is not desired by tea

producers. Small particles are separated by sieving or air flow from black tea that has a market

value. This waste called “caffeine dust” and produced at large quantities. It is totally unwanted

Corresponding Author Phone: +90 462 377 2532 Fax: +90 462 325 3196

E-mail: msokm[email protected]

International Journal of Secondary Metabolite: Vol. 4, Issue 2 (2017) pp. 78-89

residue and treated as rubbish or burned for energy production. Another waste material is also

produced during production of black tea. This material is different than caffeine dust and mainly

contains cellulosic residue of tea stalks. Çaykur sells 200.000 tones waste material in 2014 and

guaranties that they will supply 20.000 tons/year for next 10 years. According to new legislation

in Turkey damping these waste materials are not allowed and it must be consumed in an

appropriate way. Burning is one of the possible option but created massive air pollution.

On the other hand both black tea waste (BTW) and caffeine dust (CD) contain the same

compounds as black tea has: tea polyphenol, caffeine, amino acids, saponins, tannins, etc., [2].

Tea polyphenols are catechins, flavanols, phenolic acids, flavanonens, glycosides and plant

pigments [3] . The catechins can be categorized into two groups based on their structure:

epistructured catechins and non-epistructured catechins. The epistructured catechins are

epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin

gallate (ECG), of which EGCG is the major constituent and the most powerful one. Non-

epistructured catechins are gallocatechin (GC), catechin (C), gallocatechin gallate (GCG), and

catechin gallate (CG) [4]. This group of compounds is known responsible from anticarcinogenic

and antimutagenic properties of tea, as well as protective action against cardiovascular diseases.

They are good antioxidant agents and have been used to enhance the shelf-life of food products

[5]. Therefore, these waste materials can be utilized for production of valuable chemicals such

as caffeine and catechins. But an economical and eco-friendly extraction method should be used

for separation of these chemicals from tea waste. Extraction time and consumed energy is an

important issue to process the waste materials at large quantities.

Conventional solid-liquid extraction is one of the methods that are used for effective

extraction of caffeine and catechins from tea samples. Many different treatment steps were used

for extraction of caffeine and catechins that mainly consists hot water as extracting solvent [6-

12].

Other methods have been proposed for effective extraction of chemicals from tea. A

review published by Vuong et al. (2011) contains all important techniques for the extraction of

tea samples. Although hot water extraction methods are quite simple and easy they require long

extraction periods and energy that are not feasible for large scale production. Separation of

valuable chemicals from tea waste at large quantities need sophisticated techniques that: i) has

relatively lower and economical extraction periods ii) provide high extract yields and iii)

selective to target compounds.

Microwave assisted extraction (MAE) is a new techniques that requires lower extraction

period and less energy consumption. Microwave power produces high temperature regions that

ease the disruption of the cell wall and facilitates the extraction. As a result of these effects

caffeine and catechins are readily extracted into the extracting solvent [2,13]. Recently Nkhili

et al. (2009) has reported a study for extraction tea using a 600 W microwave oven. The

temperature was controlled between 80 and 100

C for 30 min and the solvent to tea ratio was

20:1 (ml/g). Under these conditions the extraction efficiency for the tea catechins was reported

to be higher than for the conventional hot water methods. Therefore, MAE of tea waste might

be a good approach to meet all requirements.

The objective of this study is to establish MAE methods for cost effective extraction of

caffeine and catechins from black tea waste (BTW) and caffeine dust (CD). Two extraction

methods, conventional hot water and microwave assisted extraction (MAE), were employed

and different extracting solvents were tested for MAE. Waste material was collected from the

same tea factory in different collection periods and subjected to extraction. After successive

separation of caffeine and catechins from extraction medium these constituents were quantified

by chromatographic analyses to determine the amount of individual catechin. The results of this

study can be used as reference for recycling of these waste materials for cost effective

Serdar, Demir & Sökmen

production of valuable chemicals. This is the first report employing MAE for extraction,

isolation and quantification of industrial important chemicals from waste materials such as

caffeine dust.

