A Review on Decaffeination of Tea Products

Zhen Wang，Qing-Sheng Li，Juan-Zhen Ni，Ci-Jie Hu，Jian-Liang Lu

Zhejiang University Tea Research Institute，866 Yuhangtang Road，Hangzhou 310058，PR China

1.Introduction

Tea(Camellia sinensis)is one of the most popular beverages consumed worldwide，and it contains many physiologically active substances.The main component catechins，such as epicatechin(EC)，epicatechin gallate(ECG)，epigallocatechin(EGC)，and epigallocatechin gallate(EGCG)，exhibit various pharmacological effects，such as antioxidative，antitumour，and antimicrobial activity.Also，an amino acid unique to tea，theanine，induces a relaxing effect and potentiates the effects of anticancer drugs.On the other hand，green tea also contains abundant caffeine(1，3，7-trimethylxanthine)，the most consumed alkaloid.It is one of the few plant products and the general public is readily familiar with，because of its occurrence in beverages such as tea and coffee，as well as various soft drinks.

Caffeine has some positive physiological effects;it has been linked with enhancement of cognitive function improvement of neuromuscular coordination，elevation of mood and relief of anxiety.But when the doses of caffeine is high，it stimulates the central nervous，affects sleep，increases blood pressure，improves the prevalence of rheumatoid arthritis，mutagenic and causes premature infants［1-4］.Therefore，to keep the tea flavors and nutritions of low caffeine tea is welcomed by consumers.

Generally，the content of caffeine in dry tea is 2%-5% ，in fresh tea leaves is 1.4%2.8%［5］.At present，the content of caffeine in decaffeinated tea is no uniform standard，countries such as Europe and the United States require caffeine content lower than 0.5%，while China，Japan demand caffeine content lower than 1%.

2.Approaches of decaffeination

The methods of decaffeination are Water extraction，Organic solvents extraction，Activated carbon(AC)［6］，Supercritical fluid extraction with carbon dioxide(SFE-CO2)，Removing caffeine with absorbents which are made of various copolymers of triallyl isocyanurate(TAIC)or vinyl acetate(VA)［7］or molecular imprinted polymers(MIPs)［8］，Reducing caffeine using microorganisms［9］，microwave irradiation［10］，and ultrasonic devices［11］，Ionic liquid extraction［12］etc.

2.1.Water extraction

2.1.1.Decaffeination with hot water

At room temperature，caffeine dissolves best in chloroform 18% (w/v)but in water the solubility is approximately 2%(w/v).However，caffeine dissolves well in boiling water，with its solubility increasing to 70%(w/v)at 100℃.So hot water can be used to remove the caffeine in tea.

Domestic removing caffeine with water began in 1995.Liang et al.［13］also studied the removal of caffeine in fresh tea leaves with hot water，through to the temperature，soaking time and the ratio of water to tea，the research found that 1:20(w/v)of tea to water under 100℃ for 3 min as the best parameters，can remove 83%of caffeine，at the same time retains 95%of catechins.Liang et al.applied for a patent that used fresh tea leaves to produce low caffeine tea powders.The method directly took the fresh tea leaves as raw material，after more than 125℃steam and 100℃ hot water treatment，chopped，extraction，concentration，spray drying or freeze drying to make low caffeine and instant tea powders.

Hot water treatment is a convenient and inexpensive decaffeination process and leaves no organic solvent residue.At the same time hot water plays a role of inhibiting the activities of certain enzymes［13］.However，a large percentage of tea catechins was lost if rolled leaves and dry tea were decaffeinated by the hot water treatment and so the process is not suitable for processing black tea.

2.1.2.Microwave-enhanced vacuum ice water extraction

On the basis of the researches of hot water decaffeination，Lou et al.［10］try to remove caffeine in low temperature(0℃)，and combined microwave technique.The experiment analysis found that the removal yield of caffeine by microwave-enhanced vacuum ice water extraction(MVIE)was 87.6%，which was significantly higher than that by hot water extraction.Moreover，the loss of tea polyphenols of green tea in the proposed method was much lower than that in the hot water extraction.

The experiment was operated as follows:The dry green tea(10 g)was mixed with water(100 mL)and was pretreated by microwave extraction.The next step was vacuum ice water extraction，and it was carried out in a vacuum drying oven.The pretreated tea sample was put into a flask containing 100 mL of ice water(ice/water=1∶1，w/w).The flask was placed into the vacuum drying oven.The air in the vessel was pumped out till the degree of vacuum was 0.1 MPa.Then the vacuum ice water extraction was performed for several hours.The optimized conditions were as follows:Solvent(mL)to solid(g)ratio was 10∶1，microwave extraction time was 6 min，microwave power was 350 W and 2.5 h of vacuum ice water extraction.

2.1.3.The application of water extraction

Vuong et al.［14］attempted to develop optimal conditions for decaffeination and spray drying procedures to produce decaffeinated and high caffeine powders from green tea(Camellia sinensis).The experiment was operated as follows:Blanching the tea leaves with water at 100℃ for 4 min at a water-to-tea ratio of 20∶1(mL/g)，then the water was decanted from the blanched tea leaves and the leaves were dried to constant weight at 70℃in a vacuum oven to give decaffeinated dried tea leaves.The decaffeinated dried tea leaves were ground，brew twice，spray drying(180℃ for the inlet temperature and 115℃ for the outlet temperature)to get the decaffeinated green tea powers(7 mg/g caffeine).

2.2.Decaffeination using liquefied dimethyl ether

Recently，attempts have been made to remove or reduce caffeine by extraction with organic solvents such as chloroform［15］.However，these solvents are toxic and easily retain in food because they exist in a liquid state at room temperature.

Kanda et al.［16］proposed an extraction technique involving the use of liquefied dimethyl ether(DME)as an extractant.Hideki et al.［17］used green tea(after hot water extraction with water content of 74.6%-76.2%)to verify the DME extraction in both laboratory-and bench-scale processes，and then made use of high-performance liquid chromatography to measure the contents distribution of caffeine and catechins in extracted residue，organic extracts，and removed water.It was found that caffeine was completely removed from the green tea leaves.Approximately 25.2%-56.0%of catechins remained in the residue after DME extraction.In particular，56.0%of epigallocatechin gallate，which has the greatest activity of all catechins remained in the residue.

The characteristics of dimethyl ether decaffeination:It is very different from the existing methods［18，19］，because the DME method can dewater(dry)and extract organic extracts from biomaterials simultaneously and directly at room temperature;this means that the heating of the extractant and the downstream hot-dry process are both omitted.Furthermore，DME is a safe solvent and does not remain at room temperature;therefore，it can be used for food processing，and the organic compounds can be used for beverage production with or without pretreatment.The dried and decaffeinated tea leaf residue can be used as non-caffeine tea food.

2.3.Removal caffeine by Supercritical carbon dioxide extraction(SFE-CO2)

The carbon dioxide decaffeination method was discovered by Kurt Zosel and patented in the early 1970s.Since then，research on decaffeination using SFE-CO2has been intensive and the resulting methods have been covered by several patents［20］and reported in other publications［21-24］.

The principle of SFE-CO2is that pressure and temperature can influence the solubility of supercritical fluid.At the critical temperature(Tc)and critical pressure(Pc)，supercritical CO2fluid has special peculiarities.Above the critical point，CO2fluid has quite a diffusion coefficient as well as gas and a low viscosity.And its density and solubility are as well as fluid.Within a certain range，there is a linear relationship between the logarithm of solubility and the logarithm of fluid density.Therefore，though controlling T and P，it can change the density，which changes the solubility of a substance，to make a selective extraction.In a supercritical state，the supercritical CO2fluid contacts with the substance to be separated，and then extracts the different ingredients selectively according to the boiling point and molecular weight，polarity of the different ingredients.Then with decompression and warming，the supercritical CO2fluid becomes ordinary gas，so that the extracted material is completely or substantially precipitated so as to achieve the purpose of separation and purification［25］.