2. Material and Methods

2.1. Standards and chemicals

Methanol, ethanol, ethyl acetate and chloroform were analytical grade from Merck. The

standard chemicals of (−)epigallocatechin (EGC), (−)epicatechin (EC), (−)epigallocatechin

gallate (EGCG), (−)epicatechin gallate (ECG) and caffeine were purchased from Sigma (St

Louis, MO, USA). Black tea waste and caffeine dust used in the experiments were collected

from Sürçay San. Co. Ltd. (Sürmene, Trabzon, Turkey). All experiments works were repeated

three times.

2.1.2. Conventional hot water extraction

Briefly, 10 g of waste material was extracted with 200 ml of pure water at 80 °C for 40

min. Residue was removed by filtration using a filter paper (pore size: 5 μm) and filtrates were

initially partitioned with chloroform to remove caffeine. Then aqueous phase was extracted

with ethyl acetate three times using 150 ml ethyl acetate in each extraction to separate catechins.

The chloroform and ethyl acetate phases were ﬁltered and the ﬁltrate was concentrated by a

rotary evaporator under reduced pressure at 55 °C.

2.1.3. Microwave assisted extraction (MAE)

Tea catechins and other components were extracted using a close microwave assisted

extraction system (MILESTONE, START S Microwave, USA). MAE parameters such as

microwave power, extraction time and extracting solvent can affect the efficiency [14]. 10 g

of sample and 200 ml extracting solvent were placed in vessel and soaked for 90 minutes at

room temperature. Presence of citric acid or ethanol facilitates the selective extraction of

catechins [4]. Therefore, three extracting solvents (water, citric acid-water or ethanol-water)

were used. Citric acid-water solvent was prepared using 0.1 M citric acid and water (1:1).

Ethanol-water was prepared diluting technical ethanol (96%) with equal volume of water. Then

sample was transferred into microwave extraction apparatus [3]. Extraction was carried out

under a controlled 600 W microwave power at 80

C temperature for 4 min irradiation time

[15]. After extraction, the flask was allowed to cool down room temperature before opening the

cap. Aqueous infusion was extracted with first chloroform then ethyl acetate as described

above.

2.1.4. Quantification of caffeine and catechins

In conventional and MAE quantification of caffeine and catechins were carried out by

weighting the chloroform and ethyl acetate fractions after evaporation the solvent and drying

the extract. Extract yields were calculated from the mass of extracts. HPLC analyses were

carried out to determine the amount of each catechins present in the mixture [16]. The

instruments used in this study was a HPLC system (Hitachi Elite Lachrom) equipped with a

Shim-pack VP-ODS C18 column (5 mm, 4.6 x 250 mm, 35 °C) at 278 nm. Solvents A (water)

and B (DMF-methanol-acetic acid mixture, 20:1:0.5) were run with 86% A for 13 minutes then

its volume was decreased to 64% within next 15 minutes and finally back to initial

concentration for another 6 min. Concentrations of catechins were quantiﬁed by their peak areas

against those of standards prepared from original compounds.

International Journal of Secondary Metabolite: Vol. 4, Issue 2 (2017) pp. 78-89

3. Results and Discussion

3.1. Extraction yields

3.1.1.Conventional Extraction

Waste materials were collected at three collection and processing times in 2013. The

average caffeine and catechin yields from the first (in May, I. Collection), second (in June, II.

Collection) and third collection period (in August-September, III. Collection) using

conventional extraction method is given in Figure 1.

Figure 1. Caffeine and catechin yields of black tea waste (BTW) and caffeine dust (CD) in three

collection periods. Data are expressed as the mean of three replicate.

Figure 1 shows that caffeine yields of both waste materials are quite high. Processed black

tea usually contains 2-4% caffeine and Serpen et al. (2012) reported the caffeine content of

black tea produced in the same region was between 1.5-1.8%. Their results were solely obtained

from HPLC analysis and separation of caffeine from tea extract had not been carried out. In

I. Collection II. Collection III.Collection

Extraction Yield %(w/w)

Caffeine

BTW

I. Collection II. Collection III.Collection

Extraction Yield %(w/w)

Caffeine

BTW

Serdar, Demir & Sökmen

BTW caffeine yields were similar at first two collection periods (6.12 and 6.72% respectively).