The decaffeination of green tea using SFE-CO2is usually done by first grinding the tea leaves into small particle sizes(＜1 mm in diameter).The ground tea leaves are then soaked in a co-solvent(e.g.，95∶1，ethanol∶water)，to enhance the extraction of the caffeine［26］，and are loaded into an extraction vessel that is sealed.Liquid carbon dioxide is pumped in at a designated pressure and the back pressure is continuously monitored.The liquid carbon dioxide is then heated and pumped into the extraction vessel to extract the caffeine from the ground tea leaves.The caffeine，dissolved in the supercritical carbon dioxide，is separated from the carbon dioxide and collected by a reduction in pressure which occurs in the separator sections of the SFE-CO2extractor.

It should be noted that the outcomes of the decaffeination process using SFE-CO2are influenced by various factors including the tea particle size，the co-solvent used to soak the tea in and the temperature，pressure and flow rate of the CO2.Kim［27］et al.using the SFE-CO2to selectively extract caffeine from green tea，found that SFECO2with water as entrainer was more selective in green tea.Hacer Icen et al.［28］made use of Turkish black tea fibers and stalks wastes to study the effect of ethanol content on the SFE-CO2.Tu et al.［29］chose the complete roasted leaves(produced in Lin'an，Hangzhou sale)as raw materials for the study of SFE-CO2.Chen［30］chose green tea，black tea，Tie Guan Yin，Pu'er tea as materials for the study with SFE-CO2through single factor experiment and orthogonal experiment.

The main limitation of the SFE-CO2decaffeination method is the high setup costs.However，this is outweighed by several advantages such as it being a fast process with no toxic residues，less degradation of the tea catechins and a high retention of the tea flavors［31］.

2.4.Microorganism and enzymatic hydrolysis

Some microorganisms isolated from the environment can degrade the caffeine，for example Stemphyllium sp.［32］，Penicillium sp.and Aspergillus sp.［33，34］.Aspergillus，Penicillium and Rhizopus can degrade the caffeine in coffee pulp.

It is assumed that the degradation of caffeine in fungi takes place via the demethylation route，with theophylline as the first formed intermediate.In this respect，the caffeine degradation pathway in filamentous fungi closely resembles that of plants.Under aerobic conditions，bacteria and fungi decompose caffeine of nucleotide precursors:a.Fungi occurs theophylline metabolic pathways that caffeine degrades to xanthine.And these decomposition pathways are alike in plants.b.Bacterial decomposes caffeine initially produced theobromine，ultimately into xanthine.And the key to this catabolism is demethylase.c.Some bacteria oxidize caffeine to uric acid first like Rhodococcus and Klebsiella，then the uric acid continues the purine metabolic pathways.d.Caffeine can also indirectly be decomposed by xanthine oxidase.Compared with the aerobic degradation，the decomposition degree of caffeine is incomplete and the period is longer under anaerobic conditions.

Application of microbial degradation of caffeine:Some bacteria use the coffee husk into solid state fermentation，so the nutritional value of cattle and other livestock's feed can be improved.The research conducted by Gummadi et al.indicates that caffeine demethylase production kinetics was growth-related.

Caffeine degradation by microbes or microbial enzymes is effective in producing useful by-products besides offering advantages in decaffeination.Theophylline，a metabolite of caffeine degradation being an adenosine antagonist，has been shown to reduce the incidence of contrast-induced nephropathy，is considered as an alternative in the treatment of chronic obtrusive pulmonary disease.

2.5.Breeding new cultivars with low caffeine content

Cultivating low caffeine tea cultivars is the most effective way to produce low caffeine tea，and excellent germplasm resource is an important material basis for genetic technology research and cultivars innovation.In China，low caffeine resources are very abundant，such as Daba tea tree(caffeine content about 0.07%)，Jinchang plant tea tree(caffeine content about 0.06%)，Yanjin niuzhai tea(caffeine content ＜1.0%)and Houzhou tea(caffeine content ＜1.0%)，etc.

2.5.1.Conventinal crossbreeding breeding low caffeine tea cultivars

The most economical，safest and effective method to take off the caffeine is conventional crossbreeding.Generally，it is believed that tea caffeine content less than 2% can be considered low caffeine-specific resources［35］.

In Japan，they got 8 plant offsprings whose caffeine content are less than 1.7%by using a low caffeine content variety hybridizing with Yabukita since 1989 and the lowest is 1.42%.

Recently，Tea Research Institute of Guangdong Academy of Agricultural Sciences cooperated with Sun Yat-sen university and they used wild nankunshan primary tea as a parent to hybrid breeding a number of cocoa new lines，including"the 1st cocoa"and"the 2nd cocoa".The suitable system results of Cocoa tea for black tea，green tea，yellow tea and oolong showed that its inclusions are richful and it has sucrose incense，flower aroma and strong taste with slightly bitter.It may come to the conclusion that cocoa is capable of being domesticated as a home-grown tea and it can be developed into a natural non-caffeine beverage different from traditional tea.

2.5.2.Genetic engineering reduced tea caffeine

Currently the pathway about the biosynthesis of caffeine in tea has been basically clear，and the full-length cDNA sequences of the three key genes(SAM synthetase，inosine monophosphate dehydrogenase and caffeine synthase gene)have also been cloned.Therefore，the most convenient way to reduce the caffeine in tea is that using antisense RNA technology or RNAi inhibits the key enzyme gene expression in caffeine biosynthesis.But the Camellia sinensis is less sensitive to Agrobacterium，so conversion rate is very low and genetic transformation techniques in tea have been a world problem stuck in vector construction and transformation of post-resistant callus screening stage［36-39］until Indian scientists Mohanpuria et al.［40，41］introduced the RNAi vectors containing caffeine synthase gene fragment into tea cotyledon callus and the root of tea tree seedings germinating a month through particle bombardment and Agrobacterium method，and obtained respectively 11 and 7 of transgenic plants contained the vected successfully.The caffeine and theobromine in these plants are respectively 44%-61%and 46%-67%lower than control group.And the result not only confirms the possibility of genetic engineering technology to reducing caffeine in tea，but also firstly confirms caffeine synthase synthesis of both caffeine and theobromine.However，tea is a natural cross-pollination of plants with self-incompatibility characteristics，which is not necessary to pass the parents'genetic traits，because the transgenic plants is heterozygous using its cotyledon induced callus to transform or tea tree seedling roots from seed germination by direct conversion.Therefore，it remains to be established an efficient genetic transformation system，using the tea tree stems，leaves and roots，and other stable genetic characteristics of the parent organization as explants.Through the system，a genetically modified tea reducing the caffeine content，and maintaining good genetic traits from parents can be found.

3.Conclusions

The hot water treatment is a safe and inexpensive method for decaffeinating green tea，however，it seems only suitable for removal caffeine from fresh leaves.Some reports showed that DME have many advantages over other organic solvents.Having preliminary explored the laboratory and small-scale industrial production of the decaffeination using DME，the efficiency of the industrial mass decaffeinated using DME requires further study.The studies of SFE-CO2are more，and the technology is relatively mature，industrial production is feasible.Caffeine degradation by microbes or microbial enzymes is effective in producing useful by-products besides offering advantages in decaffeination.The demethylase and oxidases are involved in the microbial decaffeination process.Results showed purified demethylase is very unstable.Therefore future work pertaining to caffeine and its degradation can concentrate on the purification and characterisation of this enzyme.In the cultivation process，there are many issues that need further researches to clarify.Such as，the caffeine in tea(Camellia sinensis)is mainly synthesized in the chloroplast，and then transported to the vacuole，and combines with chlorogenic acid to form complexes，which gatheres in cell vacuole，but the transport mechanism is unclear;tea caffeine contents vary with cultivars，season，climate and cultivation measurements，and in different cells，tissues and organs，the factors which will affect the synthesis and decomposition of caffeine need to be further studied.

Acknowledgements

This study was financed by the National Natural Science Foundation of China(31170644)and Tea industry＆technology innovation strategic alliance foundation of Zhejiang Province(Project No.7).

1.Lane JD，Pieper CF，Phillips-Bute BG，Bryant JE，Kuhn CM.Caffeine affects cardiovascular and neuroendocrine activation at work and home.Psychosomatic Medicine，2002，64(4):595-603.