BTW collected in the third collection period gave lower caffeine extract (4.25%). CD gave

higher caffeine yields than BTW at all collection periods (ranging between 6.72-7.31%).

Although catechin content of BTW was slightly higher than CD both waste materials

have lower catechin yields. However, catechin yield was slightly higher in BTW collected in

the third collection period (2.27%).

3.1.2 Microwave Extraction of BTW and CD

Waste material was subjected to MAE in different solvents for a 4 minutes exposure

period. Extract yields (%, w/w) obtained from black tea waste and caffeine dust is given in

Figure 2 and 3.

Figure 2. Caffeine yield of a) black tea waste (BTW) and b) caffeine dust (CD) in three collection

periods. Data are expressed as the mean of three replicate.

First of all, caffeine yields in both waste materials were high as much as conventional hot

water extraction. Extract yields varied between 4.36-7.42% in all collection periods and solvent

systems. The mass of caffeine extracts was highest in CD reaching 7.42% (w/w, 74.2 mg/g tea

waste). Caffeine content is usually between 2-4% in black or green tea that means 20-40 mg

caffeine can be obttained from 1 gram of tea material [1]. In current literature there are

published studies related to extraction of caffeine from green tea and tea waste. Water and

supercritical carbon dioxide extraction (SFE) of caffeine from tea plant waste (mainly stalks)

was reported by İçen and Gürü [12, 17, 18]. They reported that the maximum yield of caffeine

from tea stalk wastes and fiber wastes were 14.95 mg/g and 18.92 mg/g, respectively.

According to another study carried out by Shalmashi et al., (2010) supercritical water extraction

I. Collection II. Collection III.Collection

Extraction Yield %(w/w)

Water

Citric Acid

Ethanol

I. Collection II. Collection III.Collection

Extraction Yield %(w/w)

Water

Citric Acid

Ethanol

International Journal of Secondary Metabolite: Vol. 4, Issue 2 (2017) pp. 78-89

of Iranian tea waste produced 0.77% (w/w) of caffeine while conventional hot water extraction

yielded only 0.46% (w/w). Our results show that caffeine content of tea waste used in this study

is extremely high and should be considered as a good source for caffeine extraction. Using

water as extraction solvent seems to be more appropriate providing the same amount of caffeine

in a 4 minutes extraction period for all collection periods. Citric acid-water or ethanol-water

mixture does not alter the extraction yields except for third collection period of BTW. Using

ethanol as co solvent in MAE increased the caffeine yield to 6.46%.

BTW and CD yielded similar amount of catechin extracts (Figure 3). The yields of BTW

were between 1.41-2.27% for conventional extraction and 1.82-2.45% for MAE in water. Using

citric acid-water or ethanol-water solvents slightly increase the catechin yields. It was the

highest in the third collection period.

Figure 3. Catechin yield of a) black tea waste (BTW) and b) caffeine dust (CD) in three collection

periods. Data are expressed as the mean of three replicate.

MAE of caffeine dust gave higher yields than conventional hot water extraction especially

in third collection period. Ethanol-water solvent increased catechin yields in some collection

periods but there was no constant improvement. But it is clear that extraction of catechins

employing MAE is more promising since the same amount of extract was obtained after just 4

minutes treatment. Microwave seems to be facilitating the extraction of catechins into

extracting solvent via heat zones formed in waste material (Oliveira and Franca, 2002). The

temperature is localized in zones that ease the selective migration of target compounds from

the material in shorter time.

I. Collection II. Collection III.Collection

Extraction Yield % (w/w)

Water

Citric Acid

Ethanol

I. Collection II. Collection III.Collection

Extraction Yield %(w/w)

Water

Citric Acid

Ethanol

Serdar, Demir & Sökmen

Extraction yield calculated from extract mass can give a rough idea about how much

caffeine and catechin can be isolated from extracting medium. Caffeine extraction with

chloroform provides pure caffeine but separation of catechins by liquid-solid extraction is not

purely selective and ethyl acetate can remove some other principles as well as catechins.