2.Shilo L，Sabbah H，Hadari R，Kovatz S，Weinberg U，Dolev S，Shenkman L.The effects of coffee consumption on sleep and melatonin secretion.Sleep Medicine，2002，3(3):271-273.

3.Smith A.Effects of caffeine on human behavior.Food and Chemical Toxicology，2002，40(9):1243-1255.

4.Wen W，Shu XO，Jacobs JrDR，Brown JE.The associations of maternal caffeine consumption and nausea with spontaneous abortion.Epidemiology，2001，12(1):38-42.

5.Lin YS，Tsai YJ，Tsay JS，Lin JK.Factors affecting the levels of tea polyphenols and caffeine in tea leaves.Journal of Agricultural and Food Chemistry，2003，51(7):1864-1873.

6.Ye JH，Liang YR，Jin J，Liang HL，Du YY，Lu JL，Lin C.Preparation of partially decaffeinated instant green tea.Journal of Agricultural and Food Chemistry，2007，55(9);3498-3502.

7.Ma N，Wang P，Kong X，Shi R，Yuan Z，Wang C.Selective removal of caffeine from tea extracts using macroporous crosslinked polyvinyl alcohol adsorbents.Journal of Separation Science，2012，35(1):36-44.

8.Hohnloser W，Osswald B，Lingens F.Enzymological aspects of caffeine demethylation and formaldehyde oxidation by Pseudomonas putida C1.Biological Chemistry，1980，361(2):1763-1766.

9.Gummadi SN，Bhavya B，Ashok N.Physiology，biochemistry and possible applications of microbial caffeine degradation.Applied Microbiology and Biotechnology，2012，93(2):545-554.

10.Lou Z，Er C，Li J，Wang H，Zhu S，Sun J.Removal of caffeine from green tea by microwave-enhanced vacuum ice water extraction.Analytica Chimica Acta，2012，716:49-53.

11.Tang WQ，Li DC，Lv YX，Jiang JG.Extraction and removal of caffeine from green tea by ultrasonic-enhanced supercritical fluid.Journal of Food Science，2010，75(4):C363-C368.

12.Yu HN.Study on the extraction and separation of caffeine in tea-leaf by ionic liquids.Master Degree Thesis，Northeast Petroleum University，2012.13.Liang H，Liang Y，Dong J，Lu J，Xu H，Wang H.Decaffeination of fresh green tea leaf(Camellia sinensis)by hot water treatment.Food Chemistry，2007，101(4):1451-1456.

14.Vuong QV，Golding JB，Nguyen MH，Roach PD.Preparation of decaffeinated and high caffeine powders from green tea.Powder Technology，2012，233:169-175.

15.Copeland EL，Clifford MN，Williams CM.Preparation of epigallocatechin gallate from commercial green tea by caffeine precipitation and solvent partition.Food Chemistry，1998，61:81-87.

16.Kanda H，Li P.Simple extraction method of green crude from natural blue-green microalgae by dimethyl ether.Fuel，2011，90(3):1264-1266.

17.Kanda H，Li P，Makino H.Production of decaffeinated green tea leaves using liquefied dimethyl ether.Food and Bioproducts Processing，2013，379-383.

18.Kanda H，Makino H.Energy-efficient coal dewatering using liquefied dimethyl ether.Fuel，2010，89(8):2104-2109.

19.Mehr C，Biswal R，Collins J，Cochran H.Supercritical carbon dioxide extraction of caffeine from guaran.The Journal of Supercritical Fluids，1996，9(3):185-191.

20.Margolis G，Chiovini J.Decaffeination process:Google Patents，1981.

21.Huang KJ，Wu JJ，Chiu YH，Lai CY，Chang CM.Designed polar cosolvent-modified supercritical CO2removing caffeine from and retaining catechins in green tea powder using response surface methodology.Journal of Agricultural and Food Chemistry，2007，55(22):9014-9020.

22.Kim WJ，Kim JD，Oh SG.Supercritical carbon dioxide extraction of caffeine from Korean green tea.Separation Science and Technology，2007，42(14):3229-3242.

23.Park HS，Choi HK，Lee SJ，Park KW，Choi SG，Kim KH.Effect of mass transfer on the removal of caffeine from green tea by supercritical carbon dioxide.The Journal of Supercritical Fluids，2007，42(2):205-211.

24.Park HS，Im NG，Kim KH.Extraction behaviors of caffeine and chlorophylls in supercritical decaffeination of green tea leaves.Lwt-Food Science and Technology，2012，45(1):73-78.

25.Jiang KM，Liu JW，Xie PM.Review on the technology of extraction natural caffeine from tea.Yun Nan Chemical Technology，1998，4:10-14.

26.Park HS，Lee HJ，Shin MH，Lee KW，Lee H，Kim YS，Kim KH.Effects of co-solvents on the decaffeination of green tea by supercritical carbon dioxide.Food Chemistry，2007，105(3):1011-1017.

27.Kim WJ，Kim JD，Kim J，Oh SG，Lee YW.Selective caffeine removal from green tea using supercritical carbon dioxide extraction.Journal of Food Engineering，2008，89(3):303-309.

28..I?en H，Gürü M.Effect of ethanol content on supercritical carbon dioxide extraction of caffeine from tea stalk and fiber wastes.The Journal of Supercritical Fluids，2010，55(1):156-160.

29.Bingxin T，Linjiang P，Yunxiang W，Qianxin G.Optimum technology on decaffeination of green tea by supercritical carbon dioxide.Journal of Agricultural Mechanization Research，2009，9:166-168.

30.Chen XZ.The study on the preparation technology of decaffeinated tea and the quality of decaffeinated tea，which based on supercritical CO2extraction technology.Master Degree Thesis，South China University of Technology，2011.

31.Kurtzman Jr R，Schwimmer S.Caffeine removal from growth media by microorganisms.Experientia，1971，27(4):481-482.

32.Schwimmer S，Kurtzman JrRH，Heftmann E.Caffeine metabolism by Penicillium roqueforti.Archives of Biochemistry and Biophysics，1971，147(1):109-113.

33.Hakil M，Denis S，Viniegra Gonzalez G，Augur C.Degradation and product analysis of caffeine and related dimethylxanthines by filamentous fungi.Enzyme and Microbial Technology，1998，22(5):355-359.

34.Roussos S，De los Angeles Aquiahuatl M，del Refugio Trejo Hern ndez M，Perraud IG，F(xiàn)avela E，Ramakrishna，Viniegra Gonz lez G.Biotechnological management of coffee pulp-isolation，screening，characterization，selection of caffeine-degrading fungi and natural microflora present in coffee pulp and husk.Applied Microbiology and Biotechnology，1995，42(5):756-762.

35.Yang Y，Yu F，Chen L，Zeng J，Yang S，Li S，Wang Y.Elite germplasm evaluation and genetic stability of tea plants.Journal of Tea Science，2003，23(S):1-8.

36.Ogita S，Uefuji H，Yamaguchi Y，Koizumi N，Sano H.RNA interference:producing decaffeinated coffee plants.Nature，2003，423(6942);823-823.

37.Yadav SK，Ahuja PS.Towards generating caffeine-free tea by metabolic engineering.Plant Foods for Human Nutrition，2007，62(4):185-191.

38.Yu YB.Inhibition of caffeine synthase gene in tea plant and its expression in other organism.Ph.D.Degree Thesis，Anhui Agricultural University，2004.

39.Zhang GH，Liang YR，LU JL，Dong JJ.Construction of tea caffeine synthase gene RNAi vector.Journal of Tea Science，2006，26(4):243-248.

40.Mohanpuria P，Kumar V，Ahuja PS，Yadav SK.Prouducing low-caffeine tea through post-transcriptional silencing of caffeine synthase mRNA.Plant Molecular Biology，2011，76(6):523-534.

41.Mohanpuria P，Kumar V，Ahuja PS，Yadav SK.Agrobacterium-mediated silencing of caffeine synthesis through root transformation in Camellia sinensis L.Molecular Biotechnology，2011，48(3):235-243.