Quantification of individual catechins present in the extract should be done for proper

evaluation. Therefore, HPLC analyses were carried out to determine the real amount of each

catechin present in the extracts.

3.1.3. HPLC analysis

Caffeine and catechin constituents of the extracts were determined by reversed-phase

HPLC. A standard mixture is used for identification and quantification of individual catechin.

HPLC chromatogram of standard mixture containing catechins, gallic acid and caffeine is given

in Figure 4.

Figure 4. HPLC chromatogram of the catechin standard mixture. Gallic acid and caffeine were included

into the standard mixture. Retention times are as following; gallic acid (GA): 7.00; caffeine: 11.03;

epigallocatechin (EGC): 13.44; catechin (C): 16.05; epicatechin (EC): 20.44; epigallocatechin gallate

(EGCG): 22.79 min. (=280 nm, injection volume: 20 µL).

The identification of catechins was carried out by comparing their retention times to

standards and the amount of individual catechin was calculated from these chromatograms. The

amounts of individual catechins present in the extract might give a good idea to compare

extraction efficiency. Therefore quantified catechins from the HPLC chromatograms, along

with gallic acid and caffeine residue, in the extracts obtained by MAE are given in Figure 5.

International Journal of Secondary Metabolite: Vol. 4, Issue 2 (2017) pp. 78-89

Serdar, Demir & Sökmen

Figure 5. Quantification of catechins present in microwave extraction of BTW (a-c) and CD (d-f).

Quantification studies provided good information about which extraction solvent was

more fruitful for effective extraction of individual catechins. As seen from Figure 5a-c BTW

contains lower catechins than CD samples (Figure 5d-f). Concentration of EC is higher than

other catechins in both waste materials but CD has also higher amount of other catechins such

as EGC and EGCG. Extracted catechins were significantly high in the third collection period.

The amounts of extracted catechins, especially EC and EGCG, are quite high in MAE in water

and citric acid-water mediated systems. Citric acid must be providing an acidic medium for

successful separation of EC and EGCG. It should be noted that citric acid-water solvent system

extracts less caffeine than water mediated MAE. Therefore, it seems to be more selective to

extraction of catechins. The amount of catechins obtained form conventional hot water

extraction is given in Figure. 6.

International Journal of Secondary Metabolite: Vol. 4, Issue 2 (2017) pp. 78-89

Figure 6. Quantification of catechins present in conventional hot water extract of a) BTW and b) CD.

Hot water extraction provided lower catechin constituent than MAE. Extract obtained

from BTW has lower EC (ranged between 0.01-1.80 mg/g) and EGCG (0.28-0.82 mg/g) in all

collection periods (Figure 6a). In CD extracts, EC concentration was extremely low in the first

two collection periods. It increased to 5.21 mg/g in the third collection but still nearly half of

MAE of the same sample (Figure 6b).

It is clear that extract yields are not solid evidence how extraction is successful for

effective separation of these valuable chemicals. This study provides useful information related

to effective extraction of tea waste employing microwave power. Separation of catechins was

achieved in shorter extraction period (4 minutes), proposed MAE provide high extract yields

and it is selective to EC and EGCG. MAE can be used as an effective, economical and time

saving technique to obtain caffeine and catechins from tea wastes such as BTW and CD. Using

citric acid-water for extraction in MAE enhanced the EC and EGCG constituent and might be

used for selective extraction of these compounds.

The waste materials used in this study contain quite high amount of caffeine and

remarkable amount of catechins. As a result, tea waste produced in Black Sea region can be

considered as valuable bio-resource and MAE at large scale might be favored for energy cost

industrial applications.

GA Caffeine EGC C EC EGCG

mg/g BTW

I. Collection

II. Collection

III. Collection

GA Caffeine EGC C EC EGCG

mg/g CD

I. Collection

II. Collection

III. Collection

Serdar, Demir & Sökmen

Acknowledgements

The authors gratefully appreciate the financial support from Turkish Ministry of Industry

(SAN-TEZ Project, Grant No 00932.STZ.2011-1). Thanks to tea producers OrÇay, SurÇay,

Filiz Çay for their financial support.

Conflict of interest

This study does not have any conflict of interest.

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